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Research Article Molecular Analysis of a Recurrent Sarcoma Identifies a Mutation in FAF1 Georg F. Weber College of Pharmacy, University of Cincinnati, 3225 Eden Avenue, Cincinnati, OH 45267-0004, USA Correspondence should be addressed to Georg F. Weber; [email protected] Received 18 November 2014; Accepted 11 February 2015 Academic Editor: Enrique de Alava Copyright © 2015 Georg F. Weber. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A patient presented with a recurrent sarcoma (diagnosed as leiomyosarcoma) 12 years aſter the removal of an initial cancer (diagnosed as extracompartmental osteosarcoma) distally on the same limb. Following surgery, the sarcoma and unaffected muscle and bone were subjected to measurements of DNA exome sequence, RNA and protein expression, and transcription factor binding. e investigation provided corroboration of the diagnosis leiomyosarcoma, as the major upregulations in this tumor comprise muscle-specific gene products and calcium-regulating molecules (calcium is an important second messenger in smooth muscle cells). A likely culprit for the disease is the point mutation S181G in FAF1, which may cause a loss of apoptotic function consecutive to transforming DNA damage. e RNA levels of genes for drug transport and metabolism were extensively skewed in the tumor tissue as compared to muscle and bone. e results suggest that the tumor represents a recurrence of a dormant metastasis from an originally misdiagnosed neoplasm. A loss of FAF1 function could cause constitutive WNT pathway activity (consistent with the downstream inductions of IGF2BP1 and E2F1 in this cancer). While the study has informed on drug transport and drug metabolism pharmacogenetics, it has fallen short of identifying a suitable target for molecular therapy. 1. Introduction Sarcomas are cancers of mesenchymal origin [1] that com- prise about 1% of adult malignancies. Leiomyosarcomas are derived from smooth muscle cells. At most primary sites, other than the uterus or gastrointestinal tract, leiomyosar- comas are likely to originate from the tunica media of blood vessels. However, it has been postulated that primary leiomyosarcoma of the bone might also develop through advanced myogenic metaplasia of a sarcoma originating from fibroblastic tissue [2]. e disease typically occurs in the 5th to 6th decades of life, with women being affected more than men (2 : 1). is gender distribution may reflect the proliferation of smooth muscle that can occur in response to estrogen [3]. Because sarcomas tend to respond poorly to stan- dard chemotherapy, they have no good treatment options beside total excision with wide margins. However, the gradual replacement of the highly toxic conventional can- cer chemotherapy (comprising nonspecific antiproliferative agents) with molecularly targeted drugs, which was initi- ated with the market entry of neutralizing antibodies and small molecule kinase inhibitors in 1997 (Rituxan) and 2001 (Gleevec), respectively, has opened the possibility to tailor drug treatment to particular tumors. Yet, this transition has also necessitated the molecular characterization of the lesions that are causative for the transformation of healthy cells to cancerous cells because drugs need to be matched with the underlying carcinogenic defect to be effective. Here, we take a sarcoma through a comprehensive molecular analysis that applies multiple screening techniques, with the goal to identify the disease-causing defects as well as potential drug targets. 2. Materials and Methods 2.1. Patient and Tissues. A 52-year-old female patient under- went surgery for a recurrent sarcoma. Samples of skeletal muscle, bone, and tumor were obtained postsurgery. Hindawi Publishing Corporation Sarcoma Volume 2015, Article ID 839182, 20 pages http://dx.doi.org/10.1155/2015/839182

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Page 1: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

Research ArticleMolecular Analysis of a Recurrent Sarcoma Identifiesa Mutation in FAF1

Georg F Weber

College of Pharmacy University of Cincinnati 3225 Eden Avenue Cincinnati OH 45267-0004 USA

Correspondence should be addressed to Georg F Weber georgweberucedu

Received 18 November 2014 Accepted 11 February 2015

Academic Editor Enrique de Alava

Copyright copy 2015 Georg F Weber This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A patient presented with a recurrent sarcoma (diagnosed as leiomyosarcoma) 12 years after the removal of an initial cancer(diagnosed as extracompartmental osteosarcoma) distally on the same limb Following surgery the sarcoma and unaffected muscleand bone were subjected tomeasurements of DNA exome sequence RNA and protein expression and transcription factor bindingThe investigation provided corroboration of the diagnosis leiomyosarcoma as the major upregulations in this tumor comprisemuscle-specific gene products and calcium-regulating molecules (calcium is an important second messenger in smooth musclecells) A likely culprit for the disease is the point mutation S181G in FAF1 which may cause a loss of apoptotic function consecutiveto transforming DNA damage The RNA levels of genes for drug transport and metabolism were extensively skewed in the tumortissue as compared to muscle and bone The results suggest that the tumor represents a recurrence of a dormant metastasis froman originally misdiagnosed neoplasm A loss of FAF1 function could cause constitutive WNT pathway activity (consistent with thedownstream inductions of IGF2BP1 and E2F1 in this cancer)While the study has informed on drug transport and drugmetabolismpharmacogenetics it has fallen short of identifying a suitable target for molecular therapy

1 Introduction

Sarcomas are cancers of mesenchymal origin [1] that com-prise about 1 of adult malignancies Leiomyosarcomas arederived from smooth muscle cells At most primary sitesother than the uterus or gastrointestinal tract leiomyosar-comas are likely to originate from the tunica media ofblood vessels However it has been postulated that primaryleiomyosarcoma of the bone might also develop throughadvancedmyogenicmetaplasia of a sarcoma originating fromfibroblastic tissue [2] The disease typically occurs in the5th to 6th decades of life with women being affected morethan men (2 1) This gender distribution may reflect theproliferation of smooth muscle that can occur in response toestrogen [3]

Because sarcomas tend to respond poorly to stan-dard chemotherapy they have no good treatment optionsbeside total excision with wide margins However thegradual replacement of the highly toxic conventional can-cer chemotherapy (comprising nonspecific antiproliferative

agents) with molecularly targeted drugs which was initi-ated with the market entry of neutralizing antibodies andsmall molecule kinase inhibitors in 1997 (Rituxan) and 2001(Gleevec) respectively has opened the possibility to tailordrug treatment to particular tumors Yet this transition hasalso necessitated themolecular characterization of the lesionsthat are causative for the transformation of healthy cellsto cancerous cells because drugs need to be matched withthe underlying carcinogenic defect to be effective Here wetake a sarcoma through a comprehensive molecular analysisthat applies multiple screening techniques with the goal toidentify the disease-causing defects as well as potential drugtargets

2 Materials and Methods

21 Patient and Tissues A 52-year-old female patient under-went surgery for a recurrent sarcoma Samples of skeletalmuscle bone and tumor were obtained postsurgery

Hindawi Publishing CorporationSarcomaVolume 2015 Article ID 839182 20 pageshttpdxdoiorg1011552015839182

2 Sarcoma

22 DNA Exome Sequencing 1 120583g of dsDNA determined byInvitrogen Qubit high sensitivity spectrofluorometric mea-surement was sheared by sonication to an average size of300 bp on a Diagenode Bioruptor Automated library con-struction was performed on an IntegenX Apollo324 whichsize-selects fragments by double-SPRI binding with differ-ent concentrations of PEG for a high cut and a low cut Eachlibrary can be fitted with one of 48 adapters each contain-ing a different 6-base molecular barcode for high level multi-plexing After 12 cycles of PCR amplification 1 120583g of geno-mic library was recovered for exome enrichment usingthe NimbleGen EZ Exome V2 kit Enriched libraries weresequenced on an Illumina HiSeq2000 generating around 32million high quality paired end reads of 100 base each or64GB of usable sequence per sample The analysis methodsutilize the Broad Institutersquos GenomeAnalysis Toolkit (GATK)and follow a pipeline previously described [4] along withpublished modifications (httpwwwbroadinstituteorggsawikiindexphpThe Genome Analysis Toolkit) The analy-sis comprises aligning the reads that pass Illumina ChastityFilter with the Burrows-Wheeler Aligner (BWA) [5] For eachsample Picardrsquos MarkDuplicates are used to flag reads thatappear to be artifacts of PCR bias All reads that overlapknown or putative indels are realigned All base qualityscores are recalibrated to the empirical error rate derivedfrom nonpolymorphic sites The GATKrsquos Unified Genotypermodule is used to call variant sites (both single nucleotide andsmall indel) in all samples simultaneously Finally the SNVcalls are filtered using the variant quality score recalibrationmethod [4] Indel calls were filtered with a set of hard filtersas there are not enough indels in an exome to use theGaussianmethod

23 RNAseq Tissue samples were homogenized in RNazolRT (MRC) with a manual homogenizer and stored on iceuntil extractionThe RNA isolation was performed accordingto the manufacturerrsquos instructions

The Ovation RNA-Seq FFPE system (NuGen) was usedto initiate amplification at both 31015840 end as well as randomlythroughout the transcriptome in the sample 100 ng of totalRNA with RIN lt 50 was converted into a library of tem-plate molecules suitable for subsequent cluster generationand sequencing by Illumina HiSeq Total RNA was reversetranscribed and converted to double stranded cDNA witha unique DNARNA heteroduplex at one end NuGENrsquosRibo-SPIA technology was used for isothermal amplificationresulting in the rapid generation of cDNA with a sequencecomplementary to the original mRNA The cDNA was thendouble stranded and fragmented to 200 bp using CovarisS2 and a sequencing library was generated using IlluminarsquosTruSeq DNA Sample Prep Kit V2 according to standardprotocols The cDNA library was enriched by a limitednumber of 10 PCR cycles validated using an Agilent 2100Bioanalyzer and quantitated using the Quant-iT dsDNA HSKit (Invitrogen) Two individually indexed cDNA librarieswere pooled and sequenced on Illumina HiSeq to get aminimum of 90 million reads Libraries were clustered ontoa flow cell using Illuminarsquos TruSeq SR Cluster Kit v25 andsequenced 50 cycles using TruSeq SBS Kit-HS on HiSeq The

obtained sequence reads were aligned to the genome by usingthe standard Illumina sequence analysis pipeline

24 Protein-DNA Array Tissues were ground betweenfrosted glass slides and then incubated with Collagenaseand Dispase in cell culture medium at 37∘C for 45 minutesto release individual cells These cells were collected afterpassing the samples through a strainer and centrifugationNuclear extracts and cytosol were prepared using a kit fromActive Motif After protein determination DNA binding ofthe nuclear extracts was assessed with the Combo protein-DNA array (Panomics) Signal intensity was measured withthe software MetaMorph

25 Western Blotting For the analysis of individual proteinstissues were homogenized in RIPA buffer (50mM Tris-HClpH 75 150mM NaCl 1 NP-40 01 sodium dodecylsulfate) using a handheld battery-operated homogenizer10 120583g lysates were loaded per lane and electrophoresed on10 SDS-polyacrylamideminigels with reducing denaturingsample buffer The separated proteins were transferred toPVDF membranes and probed with antibody O-17 (IBC)to the C-terminus of osteopontin and anti-HCAM to thecytoplasmic domain of CD44 (Santa Cruz) and to STAT3 andphospho-STAT3 (Cell Signaling Technology) Antitubulinserves as a loading control

26 2DGel Electrophoresis andMass Spectrometry Thetumorand muscle samples were diluted to 4 and 1mgmL in 1 1diluted SDS Boiling Buffer Urea Sample Buffer before load-ing (the bone samplewas ethanol precipitated and redissolvedto 4 and 1mgmL in 1 1 diluted SDS Boiling Buffer UreaSample Buffer) Two-dimensional electrophoresis was per-formed according to the carrier ampholine method of iso-electric focusing [9 10] by Kendrick Labs Inc Isoelectricfocusing was carried out in glass tubes of inner diameter23mm using 2 pH 4ndash8 Servalytes (Serva Germany) for9600 volt-hours 1120583g (Coomassie stain) or 50 ng (silver stain)of an IEF internal standard tropomyosin was added toeach sample This protein migrates as a doublet with lowerpolypeptide spot of MW 33000 and pI 52 its position ismarked by an arrow on the stained gelsThe enclosed tube gelpH gradient plot for this set of ampholines was determinedwith a surface pH electrode

After equilibration for 10min in buffer ldquoOrdquo (10 glycerol50mm dithiothreitol 23 SDS and 00625M Tris pH 68)each tube gel was sealed to the top of a stacking gel that wason top of a 10 acrylamide slab gels (075mm thick) SDSslab gel electrophoresis was carried out for about 4 hoursat 15mAgel The following proteins (Sigma Chemical Co)were used as molecular weight standards myosin (220000)phosphorylase A (94000) catalase (60000) actin (43000)carbonic anhydrase (29000) and lysozyme (14000) Thesestandards appear along the basic edge of the Coomassie blueR-250 stained or silver-stained [11] 10 acrylamide slab gelThe gels were dried between sheets of cellophane paper withthe acid edge to the left Each of the gels was overlaid witha transparent sheet for labeling polypeptide spot differenceswithout marking the original gel (Kendrick Labs)

Sarcoma 3

(a)

(b) (c)

Figure 1 Osteosarcoma (a)Whole body scan shows abnormally intense uptake of the radionuclide (Tc) within the middiaphysis of the rightfemur No increased radionuclide uptake is seen anywhere else in the bony skeleton (b) MRI scan displays an extensively abnormal signal inthe diaphysis of the right femur It is surrounded by soft tissue involvement (c) Bone lesion displayed postoperatively

27 Polymorphism Analysis We obtained 22 formalin-fixedparaffin-embedded leiomyosarcoma specimens (stroma andparaffin had been removed from unstained slides undermicroscopic examination) through the Department ofPathology University of Cincinnati DNA was extracted withthe AllPrep DNARNA FFPE kit (Qiagen) We purchased7 frozen leiomyosarcoma tissues from Creative Bioarrayand extracted DNA with the AllPrep DNARNA Mini Kit(Qiagen) One blood sample from a leiomyosarcoma patientwas received from the University of Cincinnati TissueBank Polymorphisms in FAF1 were analyzed in the DNAusing a custom TaqMan assay with the probe ACACCA-GATTTGCCACCACCTTCATCATCT [AG] GTCAT-GCTGGGTAAGTTGTTTATATTTCCTG A TaqMan assaywith an existing probe for position minus443 in the osteopontinpromoter served as a reference assay 34 breast cancerDNA samples served as nonsarcoma control The assay wasperformed by the CCHMC DNA core

3 Results

31 Patient History In 1998 the patient was diagnosedwith high grade stage IIb osteogenic sarcoma of the right

femur which was extracompartmental Upon resection thelesion had a size of 95 times 3 times 4 cm (Figure 1) The tumorwas assessed as stage T2NxMx grade III characterized ashypercellular it showed marked cytologic atypia and a highmitotic rate Histologic features included anaplasia pleomor-phism numerous abnormal mitoses numerous giant cellsand osteoid production with focal calcifications

In 2012 a CT scan done because of hip pain revealed a43 times 34 times 31 cm lytic mass in the superior right acetabulumgrossly stable in size and configuration There was diffuseosteopenia involving the right femoral head and neck withdiffuse atrophy of the right pelvic girdle musculature Perios-titis and cortical interruptionwere associatedwith this lesion

The postsurgical pathology report identified the 57 times55 times 49 cm mass as a high grade leiomyosarcoma stagepT2bpNx Whereas a bone scan revealed intense activityin the left seventh rib a follow-up chest CT providedno indication of pulmonary masses mediastinal or hilarlymphadenopathy enlargement of axillary nodes or pleuraleffusion implying stage M0 The mitotic rate was 70 with60 necrosis The tumor caused extensive bone destructionand involvement of adjacent tissue Histologically the tumorcells stained positively for smooth muscle actin They were

4 Sarcoma

also positive for CD68 and displayed diffuse positive stainingfor vimentin but were negative for CD117 pancreatin S-100and CD34

Seven months after the surgery the patient received aPET-MRI scan for pain which revealed sixmetastatic lesionsincluding both lungs and multiple ribs She was put on three21-day cycles of Gemzar (days 1 and 8) Taxotere (day 8) andNeulasta (beginning on day 9) but was unable to continuepast the first cycle due to hospitalizations for continued andproblematic wound infections at the surgical lung biopsysites

32 DNA Exome Sequence Exome sequencing of thegenomic DNAs for tumor muscle and bone identified65546 potential sequence variants Filtering yielded 46 likelysomatic mutations in the tumor (Table 1) of which 7 (affect-ing EIF4A1 EPHA3 FAF1 IPO8 KIAA1377 LIMCH1 andNIPBL) were confirmed in the RNASeq results FAF1 asso-ciates with FAS and enhances apoptosis mediated throughthis receptor [12] The point mutation S181G (Figure 2) couldcause a loss of function in FAF1 and lead to transformationvia antiapoptosis

33 RNA Analysis Expectedly the gene expression patternsaccording to RNASeq were very different among tumormuscle and bone The cancer contained several gene prod-ucts that were overexpressed compared to both muscle andbone (Tables 2(a) and 2(b)) Among the top 30 changesin the tumormuscle and tumorbone comparisons 13 wereidentical (Table 2(c)) It is implied that these gene productsare quite unique for the tumor and likely contribute to itspathogenesis This notion is supported by the upregulationof the smooth muscle gene Ano4 which corroborates theleiomyosarcomatous nature of the cancer When limiting theanalysis to genes expressed at least at the level of 1 unit the17 genes overexpressed in the tumormuscle and tumorbonecomparisons contain several extracellular matrix proteinsimplying an active remodeling of the tumor microenviron-ment (Table 2(d)) By contrast none of the underexpressedgene products in the tumormuscle comparison matched thetumorbone comparison (not shown)

Of note the RNA level of IGF2BP1 (IMP-1 CRD-BPand ZBP-1) is highly upregulated in the tumor compared tomuscle as well as bone IGF2BP1 is a RNA-binding factorthat affects mRNA nuclear export localization stability andtranslation It regulates mRNA stability during the inte-grated cellular stress response in stress granules IGF2BP1 isa transcriptional target of the WNT pathway which is nega-tively regulated by intact FAF1 and may be unregulated byFAF1S181GThe IGF systemhas been linked to sarcoma patho-genesis [13] and may play a role in this specific cancer OtherIGF family members with increased RNA message levels inthis tumor (compared to muscle and bone) include IGFBPL1(5-6-log

2-fold) IGFL3 (6-7-log

2-fold) and IGF2BP3 (2-6-

log2-fold)The identified point mutation in FAF1 may be patho-

genetic for this cancer FAF1 is a regulator of NF-120581B activa-tion It directly binds to RelA (P65) retaining it in the cyto-plasm It can also interact with IKK120573 thus allowing for the

I120581B-mediated degradation of the transcription factors P65and P50 [6] Consistently the expression of regulators of theNF-120581B activation pathway is skewed in the tumor comparedto muscle or bone (Table 2(e))

34 Transcription Factor Binding ProteinDNA arrays mea-sure the binding activity of transcription factors Theycomprise three basic steps A set of biotin-labeled DNAbinding oligonucleotides are preincubated with a nuclearextract of interestThe proteinDNA complexes are separatedfrom the free probes The probes in the complexes arethen extracted and hybridized to prespotted membranesfollowed by HRP-based chemiluminescence detection Wemade nuclear extracts from tumor bone and muscle andtested them for DNA binding activity Binding that wasinduced in cancer but not in the normal tissues was dis-played by the transcription factors E2F1 AP3 LIII-BP PAX6ADD-1 and CCAC [14ndash19] The CCAC binding activity isconsistent with a muscle-derived tumor E2F1 may associatewith the WNT pathway-induced transcription factor LEF1resulting in transcriptional derepression of E2F1 [20] Likelyconstitutive transcription factors that are active in all 3 tissuescomprise AhRAmt GATA1 GATA2 GATA12 HIF1 andHOXD8910 (Figure 3)

35 Protein Analysis 2D gel electrophoresis of the RIPAlysates from tumor muscle and bone showed very divergentpatterns (Figure 4(a)) Two experienced analysts comparedthe protein pattern from the tumor with the protein patternfrom either bone or muscle Polypeptide spots that wereunique to the gels from the tumor were outlined (spotsunique to or relatively darker in the bone or muscle werenot indicated)The labeled proteins were extracted for identi-fication with mass spectrometry This yielded several struc-tural proteins which may reflect modification of the cellulararchitecture under rapid growth Transgelin-1 and transgelin-2 were abundant and corroborated the identity of thetumor as a leiomyosarcoma Four calcium-binding proteinswere highly expressed in the cancer In addition regulatorsof protein synthesis (40S ribosomal protein S12 glycine-tRNA ligase) protein modification (N-terminal fragmentof heat shock protein HSP 90120572 C-terminal fragment ofprotein disulfide isomerase) and protein degradation (1205721-antitrypsin proteasome activator complex subunit 2) wereidentified (Figure 4(b)) Of interest may be the C-terminalfragment of protein disulfide isomerase which not onlyhydroxylates prolines in preprocollagen but also contributesto microsomal triglyceride transfer It could be reflectiveof a skewed tumor metabolism The protein analysis wascorroborated by the mRNA levels (Figure 4(d))

Cancer markers were tested according to Western blot(Figure 4(c)) The tumor but not normal muscle expressedthe metastasis protein osteopontin and a single small form(lt75 kD) of CD44 that is likely the not alternatively splicedstandard form Unexpectedly while both tumor and muscleexpressed comparably abundant amounts of STAT3 phos-phorylation (reflective of activation) was present in themuscle but not in the tumorThe STAT3 pathway is associatedwith progression in several human cancers and this is often

Sarcoma 5

Table1DNAexom

eSNPsTh

eDNAexom

esfortum

orm

uscle

and

bone

weres

equencedTh

eresultswerefi

lteredin

thefollowingorder(1)d

ifferentgenotypeintumor

from

muscle

and

bonew

ithmuscle

andbo

nebeingidentic

alto

each

other(2)d

eletem

utations

with

lowconfi

dence(

valueo

f20or

lower)inall3

tissues(3)

deleteun

identifi

edgenes(4)d

eletem

utations

thatareh

omozygou

sreference

inthetum

or(5)

deletemutations

thathave

aMAFin

dbSN

Pgt10(6)

deletelowim

pactandmod

ifier

mutationsTh

egenen

ames

arep

arto

fthe

keyin

the

leftcolumnIn

thiscolumnresults

onbo

ldfont

representSNPs

thatwerec

onfirmed

ontheR

NAlevelbyRN

ASeqTh

edatafi

lesh

aveb

eensubm

itted

totheN

CBIsho

rtread

archive(SR

A)

underthe

accessionnu

mberS

RP052797

(biosamples

SAMN03316820SAMN03316821SAMN03316822)

Key

Chromosom

ePo

sition

Reference

Alternate

dbSN

PID

dbSN

PMAF

ESPMAF

(All)

ESPMAF

(EA)

ESPMAF

(AA)

Con

sensus

impact

Bone

muscle

geno

type

Bone

overall

depth

Bone

allele

depths

Bone

quality

Muscle

overall

depth

Muscle

allele

depths

Muscle

quality

Tumor

geno

type

Tumor

overall

depth

Tumor

allele

depths

Tumor

quality

177479

998T

EIF4

A1

177479998

CT

mdash

mdashmdash

mdashMod

erate

00

175

1840

99103

1080

9901

133

7968

99389

2592

00T

EPHA3

389259200

CT

mdash

mdashmdash

mdashMod

erate

00

43450

99117

1230

9901

723048

9915120

4545

CFA

F11

51204545

TC

mdash

mdashmdash

mdashMod

erate

00

87910

99108

1130

9901

8566

27

9912

3083

4620

AIPO8

1230834620

CA

mdash

mdashmdash

mdashMod

erate

00

68712

99120

1260

9901

807018

9911

101834

425A

KIA

A1377

11101834425

GA

mdash

mdashmdash

mdashMod

erate

00

36371

9067

700

9901

863163

9944168

2102

CLIMCH

14

41682102

GC

mdash

000

0077

0000

0227

Mod

erate

00

71740

9996

1010

9901

153

12049

99536

985326

GNIPBL

536985326

AG

mdash

mdashmdash

mdashMod

erate

00

660

1835

360

8101

201012

99377623789

ARO

BO2

377623789

AGA

mdash

mdashmdash

mdashHigh

00

22NA

5429

NA

8701

37NA

99

2217300

095A

SMARC

AL1

2217300

095

ATTG

CATC

A-AC

GTC

GTG

GA

mdash

mdashmdash

mdashHigh

00

95NA

99122

NA

9901

99NA

99

2152236045TTA

ATN

FAIP6

2152236045

TATTA

Ars3506

0021

mdashmdash

mdashmdash

High

02

19NA

124

NA

5701

17NA

383520206

69TAC

Y13

520206

69G

T

mdashmdash

mdashmdash

Mod

erate

00

130

1360

9986

900

9901

804543

999117

130734

GAKN

A9

117130734

CG

mdash

mdashmdash

mdashMod

erate

00

27280

7830

310

9001

503024

9912

6030354A

ANO2

126030354

CA

mdash

mdashmdash

mdashMod

erate

00

101

1060

99114

1200

9901

135

10445

9918

10487667

AAPC

DD1

1810487667

GA

mdash

mdashmdash

mdashMod

erate

00

43450

9938

400

9901

503816

99734118

560C

BMPE

R7

34118

560

GC

mdash

mdashmdash

mdashMod

erate

00

69720

99101

1060

9901

716314

9915

24921273

TC1

5orf2

1524921273

GT

mdash

mdashmdash

mdashMod

erate

00

12120

3638

390

9901

26226

9919

54483249

CCA

CNG8

1954483249

TC

mdash

mdashmdash

mdashMod

erate

00

147

1540

99103

1080

9901

152

13632

9910

16893265

CCU

BN10

16893265

GC

mdash

mdashmdash

mdashMod

erate

00

57600

9990

940

9901

403410

997148489854C

CUL1

7148489854

AC

mdash

mdashmdash

mdashMod

erate

00

73760

9965

680

9901

574221

9917

41566894

CDHX8

1741566894

GC

mdash

mdashmdash

mdashMod

erate

00

106

1110

9988

920

9901

124

9442

9920

61512358

TDID

O1

2061512358

GT

rs73304513

00417

0045932

000

0838

0135456

Mod

erate

00

600

182

00

601

400

2116

23703526

AER

N2

1623703526

GA

mdash

mdashmdash

mdashMod

erate

00

81850

9998

1030

9901

916041

993197880164G

FAM157A

3197880164

GCA

GCA

GCA

AG

mdash

mdashmdash

mdashMod

erate

00

21NA

2525

NA

101

31NA

614

4564

4287

GFA

NCM

144564

4287

AG

000

09mdash

mdashmdash

Mod

erate

00

13130

3331

320

8701

12102

14189637524

CGBP

71

89637524

GC

mdash

mdashmdash

mdashMod

erate

00

171

1790

99201

2110

9901

206

1616

799

742003933

CGLI3

742003933

GC

mdash

mdashmdash

mdashMod

erate

00

87910

9981

850

9901

856132

9917

4837170TGP1BA

174837170

CT

mdash

mdashmdash

mdashMod

erate

00

16160

459

9015

01

11101

114

24635161

GIRF9

1424635161

TG

mdash

mdashmdash

mdashMod

erate

00

66690

9943

450

9901

362513

9915

42133096

AJM

JD7-

PLA2G

4B15

42133096

GA

mdash

mdashmdash

mdashMod

erate

00

111

1160

9975

780

9901

807414

92

1740271678

AKA

T2A

1740271678

GA

mdash

mdashmdash

mdashMod

erate

00

65680

9958

610

9901

563032

99873848894

GKC

NB2

873848894

AG

mdash

mdashmdash

mdashMod

erate

00

15150

3324

250

6601

301517

9919

50827053

TKC

NC3

1950827053

CT

mdash

mdashmdash

mdashMod

erate

00

220

2310

99136

1430

9901

160

13046

9917

21319069

AKC

NJ12

1721319069

GA

rs76265595

mdashmdash

mdashmdash

Mod

erate

00

23241

6343

434

4001

28255

6012

53011932

CKR

T73

1253011932

TC

mdash

mdashmdash

mdashMod

erate

00

146

1530

99157

1650

9901

159

11758

99X

7500

4584

AMAG

EE2

X7500

4584

CA

mdash

mdashmdash

mdashMod

erate

00

29300

8148

500

9901

382616

995140182157A

PCDHA3

5140182157

GA

mdash

mdashmdash

mdashMod

erate

00

180

1890

99153

1610

9901

150

13234

9915

42133096

APL

A2G

4B15

42133096

GA

mdash

mdashmdash

mdashMod

erate

00

111116

099

75780

9901

807414

9210

96005840

CPL

CE1

1096005840

TC

mdash

mdashmdash

mdashMod

erate

00

81851

9973

762

9901

875540

991166816805G

POGK

1166816805

CG

mdash

mdashmdash

mdashMod

erate

00

84880

9982

860

9901

634920

99

6 Sarcoma

Table1Con

tinued

Key

Chromosom

ePo

sition

Reference

Alternate

dbSN

PID

dbSN

PMAF

ESPMAF

(All)

ESPMAF

(EA)

ESPMAF

(AA)

Con

sensus

impact

Bone

muscle

geno

type

Bone

overall

depth

Bone

allele

depths

Bone

quality

Muscle

overall

depth

Muscle

allele

depths

Muscle

quality

Tumor

geno

type

Tumor

overall

depth

Tumor

allele

depths

Tumor

quality

12111

020739

TPP

TC7

12111

020739

TCGC

Trs71083132

mdashmdash

mdashmdash

Mod

erate

00

18NA

3014

NA

1801

19NA

1019

804293

APT

BP1

19804293

CA

mdash

mdashmdash

mdashMod

erate

00

120

1261

9971

741

9901

755330

9971564

51175C

RNF32

71564

51175

GC

mdash

mdashmdash

mdashMod

erate

00

55570

9967

700

9901

594223

993520206

69TRP

11-155D

1811

3520206

69G

T

mdashmdash

mdashmdash

Mod

erate

00

130

1360

9986

900

9901

804543

9921

43838624

TUBA

SH3A

2143838624

GT

mdash

mdashmdash

mdashMod

erate

00

70730

9945

470

9901

897621

9919

58773857

AZN

F544

1958773857

GA

mdash

mdashmdash

mdashMod

erate

00

46480

9943

450

9901

715126

99516465723

CZN

F622

516465723

TC

mdash

mdashmdash

mdashMod

erate

00

17170

3914

140

3301

271415

99

Sarcoma 7

Table 2 RNA induced in the tumor (a) Transcripts overexpressed in the tumor compared to muscle but not bone (b) Transcriptsoverexpressed in the tumor compared to bone but not muscle (c) Transcripts most abundantly overexpressed in the tumor compared tomuscle and bone (d) as (a) but limited to genes expressed at least at the level of 1 unit in the reference tissue (muscle or bone) (e) Alteredexpression of genes associated with NF-120581B activity Analysis of RNASeq for the transcription factor NF-120581B and gene products that regulate itsactivity RNAmessages that are selectively associated with the TNF pathway to NF-120581B activation are marked with bold font log2-fold changeindicates the alteration in the tumor compared to muscle or bone (the respective columns indicate the expression level for each organ)

(a)

log2-fold changeMuscle

Tumor over muscleNRG1 Neuregulin 1 9909KSR2 Kinase suppressor of ras 2 9675MMP13 Matrix metallopeptidase 13 (collagenase 3) 9528SPP1 Secreted phosphoprotein 1 9098INHBA Inhibin beta A 8570COL11A1 Collagen type XI alpha 1 8564MMP9 Matrix metallopeptidase 9 (gelatinase B 92 kda gelatinase 92 kda type IV collagenase) 8552FBN2 Fibrillin 2 8495LRRC15 Leucine rich repeat containing 15 8429MMP11 Matrix metallopeptidase 11 (stromelysin 3) 8336E2F7 E2F transcription factor 7 8314PRAME Preferentially expressed antigen in melanoma 8179PTK7 PTK7 protein tyrosine kinase 7 7996DSCAM Down syndrome cell adhesion molecule 7761RGS4 Regulator of G-protein signaling 4 7633CNIH3 Cornichon homolog 3 (Drosophila) 7632OR10V1 Olfactory receptor family 10 subfamily V member 1 7610CEP55 Centrosomal protein 55 kda 7583WNT5B Wingless-type MMTV integration site family member 5B 7569CA12 Carbonic anhydrase XII 7520

GALNT5 UDP-N-acetyl-alpha-D-galactosaminepolypeptide N-acetylgalactosaminyltransferase 5(GalNAc-T5) 7517

(b)

log2-fold changeBone

Tumor over boneROS1 c-ros oncogene 1 receptor tyrosine kinase 10987GREM1 Gremlin 1 10604NPTX1 Neuronal pentraxin I 8813GJB2 Gap junction protein beta 2 26 kDa 8597CREB3L1 cAMP responsive element binding protein 3-like 1 8506KRT14 Keratin 14 8351SERPINE1 Serpin peptidase inhibitor clade E (nexin plasminogen activator inhibitor type 1) member 1 8288HOXD10 Homeobox D10 8105ALPK2 Alpha-Kinase 2 7783POU3F2 POU class 3 homeobox 2 7530POSTN Periostin osteoblast specific factor 7497NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439STC2 Stanniocalcin 2 7434WNT5A Wingless-type MMTV integration site family member 5A 7412IGFN1 Immunoglobulin-like and fibronectin type III domain containing 1 7387

8 Sarcoma

(b) Continued

log2-fold changeBone

STC1 Stanniocalcin 1 7385FAM180A Family with sequence similarity 180 member A 7315KRT17 Keratin 17 7305BBOX1 Butyrobetaine (gamma) 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 7277MAGEA1 Melanoma antigen family A 1 (directs expression of antigen MZ2-E) 7267

(c)

log2-fold changeMuscle Bone

Tumor over bonemuscle

ANO4 Anoctamin 4 (TMEM16D) transmembrane calcium-activated chloride channelfacilitates smooth muscle contraction 9937 8542

SLCO1B3 (OATP1B3) Solute carrier organic anion transporter family member 1B3 9569 9760

MARCH4 Membrane-associated ring finger (C3HC4) 4 E3 ubiquitin ligase located predominantlyto the endoplasmic reticulum 9538 7406

IGF2BP1 Insulin-like growth factor 2 mRNA binding protein 1 binds to and stabilizes mRNA 9440 9631ADAMTS16 ADAMmetallopeptidase with thrombospondin type 1 motif 16 zinc-dependent protease 9260 8450SOX11 SRY (sex determining region Y)-box 11 important in brain development 9178 9954

HAPLN1 Hyaluronan and proteoglycan link protein 1 stabilizes aggregates of aggrecan andhyaluronan giving cartilage its tensile strength and elasticity 8951 8142

MUC15 Mucin 15 cell surface associated 8801 7992HOXB9 Homeobox B9 8773 7378MAGEC2 Melanoma antigen family C 2 8693 8884

ST6GALNAC5 ST6 (alpha-N-acetyl-neuraminyl-23-beta-galactosyl-13)-N-acetylgalactosaminidealpha-26-sialyltransferase 5 7848 8038

C11orf41 Chromosome 11 open reading frame 41 7781 8141MMP1 Matrix metallopeptidase 1 (interstitial collagenase) 7455 7646

(d)

Symbol Name log2-fold change log2-fold changeMuscle Bone

Tumor over bonemuscleFN1 Fibronectin 1 7328 6293 6457 19860COL1A1 Collagen type I alpha 1 6768 3706 5868 11934CCND1 Cyclin D1 5595 3958 5098 9637RGS1 Regulator of G-protein signaling 1 5118 1056 4653 2533ITGBL1 Integrin beta-like 1 (with EGF-like repeat domains) 5071 3177 3895 12415COL1A2 Collagen type I alpha 2 4852 8756 4910 14503MXRA5 Matrix-remodelling associated 5 4834 1352 4631 2685POSTN Periostin osteoblast specific factor 4513 5870 7497 1267PLOD2 Procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 4285 1801 4023 3730COL5A2 Collagen type V alpha 2 4275 1297 4833 1517COL3A1 Collagen type III alpha 1 4003 13118 5801 6500

SEMA3C Sema domain immunoglobulin domain (Ig) shortbasic domain secreted 3954 1164 4215 1675

FBN1 Fibrillin 1 3912 6957 4018 11148AEBP1 AE binding protein 1 3808 3212 4002 4843SIK1 Salt-inducible kinase 1 3805 1219 3566 2484

Sarcoma 9

(d) Continued

Symbol Name log2-fold change log2-fold changeMuscle Bone

SERPINH1 Serpin peptidase inhibitor clade H (heat shock protein47) member 1 3706 1652 4145 2098

ANTXR1 Anthrax toxin receptor 1 3670 3789 3690 6445

(e)

Symbol Name log2-fold change log2-fold changeMuscle Bone

HELLS Helicase lymphoid-specific 5315602 0155898 minus082713 202489

TNFRSF11A Tumor necrosis factor receptor superfamily member 11a andNFKB activator 3017922 003091 1400993 0202453

NFKBID Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor delta 2655352 0 minus147615 0504341

TNFRSF25 Tumor necrosis factor receptor superfamily member 25 2432959 0046388 0163954 0490795

NFKBIE Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor epsilon 2121015 0062395 0311441 043382

TNF Tumor necrosis factor 1847997 0 minus142101 003733

TNFRSF10D Tumor necrosis factor receptor superfamily member 10d decoywith truncated death domain 1754888 0172968 minus01757 1183343

TNFRSF1B Tumor necrosis factor receptor superfamily member 1B 1473438 0709902 minus097011 6729401TNFRSF10A Tumor necrosis factor receptor superfamily member 10a 1411898 0828219 0357701 3004386

NFKBIB Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor beta 1193993 1209816 046055 3484692

TNFRSF10B Tumor necrosis factor receptor superfamily member 10b 1094157 1908597 0425446 5242006TNFRSF21 Tumor necrosis factor receptor superfamily member 21 1055762 3882038 0745815 8305711TNFRSF1A Tumor necrosis factor receptor superfamily member 1A 0875167 3790938 minus011707 130344

NFKBIZ Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor zeta 0575974 3698951 0344657 7493136

RIPK1 Receptor (TNFRSF)-interacting serine-threonine kinase 1 0494467 311749 minus108854 161392NKRF NFKB repressing factor 0475722 2156087 minus086397 9434166NFKB1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 0432959 2054532 minus065865 7568586CHUK Conserved helix-loop-helix ubiquitous kinase 0176126 2961281 minus120204 1330333RELA V-rel reticuloendotheliosis viral oncogene homolog A (avian) 0013848 1263837 0287167 1803044

NFKB2 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 2(p49p100) minus008641 0312993 0485882 0360442

NKAPP1 NFKB activating protein pseudogene 1 minus010692 0825177 minus099449 2655392

TNFRSF10C Tumor necrosis factor receptor superfamily member 10c decoywithout an intracellular domain minus0152 0064676 minus556891 6321515

NKIRAS2 NFKB inhibitor interacting Ras-like 2 minus060768 1095135 minus088758 2299946

NFKBIL1 Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor-like 1 minus08719 0141933 minus008357 0140418

NKIRAS1 NFKB inhibitor interacting Ras-like 1 minus087428 6296399 1467735 2122983TANK TRAF family member-associated NFKB activator minus0983 6777124 minus151908 1695872NKAP NFKB activating protein minus135735 1422458 minus078243 1646287

NFKBIA Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor alpha minus185483 4032743 minus031802 2395275

NKAPL NFKB activating protein-like minus557347 5875743 minus140215 0534643

10 Sarcoma

FAS associating region

DED interacting region

UB12 DZM

UB2domain

UASdomain

UBXdomain

UBAdomain

S289 S291

Aurora ACK II

S181

AKT1ATMGSK3

AuroraPKG

PLK1RSK

STK4CHK1

650566481336260205169110475

(a)

MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRM

6

1

101

100

200

300

400

500

600

650

201

301

401

501

601

667855899998876467765678888887578758999986467 88 8888888888888988888899988888889888877767888777888757

2222454345655744642554435764462443558468643653433344333424435334233333332333332333354335333344334354

LDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITH

9999997797689998787758999999999859996577754788888888875333467888868987688888888888877888855675799987

9585847535375857543447459666955563844544585374435443365845846434457567533353232334334444433337484944

REVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDAT

5888757887577654788887766654677776555468877888765455546887766555688888877777777777776556788888767766

5344456484742545656645594364384534437445444342323455453535435463434323333343434333333334333335433333

EFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESI

5666776654456777888888888888866889999999998848888876677778999999998776686899998589875579999987486899

4635343574354322343434533333343548479747945564424647635575478569766456468999999754233454687457844669

VSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDE

9999888589997557877545444345677876335554467888876899986799876447777677888999999998877555778887777778

6778547999996674433343444333333335466456434433487999895353333464346434365468755846444545454565446565

REARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVF

8888888887788877766545557777777666777777777776678877777766578888888888885799998589975788758987589999

4545445564445344433443354555556653665456544454434444444443344334333333446859596743354745795535796898

DFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKE

99997799988779985788754577888765665678898589998589

67974525444795877564635844444476875837446989897366

(b)

Figure 2 FAF1 structure (a) A schematic of functional domains in FAF1 with annotations (adapted from [6ndash8]) There are two ubiquitin-like domains flanking the S181 site The PDB structure 2DZM identifies the N-terminal one while the C-terminal prediction is by sequencesimilarity Whereas the mutation S181G is not expected to disrupt the protein structure per se this amino acid has a high score as a possiblephosphorylation site for a number of kinases involved inDNAdamage repair (b) Secondary structure prediction of FAF1The residues aroundS181 appear unstructured

reflected in STAT3 constitutive phosphorylation The lack ofphosphorylation in this case suggests that the leiomyosar-coma may not depend on the STAT3 pathway

36 Pharmacogenetic Evaluation Predicting the sensitivityto anticancer drugs is a main goal of molecular analysisFor this the over- or underexpression of genes for drugtransport and metabolism is of key importance Analysis ofthe RNASeq data for these groups of gene products identified

a surprisingly large number of deregulations compared tomuscle or bone (Tables 3(a) and 3(b)) Those alterationsmay affect choices for drug treatment For example the highlevels of glutathione S-transferase may render carmustinethioTEPA cisplatin chlorambucil melphalan nitrogenmus-tard phosphoramide mustard acrolein or steroids ineffec-tive The overexpression ofN-acetyltransferase may compro-mise 5-fluorouracil or taxolThemodest upregulation of onlytwo export transporters (ABC-transporters) and specifically

Sarcoma 11

Bone Tumor

Muscle

(a)

Transcription factor Description Reference

E2F1 Transcriptional activation of cell cycle progression is selectively activated in response to specific cues key downstream target of RB1 can promote proliferation or apoptosis when activated [14]

AP-33 interact in a mutually exclusive manner [15]

CCAC CCAC box is an essential element for muscle-specific transcription from the myoglobin promoter [16]

LIII-BP The L-III transcriptional regulatory element (a carbohydrate-response element) is in the pyruvate kinase L gene necessary for cell type-specific expression and transcriptional stimulation by carbohydrates [17]

ADD-1 (SREBP-1) activates transcription from sterol-regulated elements and from an E-box sequence present in the promoter of fatty acid synthase [18]

PAX6 Member of the paired box gene family transcriptional regulator involved in developmental processes [19]

48kD transcription factor activator protein-3 binds to the core element and recognizes the SV40 enhancer and AP-2 and AP-

(b)

Figure 3 Transcription factor activation Nuclear extracts from tumor muscle and bone were tested for DNA binding activity on a protein-DNA array (a) Transcription factor binding that was high in all 3 tissues and is therefore considered constitutively active is circled in yellow(left to right top to bottom AhRAmt GATA1 GATA2 GATA12 HIF1 and HOXD8910) Transcription factors that show high binding inthe cancer but not in muscle or bone are circled in red (left to right top to bottom E2F1 AP3 LIII-BP PAX6 ADD-1 and CCAC) (b) Thefunctions of transcription factors that display high DNA binding in the cancer but not in muscle or bone are described

the lack of ABCB1 overexpression is favorable for avoidingdrug resistance

37 Population Analysis The above-described results indi-cated that a FAF1 mutation which replaces serine in position181 thus preventing FAF1 phosphorylation and activationmay be a driver for leiomyosarcomagenesis A custom Taq-Man assay confirmed the presence of the somatic mutationin the patient To assess whether this single nucleotidereplacement is common in this type of cancer we analyzedDNA from 29 leiomyosarcomas and 1 blood sample from aleiomyosarcoma patient For comparison to nonsarcomatoustumors 34 breast cancers served as a reference None of themdisplayed amutation in the same locus By contrast there wasa distribution across all leiomyosarcomas in the osteopontin

promoter position minus443 (used as a reference) with 12 CC 10TC and 9 TT

4 Discussion

TheFAF1mutation identified as the likely cause for the cancerunder study gives room for an explanation of the sarcomatoustransformation (Figure 5) DNA damage to mesenchymalcells occurs persistently in an oxidizing environment at 37∘CThese insults are rarely transforming and such an occurrencewould trigger the initiation of programmed cell death inapoptosis-competent cells Intact FAF1 associates with FASand enhances apoptosis mediated through this receptor [12]A loss of function in FAF1 could lead to transformation viaantiapoptosis Whereas the mutation S181G is not expected

12 Sarcoma

Muscle

Bone

Tumor

220

94

6043

29

14

220

94

60

43

29

14

220

94

60

43

29

14

1

7

6

23

24

21

22

25

26

89 1011 12

1516 1719

pH 4 8 pH 4 8

8pH 4

(a)

SwissProt or NCBISpot number Protein identified accession Description

(Structural)6 Transgelin Q01995 Smooth muscle-specific cross-links actin24 Transgelin-2 P37802 Smooth muscle-specific cross-links actin

Vimentin P08670 Intermediate filament in mesenchymal tissue1 Filamin-A P21333 Actin-binding protein regulates the cytoskeleton 21 P06709 Cytoskeleton

(Calcium homeostasis)8 9 Calumenin O43852 Calcium-binding protein in the ER and golgi apparatus26 Protein S100-A11 P31949 Calcium-binding EF hand protein10 Reticulocalbin-3 Q96D15 Calcium-binding protein located in the ER12 P11021 Heat shock 70-kD protein 5 ER lumenal calcium-binding

(Protein processing)25 40S ribosomal protein S12 P25398 Core component of the ribosomal accuracy center7 Glycine-tRNA ligase P41250 Catalyzes the attachment of glycine to tRNA19 P01009 Protease inhibitor23 P01009 Protease inhibitor21 Proteasome activator complex subunit 2 Q9UL46 Proteasome activation10 P07900 Chaperone

(Other)7 Serum albumin P02768 Carrier protein in blood22 Protein disulfide isomerase (C-terminal fragment) P07237 Prolyl hydroxylation in collagen microsomal triglyceride transfer

120573 actin (C-terminal fragment)

78kD glucose-regulated protein

120572-1-antitrypsin120572-1-antitrypsin (C-terminal fragment)

Heat shock protein HSP 90120572 (N-terminal fragment)

11 15ndash17

(b)

Figure 4 Continued

Sarcoma 13

Tumor Muscle

OPN

CD44

STAT3

P-STAT3

Tubulin

75

75

50

15010075

10075

50

(c)

Tumor bone Tumor muscleGene ID Symbol Name Fold change Expression Fold change Expression6876 TAGLN Transgelin 3274 2455 3087 15088407 TAGLN2 Transgelin-2 2164 7338 6975 13037431 VIM Vimentin 1653 295010 4061 696042316 FLNA Filamin A alpha 1304 7791 9005 065160 ACTB Actin beta 0656 973187 5491 67368813 CALU Calumenin 7601 19328 12063 70596282 S100A11 S100 calcium binding protein A11 0931 158914 5071 1686357333 RCN3 Reticulocalbin 3 EF-hand calcium binding domain 2439 0604 6412 01223309 HSPA5 1068 92754 2607 220356696 SPP1 Secreted phosphoprotein 1 7572 46508 547929 0355960 CD44 CD44 molecule (Indian blood group) 2053 26708 15436 20566774 STAT3 Signal transducer and activator of transcription 3 0617 46534 0832 200165925 RB1 Retinoblastoma 1 0736 14772 0442 14268

Heat shock 70kDa protein 5 (glucose-regulated protein 78kDa)

(d)

Figure 4 Protein overexpression (a) 2D protein gel electrophoresis of lysates from muscle (upper left) tumor (upper right) and bone(bottom) in RIPA buffer Red circles indicate the spots that were identified as overexpressed and were further analyzed by mass spectrometry(b) Proteins identified in 2D gel electrophoresis as overexpressed in the tumor in comparison to muscle and bone were analyzed for theiridentity by mass spectrometry The left column indicates the spot number corresponding to the 2D gel The next column contains theprotein name followed by the accession number and a description of the protein functionThe protein functions are grouped into structuralcalcium homeostasis and various others (c) Western blot 10 120583g lysates of tumor muscle and bone in RIPA buffer were loaded per laneand electrophoresed on 10 SDS-polyacrylamide minigels with reducing denaturing sample buffer After transfer to PVDF membranesthey were probed for markers of cancer progression including osteopontin CD44 STAT3 and phospho-STAT3 Antitubulin served as aloading control (d) RNA levels corresponding to the proteins found to be affected in the sarcoma With four exceptions in the tumor-bonecomparison (gray font) upregulated proteins are associated with increased RNA levels STAT3 is not increased on the protein or RNA levelThe reduced level of RB1 expression is consistent with the elevated DNA binding activity of E2F1

to disrupt the structure of the protein this site does scorehigh as a possible phosphorylation site for a number ofkinases involved in DNA damage repair supporting thehypothesis that the cancer cells containing thismutation havelost their ability to respond to transforming DNA damagewith programmed cell death FAF1 antagonizes WNT signal-ing by promoting 120573-catenin degradation in the proteasome[21] a function that may be lost after the point mutationThe elevated DNA binding activity of the protooncogenictranscription factor E2F1 (see Figure 3) could be caused byits interaction with LEF-1 [20] consecutive to persistent

WNT signaling The RNA level of IGF2BP1 (IMP-1) a stress-responsive regulator of mRNA stability is highly upregulatedin the tumor compared to muscle as well as bone (seeTable 2(c)) IGF2BP1 is a transcriptional target of the WNTpathway that regulates NF-120581B activity (see Table 2(e)) andis antiapoptotic [22 23] IGF2BP1 may be upregulated asa consequence of mutated FAF1 not being able to suppressWNT signaling in this specific cancerOf noteWNTpathwayoveractivity may not be required for transformation ratherthe persistence of a WNT pathway signal due to the lack of aFAF1-mediated termination signal may suffice

14 Sarcoma

WNTFrizzled

Axin Dvl

igf2bp1

FAF1

P65 P50

IKK

FAS

120573-Catenin

LEF + E2F1

I120581B

Figure 5 Possible transforming pathway The point mutation inFAF1 is believed to cause a loss of function (crossed out in red)This may lead to an overactivity of the WNT signaling pathwayConsistently E2F1 (activated by LEF1) and igf2bp1 (a transcriptionaltarget of the WNT pathway) have been identified to be upregulatedin the molecular analysis In contrast to the negative regulator FAF1IGF2BP1 is a positive regulator of NF-120581B activityThe overactivity ofthe NF-120581B pathway and the reduced efficacy of FAS signaling can betransforming

The role ofWNT signaling in sarcoma has been subject todebate (eg [24]) In metastatic leiomyosarcoma 120573-Cateninmay accumulate in the nucleus despite a relatively weakexpression of WNT [25] This may be due to WNT signalactivation via noncanonical ligands [26] or to 120573-Cateninbinding the nuclear receptor NR4A2 and releasing it from thecorepressor protein LEF-1 [27] Of note in the cancer understudy here the mRNA level of NR4A2 was overexpressed10-fold compared to bone and 3-fold compared to muscleThe results from this study are consistent with the possibilitythat a lack of termination in the WNT signal rather than itsoveractivation could contribute to sarcomatous transforma-tion Such a mechanism may be reflected in an upregulationof downstream targets even though overexpression of WNTpathway components is not detectable

Other mutations beside FAF1 are less likely to becausative for the cancer EPHA3 was revealed as mutated inthis cancer by DNA exome sequencing and RNASeq EPHA3is a receptor tyrosine kinase that is frequentlymutated in lungcancer Tumor-suppressive effects of wild-type EPHA3 can beoverridden by dominant negative EPHA3 somatic mutations[28]Thismechanism is unlikely to play a role in this sarcomaas the detected mutation is located far N-terminally on theextracellular Ephrin binding domain not on the intracellularkinase domain

FAF1 has been described to act as a tumor suppressor gene[29] Its depletion due to chromosome breakage can affectprognosis in glioblastoma patients [30 31] Single nucleotidepolymorphisms in FAF1 are associated with a risk for gastriccancer [32] While numerous FAF1 mutations are associatedwith various cancers none of these genetic changes in theTCGA database affects the amino acid position 181 (Table 4)

The major upregulations identified in this tumor com-prise muscle-specific gene products (transcription factors

CAAC binding proteomics transgelin transgelin-2 RNAanoctamin-4 and immunohistochemistry smooth mus-cle actin) and calcium-regulating molecules (proteomicscalumenin S100-A11 reticulocalbin-3 and 78 kD glucose-regulated protein) The muscle-specific gene products con-firm this recurrent sarcoma as a leiomyosarcoma (the firsttumor distal to the site of the recurring one had beendiagnosed as an osteosarcoma) Calcium is one of the majorsecond messengers in smooth muscle cells Its uptake isregulated by potential-sensitive ion channels in the cellmembrane and by the activities of various receptors Calciumis stored in the sarcoplasmic reticulum from where it canbe released to facilitate actin-myosin interaction and tensiongeneration Phosphorylation of the myosin light chain by acalmodulin-regulated enzyme is important for contractionThe upregulation of gene products associated with migrationand invasion (osteopontin MMP1 vimentin filamin-A and120573-actin) and gene products for extracellularmatrixmoleculesand their modulators (fibronectin collagen ITGBL1 andMXRA5) reflects the invasive nature of this cancer

The recurrence of a sarcoma after 14 years has twoprobable explanations either it is due to a cancer pre-disposition syndrome based on a germ-line mutation (theage of the patient weakens this hypothesis) or the secondtumor is a metastatic colony of the first that was reactivatedafter dormancy The location of the sarcoma in the sameextremity and proximal to a preceding mesenchymal cancer(and therefore in its natural path of dissemination) impliedthe probability that this was a relapse in a metastatic siteThe different histologic assessment as osteosarcoma in thefirst occurrence and leiomyosarcoma as the second cancerdoes not necessarily negate that Mixed histology [33 34]and transdifferentiation [35ndash37] have been described forsarcomatous tumors Of note however in this scenarioosteosarcoma seems to more commonly follow leiomyosar-coma than precede it Material from the first cancer of thispatient was not accessible to us It is very plausible that thiscould have been a mineralized leiomyosarcoma In thosetumors the differential diagnosis from osteosarcoma can bedifficult [38] The extracompartmental location of the firsttumor supports this interpretation

On the molecular pathology level sarcomas fall intotwo groups comprising tumors with simple karyotypes(with pathogenetic translocations or specific genetic muta-tions) and tumors with very complex karyotypes (overtchromosome and genomic instability with numerous gainsand losses) [39] Some molecular alterations that lead tocarcinogenesis can be defined in absolute termsThey includegain-of-function mutations or chromosome translocationsthat transformprotooncogenes to oncogenes However otherchanges are relative to the normal tissue of origin suchas pathway overactivity or overexpression on the proteinor RNA levels We have combined the analysis of absolutechanges (DNA exome sequence RNA sequence) with theanalysis of relative changes using skeletal muscle and bone asreference organs (protein-DNA array 2D gel electrophoresisand RNA expression levels) This choice was determined inpart by tissue availability after surgery andwas intended to aidin the distinction of osteosarcoma from myosarcoma While

Sarcoma 15

Table 3 Deregulation of genes for drug disposition Analysis of RNASeq for at least twofold overexpression (italic font) or underexpression(bold font) of genes for (a) transport and (b) metabolism in tumor compared to muscle and bone For the export transporters (ABCtransporters) only overexpression is considered relevant for drug resistance Not shown in the table are the underexpressed genes (ABCA7ABCA8 ABCA13 ABCB6 ABCB10 ABCC6 ABCC6P1 ABCC6P2 ABCC8 ABCC11 ABCD2 ABCG2 and ABCG5)

(a) Transport

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

650655 ABCA17P ATP-binding cassette subfamily A (ABC1) member 17 pseudogene 1360 211624 ABCA4 ATP-binding cassette subfamily A (ABC1) member 4 2038 2802

6555 SLC10A2 Solute carrier family 10 (sodiumbile acid cotransporter family)member 2 minus1962 minus2112

345274 SLC10A6 Solute carrier family 10 (sodiumbile acid cotransporter family)member 6 2623 3240

6563 SLC14A1 Solute carrier family 14 (urea transporter) member 1 (Kidd bloodgroup) minus3074 minus3152

6565 SLC15A2 Solute carrier family 15 (H+peptide transporter) member 2 minus2027 minus2152

6566 SLC16A1 Solute carrier family 16 member 1 (monocarboxylic acid transporter1) 1401 2170

117247 SLC16A10 Solute carrier family 16 member 10 (aromatic amino acid transporter) 2113 2848

6567 SLC16A2 Solute carrier family 16 member 2 (monocarboxylic acid transporter8) 3242 3848

10786 SLC17A3 Solute carrier family 17 (sodium phosphate) member 3 minus2868 minus29596571 SLC18A2 Solute carrier family 18 (vesicular monoamine) member 2 minus2087 minus21946573 SLC19A1 Solute carrier family 19 (folate transporter) member 1 minus2099 minus220310560 SLC19A2 Solute carrier family 19 (thiamine transporter) member 2 1820 254880704 SLC19A3 Solute carrier family 19 member 3 minus3331 minus3478387775 SLC22A10 Solute carrier family 22 member 10 5711 60859390 SLC22A13 Solute carrier family 22 (organic anion transporter) member 13 2623 3217

85413 SLC22A16 Solute carrier family 22 (organic cationcarnitine transporter)member 16 minus8101 minus7299

51310 SLC22A17 Solute carrier family 22 member 17 1475 21755002 SLC22A18 Solute carrier family 22 member 18 2038 27226582 SLC22A2 Solute carrier family 22 (organic cation transporter) member 2 4793 5216

6581 SLC22A3 Solute carrier family 22 (extraneuronal monoamine transporter)member 3 2554 3182

6583 SLC22A4 Solute carrier family 22 (organic cationergothioneine transporter)member 4 minus3603 minus3776

151295 SLC23A3 Solute carrier family 23 (nucleobase transporters) member 3 2109 284810478 SLC25A17 Solute carrier family 25 (mitochondrial carrier) member 17 1311 207783733 SLC25A18 Solute carrier family 25 (mitochondrial carrier) member 18 1846 2570

788 SLC25A20 Solute carrier family 25 (carnitineacylcarnitine translocase) member20 minus2105 minus2207

89874 SLC25A21 Solute carrier family 25 (mitochondrial oxodicarboxylate carrier)member 21 minus2962 minus3105

51312 SLC25A37 Solute carrier family 25 member 37 minus4633 minus486651629 SLC25A39 Solute carrier family 25 member 39 minus2585 minus2737203427 SLC25A43 Solute carrier family 25 member 43 1325 208865012 SLC26A10 Solute carrier family 26 member 10 3896 4472115111 SLC26A7 Solute carrier family 26 member 7 3431 4018116369 SLC26A8 Solute carrier family 26 member 8 minus5354 minus5536115019 SLC26A9 Solute carrier family 26 member 9 1623 241911001 SLC27A2 Solute carrier family 27 (fatty acid transporter) member 2 minus4278 minus4487

16 Sarcoma

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

64078 SLC28A3 Solute carrier family 28 (sodium-coupled nucleoside transporter)member 3 minus4543 minus4812

222962 SLC29A4 Solute carrier family 29 (nucleoside transporters) member 4 minus1962 minus204581031 SLC2A10 Solute carrier family 2 (facilitated glucose transporter) member 10 2532 3170

6518 SLC2A5 Solute carrier family 2 (facilitated glucosefructose transporter)member 5 minus3647 minus3821

55532 SLC30A10 Solute carrier family 30 member 10 minus3547 minus37257782 SLC30A4 Solute carrier family 30 (zinc transporter) member 4 2832 33726569 SLC34A1 Solute carrier family 34 (sodium phosphate) member 1 minus4716 minus4941340146 SLC35D3 Solute carrier family 35 member D3 minus5662 minus590654733 SLC35F2 Solute carrier family 35 member F2 1433 2170206358 SLC36A1 Solute carrier family 36 (protonamino acid symporter) member 1 minus2265 minus2345285641 SLC36A3 Solute carrier family 36 (protonamino acid symporter) member 3 minus2284 minus239354020 SLC37A1 Solute carrier family 37 (glycerol-3-phosphate transporter) member 1 minus2112 minus22122542 SLC37A4 Solute carrier family 37 (glucose-6-phosphate transporter) member 4 minus2117 minus2219151258 SLC38A11 Solute carrier family 38 member 11 2410 308555089 SLC38A4 Solute carrier family 38 member 4 1837 254892745 SLC38A5 Solute carrier family 38 member 5 minus1994 minus215291252 SLC39A13 Solute carrier family 39 (zinc transporter) member 13 1301 207023516 SLC39A14 Solute carrier family 39 (zinc transporter) member 14 2569 318829985 SLC39A3 Solute carrier family 39 (zinc transporter) member 3 minus2032 minus2152283375 SLC39A5 Solute carrier family 39 (metal ion transporter) member 5 1623 23707922 SLC39A7 Solute carrier family 39 (zinc transporter) member 7 1273 205830061 SLC40A1 Solute carrier family 40 (iron-regulated transporter) member 1 minus3612 minus379684102 SLC41A2 Solute carrier family 41 member 2 1293 20708501 SLC43A1 Solute carrier family 43 member 1 minus2013 minus215257153 SLC44A2 Solute carrier family 44 member 2 minus2047 minus215250651 SLC45A1 Solute carrier family 45 member 1 2038 2722146802 SLC47A2 Solute carrier family 47 member 2 minus1962 minus20266521 SLC4A1 Solute carrier family 4 anion exchanger member 1 minus6513 minus645357282 SLC4A10 Solute carrier family 4 sodium bicarbonate transporter member 10 minus2640 minus275383959 SLC4A11 Solute carrier family 4 sodium borate transporter member 11 1846 25696508 SLC4A3 Solute carrier family 4 anion exchanger member 3 1261 20458671 SLC4A4 Solute carrier family 4 sodium bicarbonate cotransporter member 4 2445 31139497 SLC4A7 Solute carrier family 4 sodium bicarbonate cotransporter member 7 2717 33076523 SLC5A1 Solute carrier family 5 (sodiumglucose cotransporter) member 1 minus2547 minus2705159963 SLC5A12 Solute carrier family 5 (sodiumglucose cotransporter) member 12 4753 52066527 SLC5A4 Solute carrier family 5 (low affinity glucose cotransporter) member 4 minus3969 minus4249

6540 SLC6A13 Solute carrier family 6 (neurotransmitter transporter GABA)member 13 1328 2092

55117 SLC6A15 Solute carrier family 6 (neutral amino acid transporter) member 15 1623 2333388662 SLC6A17 Solute carrier family 6 member 17 2038 272854716 SLC6A20 Solute carrier family 6 (proline IMINO transporter) member 20 1697 2433

6532 SLC6A4 Solute carrier family 6 (neurotransmitter transporter serotonin)member 4 minus2512 minus2611

6534 SLC6A7 Solute carrier family 6 (neurotransmitter transporter L-proline)member 7 2623 3228

56301 SLC7A10 Solute carrier family 7 (neutral amino acid transporter y+ system)member 10 minus3421 minus3603

Sarcoma 17

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

6547 SLC8A3 Solute carrier family 8 (sodiumcalcium exchanger) member 3 minus2204 minus2309285335 SLC9A10 Solute carrier family 9 member 10 1208 20006549 SLC9A2 Solute carrier family 9 (sodiumhydrogen exchanger) member 2 2038 2706

9368 SLC9A3R1 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 1 minus2760 minus2846

9351 SLC9A3R2 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 2 1889 2619

84679 SLC9A7 Solute carrier family 9 (sodiumhydrogen exchanger) member 7 3347 393510599 SLCO1B1 Solute carrier organic anion transporter family member 1B1 3360 397728234 SLCO1B3 Solute carrier organic anion transporter family member 1B3 9760 95696578 SLCO2A1 Solute carrier organic anion transporter family member 2A1 2432 309628232 SLCO3A1 Solute carrier organic anion transporter family member 3A1 minus2032 minus2152353189 SLCO4C1 Solute carrier organic anion transporter family member 4C1 minus6850 minus6611

(b) Metabolism

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold Cchange

1583 CYP11A1 Cytochrome P450 family 11 subfamily A polypeptide 1 1623 23961589 CYP21A2 Cytochrome P450 family 21 subfamily A polypeptide 2 minus1962 minus20361591 CYP24A1 Cytochrome P450 family 24 subfamily A polypeptide 1 5208 55771592 CYP26A1 Cytochrome P450 family 26 subfamily A polypeptide 1 2623 32571594 CYP27B1 Cytochrome P450 family 27 subfamily B polypeptide 1 1846 2585339761 CYP27C1 Cytochrome P450 family 27 subfamily C polypeptide 1 3585 41781553 CYP2A13 Cytochrome P450 family 2 subfamily A polypeptide 13 minus1962 minus20351580 CYP4B1 Cytochrome P450 family 4 subfamily B polypeptide 1 minus4820 minus506566002 CYP4F12 Cytochrome P450 family 4 subfamily F polypeptide 12 minus6070 minus61958529 CYP4F2 Cytochrome P450 family 4 subfamily F polypeptide 2 minus7769 minus70604051 CYP4F3 Cytochrome P450 family 4 subfamily F polypeptide 3 minus8244 minus749111283 CYP4F8 Cytochrome P450 family 4 subfamily F polypeptide 8 minus5006 minus5270260293 CYP4X1 Cytochrome P450 family 4 subfamily X polypeptide 1 minus2476 minus25809420 CYP7B1 Cytochrome P450 family 7 subfamily B polypeptide 1 2502 31702326 FMO1 Flavin containing monooxygenase 1 4360 48592327 FMO2 Flavin containing monooxygenase 2 (nonfunctional) minus4074 minus43372328 FMO3 Flavin containing monooxygenase 3 minus4036 minus4309388714 FMO6P Flavin containing monooxygenase 6 pseudogene minus2569 minus2737493869 GPX8 Glutathione peroxidase 8 (putative) 2814 33712938 GSTA1 Glutathione S-transferase alpha 1 1623 23632939 GSTA2 Glutathione S-transferase alpha 2 2038 27412941 GSTA4 Glutathione S-transferase alpha 4 1492 21882953 GSTT2 Glutathione S-transferase theta 2 4682 5170653689 GSTT2B Glutathione S-transferase theta 2B (genepseudogene) 3739 430784779 NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439 79969027 NAT8 N-acetyltransferase 8 (GCN5-related putative) minus2769 minus29207358 UGDH UDP-glucose 6-dehydrogenase 2333 301855757 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 1604 227110720 UGT2B11 UDP glucuronosyltransferase 2 family polypeptide B11 minus3586 minus37687367 UGT2B17 UDP glucuronosyltransferase 2 family polypeptide B17 minus2836 minus295954490 UGT2B28 UDP glucuronosyltransferase 2 family polypeptide B28 minus3132 minus3284167127 UGT3A2 UDP glycosyltransferase 3 family polypeptide A2 minus4716 minus4907

18 Sarcoma

Table 4 FAF1 mutations in various cancers FAF1 mutations listed in the TCGA data base were identified without restriction to any type ofcancer For the location of the affected domains on the protein compare Figure 2

AA Mutation Cancer DomainN4S Missense Cutaneous melanomaI10S Missense Stomach adenocarcinoma UBAE21lowast Nonsense Cutaneous melanoma UBAE25K Missense Uterine endometrioid carcinoma UBAV38 splice Splice Stomach adenocarcinoma UBAP86fs FS del Colorectal adenocarcinomaG123 splice Splice Uterine endometrioid carcinoma UB1P136H Missense Colorectal adenocarcinoma UB1T147M Missense Brain lower grade glioma UB1D149Y Missense Uterine endometrioid carcinoma UB1L159V Missense Lung adenocarcinoma UB1K163N Missense Uterine endometrioid carcinoma UB1L170F Missense Cutaneous melanomaG184 splice Splice Colorectal cancerQ187H Missense Stomach adenocarcinomaS214N Missense Colorectal adenocarcinoma UB2R222I Missense Uterine endometrioid carcinoma UB2E238D Missense Lung adenocarcinoma UB2P241S Missense Uterine endometrioid carcinoma UB2T245A Missense Renal clear cell carcinoma UB2M249V Missense Uterine endometrioid carcinoma UB2E280K Missense Brain lower grade gliomaG293lowast Nonsense Colorectal cancerT300I Missense Colorectal adenocarcinomaD305H Missense Lung adenocarcinomaE308Q Missense Lung adenocarcinomaA316V Missense Stomach adenocarcinomaK319fs FS ins Head and neck squamous cell carcinomaR344G Missense Stomach adenocarcinoma UASF353I Missense Cutaneous melanoma UASL379V Missense Breast invasive carcinoma UASC396F Missense Cutaneous melanoma UASS459lowast Nonsense Uterine endometrioid carcinoma UASG469 splice Splice Glioblastoma multiforme UASR509G Missense Lung squamous cell carcinomaE510fs FS del Cutaneous melanomaR516C Missense Uterine endometrioid carcinomaA534V Missense Stomach adenocarcinomaF537L Missense Stomach adenocarcinomaE551lowast Nonsense Lung adenocarcinomaR554W Missense Colorectal cancerS582I Missense Lung adenocarcinoma UBXF585L Missense Uterine endometrioid carcinoma UBXE587lowast Nonsense Lung adenocarcinoma UBXA592V Missense Stomach adenocarcinoma UBXW610lowast Nonsense Breast invasive carcinoma UBXD611Y Missense Lung adenocarcinoma UBXE635fs FS del Brain lower grade glioma UBXP640fs FS del Cutaneous melanoma UBX

Sarcoma 19

a more accurate reference point for a leiomyosarcoma wouldhave been smooth muscle we believe that the comparison tostriated muscle is sufficient to allow the assessment of tumorspecific changes We have measured RNA DNA and proteinwith various assays Similar assessments in the future shouldalso include chromosome analysis for possible translocations

In the first-line defense against cancer the gradualreplacement of conventional chemotherapy with molecularlytargeted agents has opened the possibility to tailor drug treat-ments to particular tumors This transition necessitates thecharacterization of the molecular lesions that are causativefor the transformation of healthy cells into cancerous cellsbecause drugs need to be matched with the underlying carci-nogenic defect to be effective An additional caveat causedby unique genetic changes in the primary tumor can affectdrug transport and metabolism and needs to be taken intoconsideration In this case the RNA levels for genes that regu-late transport andmetabolismwere extensively skewed in thetumor tissue as compared to muscle and bone (see Table 3)implying potential challenges to chemotherapy An advancedmolecular treatment strategy for cancer will rely on themolecular definition of drug target drug transport and drugmetabolism pharmacogenetics in the primary tumor Con-secutive to cancer dissemination it will also require adjust-ments to account for the genetic changes in the metastases

The cost of health care has been under increasing scrutinyThe treatment of cancer patients is expensive in particularwhen hospitalization is required and further in cases ofend-of-life care Avoidable expenditures are generated bysuboptimal treatment decisions that result in low efficacy orhigh toxicity of anticancer regimens Molecular medicine hasthe potential to preempt those problems and reduce wastefulspending Yet it requires the upfront cost of molecular cancerexamination The analysis performed in this study requiredabout $11000- in nonsalary expenses to perform While ithas not identified a drug target it has specified possibleconfines for drug treatment The costs for molecular analysisneed to be weighed against the societal cost derived fromlost productivity in the workforce disrupted lives of familiesand premature deaths While currently limited drug optionsconstitute the major constraint to the approach taken herethe foreseeable future will bring an increasing spectrum ofmolecularly targeted drugs along with faster and cheapertechnologies for the molecular assessment of cancers Theywill facilitate the clinical translation of our approach

Consent

The patient provided informed consent

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by DOD Grant PR094070under University of Cincinnati IRB protocol 04-01-29-01The

author is grateful to Dr Rhett Kovall for helping with thestructural analysis of FAF1

References

[1] G F Weber Molecular Mechanisms of Cancer Springer Dor-drecht The Netherlands 2007

[2] N G Sanerkin ldquoPrimary leiomyosarcoma of the bone and itscomparison with fibrosarcomardquoCancer vol 44 no 4 pp 1375ndash1387 1979

[3] J M Weaver and J A Abraham ldquoLeiomyosarcoma of the Boneand Soft Tissue A Reviewrdquo httpsarcomahelporglearningcenterleiomyosarcomahtml

[4] M A Depristo E Banks R Poplin et al ldquoA framework forvariation discovery and genotyping using next-generationDNAsequencing datardquo Nature Genetics vol 43 no 5 pp 491ndash5012011

[5] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[6] C W Menges D A Altomare and J R Testa ldquoFAS-associatedfactor 1 (FAF1) diverse functions and implications for oncoge-nesisrdquo Cell Cycle vol 8 no 16 pp 2528ndash2534 2009

[7] M Kim J H Lee S Y Lee E Kim and J Chung ldquoCaspar asuppressor of antibacterial immunity in Drosophilardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 44 pp 16358ndash16363 2006

[8] J Song K P Joon J-J Lee et al ldquoStructure and interaction ofubiquitin-associated domain of human Fas-associated factor 1rdquoProtein Science vol 18 no 11 pp 2265ndash2276 2009

[9] P H OrsquoFarrell ldquoHigh resolution two dimensional electrophore-sis of proteinsrdquo Journal of Biological Chemistry vol 250 no 10pp 4007ndash4021 1975

[10] A Burgess-Cassler J J Johansen D A Santek J R Ideand N C Kendrick ldquoComputerized quantitative analysis ofCoomassie-Blue-stained serum proteins separated by two-dimensional electrophoresisrdquo Clinical Chemistry vol 35 no 12pp 2297ndash2304 1989

[11] B R Oakley D R Kirsch and N RMorris ldquoA simplified ultra-sensitive silver stain for detecting proteins in polyacrylamidegelsrdquo Analytical Biochemistry vol 105 no 2 pp 361ndash363 1980

[12] K Chu X Niu and L T Williams ldquoA Fas-associated proteinfactor FAF1 potentiates Fas-mediated apoptosisrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 25 pp 11894ndash11898 1995

[13] B Rikhof S de Jong A J H Suurmeijer C Meijer and W TA van der Graaf ldquoThe insulin-like growth factor system andsarcomasrdquoThe Journal of Pathology vol 217 no 4 pp 469ndash4822009

[14] RGirling J F Partridge L R Bandara et al ldquoAnew componentof the transcription factor DRTF1E2Frdquo Nature vol 362 no6415 pp 83ndash87 1993

[15] F Mercurio and M Karin ldquoTranscription factors AP-3 andAP-2 interact with the SV40 enhancer in a mutually exclusivemannerrdquo EMBO Journal vol 8 no 5 pp 1455ndash1460 1989

[16] R Bassel-Duby M D Hernandez M A Gonzalez J KKrueger and R S Williams ldquoA 40-kilodalton protein bindsspecifically to an upstream sequence element essential for mus-cle-specific transcription of the human myoglobin promoterrdquoMolecular and Cellular Biology vol 12 no 11 pp 5024ndash50321992

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

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Page 2: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

2 Sarcoma

22 DNA Exome Sequencing 1 120583g of dsDNA determined byInvitrogen Qubit high sensitivity spectrofluorometric mea-surement was sheared by sonication to an average size of300 bp on a Diagenode Bioruptor Automated library con-struction was performed on an IntegenX Apollo324 whichsize-selects fragments by double-SPRI binding with differ-ent concentrations of PEG for a high cut and a low cut Eachlibrary can be fitted with one of 48 adapters each contain-ing a different 6-base molecular barcode for high level multi-plexing After 12 cycles of PCR amplification 1 120583g of geno-mic library was recovered for exome enrichment usingthe NimbleGen EZ Exome V2 kit Enriched libraries weresequenced on an Illumina HiSeq2000 generating around 32million high quality paired end reads of 100 base each or64GB of usable sequence per sample The analysis methodsutilize the Broad Institutersquos GenomeAnalysis Toolkit (GATK)and follow a pipeline previously described [4] along withpublished modifications (httpwwwbroadinstituteorggsawikiindexphpThe Genome Analysis Toolkit) The analy-sis comprises aligning the reads that pass Illumina ChastityFilter with the Burrows-Wheeler Aligner (BWA) [5] For eachsample Picardrsquos MarkDuplicates are used to flag reads thatappear to be artifacts of PCR bias All reads that overlapknown or putative indels are realigned All base qualityscores are recalibrated to the empirical error rate derivedfrom nonpolymorphic sites The GATKrsquos Unified Genotypermodule is used to call variant sites (both single nucleotide andsmall indel) in all samples simultaneously Finally the SNVcalls are filtered using the variant quality score recalibrationmethod [4] Indel calls were filtered with a set of hard filtersas there are not enough indels in an exome to use theGaussianmethod

23 RNAseq Tissue samples were homogenized in RNazolRT (MRC) with a manual homogenizer and stored on iceuntil extractionThe RNA isolation was performed accordingto the manufacturerrsquos instructions

The Ovation RNA-Seq FFPE system (NuGen) was usedto initiate amplification at both 31015840 end as well as randomlythroughout the transcriptome in the sample 100 ng of totalRNA with RIN lt 50 was converted into a library of tem-plate molecules suitable for subsequent cluster generationand sequencing by Illumina HiSeq Total RNA was reversetranscribed and converted to double stranded cDNA witha unique DNARNA heteroduplex at one end NuGENrsquosRibo-SPIA technology was used for isothermal amplificationresulting in the rapid generation of cDNA with a sequencecomplementary to the original mRNA The cDNA was thendouble stranded and fragmented to 200 bp using CovarisS2 and a sequencing library was generated using IlluminarsquosTruSeq DNA Sample Prep Kit V2 according to standardprotocols The cDNA library was enriched by a limitednumber of 10 PCR cycles validated using an Agilent 2100Bioanalyzer and quantitated using the Quant-iT dsDNA HSKit (Invitrogen) Two individually indexed cDNA librarieswere pooled and sequenced on Illumina HiSeq to get aminimum of 90 million reads Libraries were clustered ontoa flow cell using Illuminarsquos TruSeq SR Cluster Kit v25 andsequenced 50 cycles using TruSeq SBS Kit-HS on HiSeq The

obtained sequence reads were aligned to the genome by usingthe standard Illumina sequence analysis pipeline

24 Protein-DNA Array Tissues were ground betweenfrosted glass slides and then incubated with Collagenaseand Dispase in cell culture medium at 37∘C for 45 minutesto release individual cells These cells were collected afterpassing the samples through a strainer and centrifugationNuclear extracts and cytosol were prepared using a kit fromActive Motif After protein determination DNA binding ofthe nuclear extracts was assessed with the Combo protein-DNA array (Panomics) Signal intensity was measured withthe software MetaMorph

25 Western Blotting For the analysis of individual proteinstissues were homogenized in RIPA buffer (50mM Tris-HClpH 75 150mM NaCl 1 NP-40 01 sodium dodecylsulfate) using a handheld battery-operated homogenizer10 120583g lysates were loaded per lane and electrophoresed on10 SDS-polyacrylamideminigels with reducing denaturingsample buffer The separated proteins were transferred toPVDF membranes and probed with antibody O-17 (IBC)to the C-terminus of osteopontin and anti-HCAM to thecytoplasmic domain of CD44 (Santa Cruz) and to STAT3 andphospho-STAT3 (Cell Signaling Technology) Antitubulinserves as a loading control

26 2DGel Electrophoresis andMass Spectrometry Thetumorand muscle samples were diluted to 4 and 1mgmL in 1 1diluted SDS Boiling Buffer Urea Sample Buffer before load-ing (the bone samplewas ethanol precipitated and redissolvedto 4 and 1mgmL in 1 1 diluted SDS Boiling Buffer UreaSample Buffer) Two-dimensional electrophoresis was per-formed according to the carrier ampholine method of iso-electric focusing [9 10] by Kendrick Labs Inc Isoelectricfocusing was carried out in glass tubes of inner diameter23mm using 2 pH 4ndash8 Servalytes (Serva Germany) for9600 volt-hours 1120583g (Coomassie stain) or 50 ng (silver stain)of an IEF internal standard tropomyosin was added toeach sample This protein migrates as a doublet with lowerpolypeptide spot of MW 33000 and pI 52 its position ismarked by an arrow on the stained gelsThe enclosed tube gelpH gradient plot for this set of ampholines was determinedwith a surface pH electrode

After equilibration for 10min in buffer ldquoOrdquo (10 glycerol50mm dithiothreitol 23 SDS and 00625M Tris pH 68)each tube gel was sealed to the top of a stacking gel that wason top of a 10 acrylamide slab gels (075mm thick) SDSslab gel electrophoresis was carried out for about 4 hoursat 15mAgel The following proteins (Sigma Chemical Co)were used as molecular weight standards myosin (220000)phosphorylase A (94000) catalase (60000) actin (43000)carbonic anhydrase (29000) and lysozyme (14000) Thesestandards appear along the basic edge of the Coomassie blueR-250 stained or silver-stained [11] 10 acrylamide slab gelThe gels were dried between sheets of cellophane paper withthe acid edge to the left Each of the gels was overlaid witha transparent sheet for labeling polypeptide spot differenceswithout marking the original gel (Kendrick Labs)

Sarcoma 3

(a)

(b) (c)

Figure 1 Osteosarcoma (a)Whole body scan shows abnormally intense uptake of the radionuclide (Tc) within the middiaphysis of the rightfemur No increased radionuclide uptake is seen anywhere else in the bony skeleton (b) MRI scan displays an extensively abnormal signal inthe diaphysis of the right femur It is surrounded by soft tissue involvement (c) Bone lesion displayed postoperatively

27 Polymorphism Analysis We obtained 22 formalin-fixedparaffin-embedded leiomyosarcoma specimens (stroma andparaffin had been removed from unstained slides undermicroscopic examination) through the Department ofPathology University of Cincinnati DNA was extracted withthe AllPrep DNARNA FFPE kit (Qiagen) We purchased7 frozen leiomyosarcoma tissues from Creative Bioarrayand extracted DNA with the AllPrep DNARNA Mini Kit(Qiagen) One blood sample from a leiomyosarcoma patientwas received from the University of Cincinnati TissueBank Polymorphisms in FAF1 were analyzed in the DNAusing a custom TaqMan assay with the probe ACACCA-GATTTGCCACCACCTTCATCATCT [AG] GTCAT-GCTGGGTAAGTTGTTTATATTTCCTG A TaqMan assaywith an existing probe for position minus443 in the osteopontinpromoter served as a reference assay 34 breast cancerDNA samples served as nonsarcoma control The assay wasperformed by the CCHMC DNA core

3 Results

31 Patient History In 1998 the patient was diagnosedwith high grade stage IIb osteogenic sarcoma of the right

femur which was extracompartmental Upon resection thelesion had a size of 95 times 3 times 4 cm (Figure 1) The tumorwas assessed as stage T2NxMx grade III characterized ashypercellular it showed marked cytologic atypia and a highmitotic rate Histologic features included anaplasia pleomor-phism numerous abnormal mitoses numerous giant cellsand osteoid production with focal calcifications

In 2012 a CT scan done because of hip pain revealed a43 times 34 times 31 cm lytic mass in the superior right acetabulumgrossly stable in size and configuration There was diffuseosteopenia involving the right femoral head and neck withdiffuse atrophy of the right pelvic girdle musculature Perios-titis and cortical interruptionwere associatedwith this lesion

The postsurgical pathology report identified the 57 times55 times 49 cm mass as a high grade leiomyosarcoma stagepT2bpNx Whereas a bone scan revealed intense activityin the left seventh rib a follow-up chest CT providedno indication of pulmonary masses mediastinal or hilarlymphadenopathy enlargement of axillary nodes or pleuraleffusion implying stage M0 The mitotic rate was 70 with60 necrosis The tumor caused extensive bone destructionand involvement of adjacent tissue Histologically the tumorcells stained positively for smooth muscle actin They were

4 Sarcoma

also positive for CD68 and displayed diffuse positive stainingfor vimentin but were negative for CD117 pancreatin S-100and CD34

Seven months after the surgery the patient received aPET-MRI scan for pain which revealed sixmetastatic lesionsincluding both lungs and multiple ribs She was put on three21-day cycles of Gemzar (days 1 and 8) Taxotere (day 8) andNeulasta (beginning on day 9) but was unable to continuepast the first cycle due to hospitalizations for continued andproblematic wound infections at the surgical lung biopsysites

32 DNA Exome Sequence Exome sequencing of thegenomic DNAs for tumor muscle and bone identified65546 potential sequence variants Filtering yielded 46 likelysomatic mutations in the tumor (Table 1) of which 7 (affect-ing EIF4A1 EPHA3 FAF1 IPO8 KIAA1377 LIMCH1 andNIPBL) were confirmed in the RNASeq results FAF1 asso-ciates with FAS and enhances apoptosis mediated throughthis receptor [12] The point mutation S181G (Figure 2) couldcause a loss of function in FAF1 and lead to transformationvia antiapoptosis

33 RNA Analysis Expectedly the gene expression patternsaccording to RNASeq were very different among tumormuscle and bone The cancer contained several gene prod-ucts that were overexpressed compared to both muscle andbone (Tables 2(a) and 2(b)) Among the top 30 changesin the tumormuscle and tumorbone comparisons 13 wereidentical (Table 2(c)) It is implied that these gene productsare quite unique for the tumor and likely contribute to itspathogenesis This notion is supported by the upregulationof the smooth muscle gene Ano4 which corroborates theleiomyosarcomatous nature of the cancer When limiting theanalysis to genes expressed at least at the level of 1 unit the17 genes overexpressed in the tumormuscle and tumorbonecomparisons contain several extracellular matrix proteinsimplying an active remodeling of the tumor microenviron-ment (Table 2(d)) By contrast none of the underexpressedgene products in the tumormuscle comparison matched thetumorbone comparison (not shown)

Of note the RNA level of IGF2BP1 (IMP-1 CRD-BPand ZBP-1) is highly upregulated in the tumor compared tomuscle as well as bone IGF2BP1 is a RNA-binding factorthat affects mRNA nuclear export localization stability andtranslation It regulates mRNA stability during the inte-grated cellular stress response in stress granules IGF2BP1 isa transcriptional target of the WNT pathway which is nega-tively regulated by intact FAF1 and may be unregulated byFAF1S181GThe IGF systemhas been linked to sarcoma patho-genesis [13] and may play a role in this specific cancer OtherIGF family members with increased RNA message levels inthis tumor (compared to muscle and bone) include IGFBPL1(5-6-log

2-fold) IGFL3 (6-7-log

2-fold) and IGF2BP3 (2-6-

log2-fold)The identified point mutation in FAF1 may be patho-

genetic for this cancer FAF1 is a regulator of NF-120581B activa-tion It directly binds to RelA (P65) retaining it in the cyto-plasm It can also interact with IKK120573 thus allowing for the

I120581B-mediated degradation of the transcription factors P65and P50 [6] Consistently the expression of regulators of theNF-120581B activation pathway is skewed in the tumor comparedto muscle or bone (Table 2(e))

34 Transcription Factor Binding ProteinDNA arrays mea-sure the binding activity of transcription factors Theycomprise three basic steps A set of biotin-labeled DNAbinding oligonucleotides are preincubated with a nuclearextract of interestThe proteinDNA complexes are separatedfrom the free probes The probes in the complexes arethen extracted and hybridized to prespotted membranesfollowed by HRP-based chemiluminescence detection Wemade nuclear extracts from tumor bone and muscle andtested them for DNA binding activity Binding that wasinduced in cancer but not in the normal tissues was dis-played by the transcription factors E2F1 AP3 LIII-BP PAX6ADD-1 and CCAC [14ndash19] The CCAC binding activity isconsistent with a muscle-derived tumor E2F1 may associatewith the WNT pathway-induced transcription factor LEF1resulting in transcriptional derepression of E2F1 [20] Likelyconstitutive transcription factors that are active in all 3 tissuescomprise AhRAmt GATA1 GATA2 GATA12 HIF1 andHOXD8910 (Figure 3)

35 Protein Analysis 2D gel electrophoresis of the RIPAlysates from tumor muscle and bone showed very divergentpatterns (Figure 4(a)) Two experienced analysts comparedthe protein pattern from the tumor with the protein patternfrom either bone or muscle Polypeptide spots that wereunique to the gels from the tumor were outlined (spotsunique to or relatively darker in the bone or muscle werenot indicated)The labeled proteins were extracted for identi-fication with mass spectrometry This yielded several struc-tural proteins which may reflect modification of the cellulararchitecture under rapid growth Transgelin-1 and transgelin-2 were abundant and corroborated the identity of thetumor as a leiomyosarcoma Four calcium-binding proteinswere highly expressed in the cancer In addition regulatorsof protein synthesis (40S ribosomal protein S12 glycine-tRNA ligase) protein modification (N-terminal fragmentof heat shock protein HSP 90120572 C-terminal fragment ofprotein disulfide isomerase) and protein degradation (1205721-antitrypsin proteasome activator complex subunit 2) wereidentified (Figure 4(b)) Of interest may be the C-terminalfragment of protein disulfide isomerase which not onlyhydroxylates prolines in preprocollagen but also contributesto microsomal triglyceride transfer It could be reflectiveof a skewed tumor metabolism The protein analysis wascorroborated by the mRNA levels (Figure 4(d))

Cancer markers were tested according to Western blot(Figure 4(c)) The tumor but not normal muscle expressedthe metastasis protein osteopontin and a single small form(lt75 kD) of CD44 that is likely the not alternatively splicedstandard form Unexpectedly while both tumor and muscleexpressed comparably abundant amounts of STAT3 phos-phorylation (reflective of activation) was present in themuscle but not in the tumorThe STAT3 pathway is associatedwith progression in several human cancers and this is often

Sarcoma 5

Table1DNAexom

eSNPsTh

eDNAexom

esfortum

orm

uscle

and

bone

weres

equencedTh

eresultswerefi

lteredin

thefollowingorder(1)d

ifferentgenotypeintumor

from

muscle

and

bonew

ithmuscle

andbo

nebeingidentic

alto

each

other(2)d

eletem

utations

with

lowconfi

dence(

valueo

f20or

lower)inall3

tissues(3)

deleteun

identifi

edgenes(4)d

eletem

utations

thatareh

omozygou

sreference

inthetum

or(5)

deletemutations

thathave

aMAFin

dbSN

Pgt10(6)

deletelowim

pactandmod

ifier

mutationsTh

egenen

ames

arep

arto

fthe

keyin

the

leftcolumnIn

thiscolumnresults

onbo

ldfont

representSNPs

thatwerec

onfirmed

ontheR

NAlevelbyRN

ASeqTh

edatafi

lesh

aveb

eensubm

itted

totheN

CBIsho

rtread

archive(SR

A)

underthe

accessionnu

mberS

RP052797

(biosamples

SAMN03316820SAMN03316821SAMN03316822)

Key

Chromosom

ePo

sition

Reference

Alternate

dbSN

PID

dbSN

PMAF

ESPMAF

(All)

ESPMAF

(EA)

ESPMAF

(AA)

Con

sensus

impact

Bone

muscle

geno

type

Bone

overall

depth

Bone

allele

depths

Bone

quality

Muscle

overall

depth

Muscle

allele

depths

Muscle

quality

Tumor

geno

type

Tumor

overall

depth

Tumor

allele

depths

Tumor

quality

177479

998T

EIF4

A1

177479998

CT

mdash

mdashmdash

mdashMod

erate

00

175

1840

99103

1080

9901

133

7968

99389

2592

00T

EPHA3

389259200

CT

mdash

mdashmdash

mdashMod

erate

00

43450

99117

1230

9901

723048

9915120

4545

CFA

F11

51204545

TC

mdash

mdashmdash

mdashMod

erate

00

87910

99108

1130

9901

8566

27

9912

3083

4620

AIPO8

1230834620

CA

mdash

mdashmdash

mdashMod

erate

00

68712

99120

1260

9901

807018

9911

101834

425A

KIA

A1377

11101834425

GA

mdash

mdashmdash

mdashMod

erate

00

36371

9067

700

9901

863163

9944168

2102

CLIMCH

14

41682102

GC

mdash

000

0077

0000

0227

Mod

erate

00

71740

9996

1010

9901

153

12049

99536

985326

GNIPBL

536985326

AG

mdash

mdashmdash

mdashMod

erate

00

660

1835

360

8101

201012

99377623789

ARO

BO2

377623789

AGA

mdash

mdashmdash

mdashHigh

00

22NA

5429

NA

8701

37NA

99

2217300

095A

SMARC

AL1

2217300

095

ATTG

CATC

A-AC

GTC

GTG

GA

mdash

mdashmdash

mdashHigh

00

95NA

99122

NA

9901

99NA

99

2152236045TTA

ATN

FAIP6

2152236045

TATTA

Ars3506

0021

mdashmdash

mdashmdash

High

02

19NA

124

NA

5701

17NA

383520206

69TAC

Y13

520206

69G

T

mdashmdash

mdashmdash

Mod

erate

00

130

1360

9986

900

9901

804543

999117

130734

GAKN

A9

117130734

CG

mdash

mdashmdash

mdashMod

erate

00

27280

7830

310

9001

503024

9912

6030354A

ANO2

126030354

CA

mdash

mdashmdash

mdashMod

erate

00

101

1060

99114

1200

9901

135

10445

9918

10487667

AAPC

DD1

1810487667

GA

mdash

mdashmdash

mdashMod

erate

00

43450

9938

400

9901

503816

99734118

560C

BMPE

R7

34118

560

GC

mdash

mdashmdash

mdashMod

erate

00

69720

99101

1060

9901

716314

9915

24921273

TC1

5orf2

1524921273

GT

mdash

mdashmdash

mdashMod

erate

00

12120

3638

390

9901

26226

9919

54483249

CCA

CNG8

1954483249

TC

mdash

mdashmdash

mdashMod

erate

00

147

1540

99103

1080

9901

152

13632

9910

16893265

CCU

BN10

16893265

GC

mdash

mdashmdash

mdashMod

erate

00

57600

9990

940

9901

403410

997148489854C

CUL1

7148489854

AC

mdash

mdashmdash

mdashMod

erate

00

73760

9965

680

9901

574221

9917

41566894

CDHX8

1741566894

GC

mdash

mdashmdash

mdashMod

erate

00

106

1110

9988

920

9901

124

9442

9920

61512358

TDID

O1

2061512358

GT

rs73304513

00417

0045932

000

0838

0135456

Mod

erate

00

600

182

00

601

400

2116

23703526

AER

N2

1623703526

GA

mdash

mdashmdash

mdashMod

erate

00

81850

9998

1030

9901

916041

993197880164G

FAM157A

3197880164

GCA

GCA

GCA

AG

mdash

mdashmdash

mdashMod

erate

00

21NA

2525

NA

101

31NA

614

4564

4287

GFA

NCM

144564

4287

AG

000

09mdash

mdashmdash

Mod

erate

00

13130

3331

320

8701

12102

14189637524

CGBP

71

89637524

GC

mdash

mdashmdash

mdashMod

erate

00

171

1790

99201

2110

9901

206

1616

799

742003933

CGLI3

742003933

GC

mdash

mdashmdash

mdashMod

erate

00

87910

9981

850

9901

856132

9917

4837170TGP1BA

174837170

CT

mdash

mdashmdash

mdashMod

erate

00

16160

459

9015

01

11101

114

24635161

GIRF9

1424635161

TG

mdash

mdashmdash

mdashMod

erate

00

66690

9943

450

9901

362513

9915

42133096

AJM

JD7-

PLA2G

4B15

42133096

GA

mdash

mdashmdash

mdashMod

erate

00

111

1160

9975

780

9901

807414

92

1740271678

AKA

T2A

1740271678

GA

mdash

mdashmdash

mdashMod

erate

00

65680

9958

610

9901

563032

99873848894

GKC

NB2

873848894

AG

mdash

mdashmdash

mdashMod

erate

00

15150

3324

250

6601

301517

9919

50827053

TKC

NC3

1950827053

CT

mdash

mdashmdash

mdashMod

erate

00

220

2310

99136

1430

9901

160

13046

9917

21319069

AKC

NJ12

1721319069

GA

rs76265595

mdashmdash

mdashmdash

Mod

erate

00

23241

6343

434

4001

28255

6012

53011932

CKR

T73

1253011932

TC

mdash

mdashmdash

mdashMod

erate

00

146

1530

99157

1650

9901

159

11758

99X

7500

4584

AMAG

EE2

X7500

4584

CA

mdash

mdashmdash

mdashMod

erate

00

29300

8148

500

9901

382616

995140182157A

PCDHA3

5140182157

GA

mdash

mdashmdash

mdashMod

erate

00

180

1890

99153

1610

9901

150

13234

9915

42133096

APL

A2G

4B15

42133096

GA

mdash

mdashmdash

mdashMod

erate

00

111116

099

75780

9901

807414

9210

96005840

CPL

CE1

1096005840

TC

mdash

mdashmdash

mdashMod

erate

00

81851

9973

762

9901

875540

991166816805G

POGK

1166816805

CG

mdash

mdashmdash

mdashMod

erate

00

84880

9982

860

9901

634920

99

6 Sarcoma

Table1Con

tinued

Key

Chromosom

ePo

sition

Reference

Alternate

dbSN

PID

dbSN

PMAF

ESPMAF

(All)

ESPMAF

(EA)

ESPMAF

(AA)

Con

sensus

impact

Bone

muscle

geno

type

Bone

overall

depth

Bone

allele

depths

Bone

quality

Muscle

overall

depth

Muscle

allele

depths

Muscle

quality

Tumor

geno

type

Tumor

overall

depth

Tumor

allele

depths

Tumor

quality

12111

020739

TPP

TC7

12111

020739

TCGC

Trs71083132

mdashmdash

mdashmdash

Mod

erate

00

18NA

3014

NA

1801

19NA

1019

804293

APT

BP1

19804293

CA

mdash

mdashmdash

mdashMod

erate

00

120

1261

9971

741

9901

755330

9971564

51175C

RNF32

71564

51175

GC

mdash

mdashmdash

mdashMod

erate

00

55570

9967

700

9901

594223

993520206

69TRP

11-155D

1811

3520206

69G

T

mdashmdash

mdashmdash

Mod

erate

00

130

1360

9986

900

9901

804543

9921

43838624

TUBA

SH3A

2143838624

GT

mdash

mdashmdash

mdashMod

erate

00

70730

9945

470

9901

897621

9919

58773857

AZN

F544

1958773857

GA

mdash

mdashmdash

mdashMod

erate

00

46480

9943

450

9901

715126

99516465723

CZN

F622

516465723

TC

mdash

mdashmdash

mdashMod

erate

00

17170

3914

140

3301

271415

99

Sarcoma 7

Table 2 RNA induced in the tumor (a) Transcripts overexpressed in the tumor compared to muscle but not bone (b) Transcriptsoverexpressed in the tumor compared to bone but not muscle (c) Transcripts most abundantly overexpressed in the tumor compared tomuscle and bone (d) as (a) but limited to genes expressed at least at the level of 1 unit in the reference tissue (muscle or bone) (e) Alteredexpression of genes associated with NF-120581B activity Analysis of RNASeq for the transcription factor NF-120581B and gene products that regulate itsactivity RNAmessages that are selectively associated with the TNF pathway to NF-120581B activation are marked with bold font log2-fold changeindicates the alteration in the tumor compared to muscle or bone (the respective columns indicate the expression level for each organ)

(a)

log2-fold changeMuscle

Tumor over muscleNRG1 Neuregulin 1 9909KSR2 Kinase suppressor of ras 2 9675MMP13 Matrix metallopeptidase 13 (collagenase 3) 9528SPP1 Secreted phosphoprotein 1 9098INHBA Inhibin beta A 8570COL11A1 Collagen type XI alpha 1 8564MMP9 Matrix metallopeptidase 9 (gelatinase B 92 kda gelatinase 92 kda type IV collagenase) 8552FBN2 Fibrillin 2 8495LRRC15 Leucine rich repeat containing 15 8429MMP11 Matrix metallopeptidase 11 (stromelysin 3) 8336E2F7 E2F transcription factor 7 8314PRAME Preferentially expressed antigen in melanoma 8179PTK7 PTK7 protein tyrosine kinase 7 7996DSCAM Down syndrome cell adhesion molecule 7761RGS4 Regulator of G-protein signaling 4 7633CNIH3 Cornichon homolog 3 (Drosophila) 7632OR10V1 Olfactory receptor family 10 subfamily V member 1 7610CEP55 Centrosomal protein 55 kda 7583WNT5B Wingless-type MMTV integration site family member 5B 7569CA12 Carbonic anhydrase XII 7520

GALNT5 UDP-N-acetyl-alpha-D-galactosaminepolypeptide N-acetylgalactosaminyltransferase 5(GalNAc-T5) 7517

(b)

log2-fold changeBone

Tumor over boneROS1 c-ros oncogene 1 receptor tyrosine kinase 10987GREM1 Gremlin 1 10604NPTX1 Neuronal pentraxin I 8813GJB2 Gap junction protein beta 2 26 kDa 8597CREB3L1 cAMP responsive element binding protein 3-like 1 8506KRT14 Keratin 14 8351SERPINE1 Serpin peptidase inhibitor clade E (nexin plasminogen activator inhibitor type 1) member 1 8288HOXD10 Homeobox D10 8105ALPK2 Alpha-Kinase 2 7783POU3F2 POU class 3 homeobox 2 7530POSTN Periostin osteoblast specific factor 7497NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439STC2 Stanniocalcin 2 7434WNT5A Wingless-type MMTV integration site family member 5A 7412IGFN1 Immunoglobulin-like and fibronectin type III domain containing 1 7387

8 Sarcoma

(b) Continued

log2-fold changeBone

STC1 Stanniocalcin 1 7385FAM180A Family with sequence similarity 180 member A 7315KRT17 Keratin 17 7305BBOX1 Butyrobetaine (gamma) 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 7277MAGEA1 Melanoma antigen family A 1 (directs expression of antigen MZ2-E) 7267

(c)

log2-fold changeMuscle Bone

Tumor over bonemuscle

ANO4 Anoctamin 4 (TMEM16D) transmembrane calcium-activated chloride channelfacilitates smooth muscle contraction 9937 8542

SLCO1B3 (OATP1B3) Solute carrier organic anion transporter family member 1B3 9569 9760

MARCH4 Membrane-associated ring finger (C3HC4) 4 E3 ubiquitin ligase located predominantlyto the endoplasmic reticulum 9538 7406

IGF2BP1 Insulin-like growth factor 2 mRNA binding protein 1 binds to and stabilizes mRNA 9440 9631ADAMTS16 ADAMmetallopeptidase with thrombospondin type 1 motif 16 zinc-dependent protease 9260 8450SOX11 SRY (sex determining region Y)-box 11 important in brain development 9178 9954

HAPLN1 Hyaluronan and proteoglycan link protein 1 stabilizes aggregates of aggrecan andhyaluronan giving cartilage its tensile strength and elasticity 8951 8142

MUC15 Mucin 15 cell surface associated 8801 7992HOXB9 Homeobox B9 8773 7378MAGEC2 Melanoma antigen family C 2 8693 8884

ST6GALNAC5 ST6 (alpha-N-acetyl-neuraminyl-23-beta-galactosyl-13)-N-acetylgalactosaminidealpha-26-sialyltransferase 5 7848 8038

C11orf41 Chromosome 11 open reading frame 41 7781 8141MMP1 Matrix metallopeptidase 1 (interstitial collagenase) 7455 7646

(d)

Symbol Name log2-fold change log2-fold changeMuscle Bone

Tumor over bonemuscleFN1 Fibronectin 1 7328 6293 6457 19860COL1A1 Collagen type I alpha 1 6768 3706 5868 11934CCND1 Cyclin D1 5595 3958 5098 9637RGS1 Regulator of G-protein signaling 1 5118 1056 4653 2533ITGBL1 Integrin beta-like 1 (with EGF-like repeat domains) 5071 3177 3895 12415COL1A2 Collagen type I alpha 2 4852 8756 4910 14503MXRA5 Matrix-remodelling associated 5 4834 1352 4631 2685POSTN Periostin osteoblast specific factor 4513 5870 7497 1267PLOD2 Procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 4285 1801 4023 3730COL5A2 Collagen type V alpha 2 4275 1297 4833 1517COL3A1 Collagen type III alpha 1 4003 13118 5801 6500

SEMA3C Sema domain immunoglobulin domain (Ig) shortbasic domain secreted 3954 1164 4215 1675

FBN1 Fibrillin 1 3912 6957 4018 11148AEBP1 AE binding protein 1 3808 3212 4002 4843SIK1 Salt-inducible kinase 1 3805 1219 3566 2484

Sarcoma 9

(d) Continued

Symbol Name log2-fold change log2-fold changeMuscle Bone

SERPINH1 Serpin peptidase inhibitor clade H (heat shock protein47) member 1 3706 1652 4145 2098

ANTXR1 Anthrax toxin receptor 1 3670 3789 3690 6445

(e)

Symbol Name log2-fold change log2-fold changeMuscle Bone

HELLS Helicase lymphoid-specific 5315602 0155898 minus082713 202489

TNFRSF11A Tumor necrosis factor receptor superfamily member 11a andNFKB activator 3017922 003091 1400993 0202453

NFKBID Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor delta 2655352 0 minus147615 0504341

TNFRSF25 Tumor necrosis factor receptor superfamily member 25 2432959 0046388 0163954 0490795

NFKBIE Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor epsilon 2121015 0062395 0311441 043382

TNF Tumor necrosis factor 1847997 0 minus142101 003733

TNFRSF10D Tumor necrosis factor receptor superfamily member 10d decoywith truncated death domain 1754888 0172968 minus01757 1183343

TNFRSF1B Tumor necrosis factor receptor superfamily member 1B 1473438 0709902 minus097011 6729401TNFRSF10A Tumor necrosis factor receptor superfamily member 10a 1411898 0828219 0357701 3004386

NFKBIB Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor beta 1193993 1209816 046055 3484692

TNFRSF10B Tumor necrosis factor receptor superfamily member 10b 1094157 1908597 0425446 5242006TNFRSF21 Tumor necrosis factor receptor superfamily member 21 1055762 3882038 0745815 8305711TNFRSF1A Tumor necrosis factor receptor superfamily member 1A 0875167 3790938 minus011707 130344

NFKBIZ Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor zeta 0575974 3698951 0344657 7493136

RIPK1 Receptor (TNFRSF)-interacting serine-threonine kinase 1 0494467 311749 minus108854 161392NKRF NFKB repressing factor 0475722 2156087 minus086397 9434166NFKB1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 0432959 2054532 minus065865 7568586CHUK Conserved helix-loop-helix ubiquitous kinase 0176126 2961281 minus120204 1330333RELA V-rel reticuloendotheliosis viral oncogene homolog A (avian) 0013848 1263837 0287167 1803044

NFKB2 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 2(p49p100) minus008641 0312993 0485882 0360442

NKAPP1 NFKB activating protein pseudogene 1 minus010692 0825177 minus099449 2655392

TNFRSF10C Tumor necrosis factor receptor superfamily member 10c decoywithout an intracellular domain minus0152 0064676 minus556891 6321515

NKIRAS2 NFKB inhibitor interacting Ras-like 2 minus060768 1095135 minus088758 2299946

NFKBIL1 Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor-like 1 minus08719 0141933 minus008357 0140418

NKIRAS1 NFKB inhibitor interacting Ras-like 1 minus087428 6296399 1467735 2122983TANK TRAF family member-associated NFKB activator minus0983 6777124 minus151908 1695872NKAP NFKB activating protein minus135735 1422458 minus078243 1646287

NFKBIA Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor alpha minus185483 4032743 minus031802 2395275

NKAPL NFKB activating protein-like minus557347 5875743 minus140215 0534643

10 Sarcoma

FAS associating region

DED interacting region

UB12 DZM

UB2domain

UASdomain

UBXdomain

UBAdomain

S289 S291

Aurora ACK II

S181

AKT1ATMGSK3

AuroraPKG

PLK1RSK

STK4CHK1

650566481336260205169110475

(a)

MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRM

6

1

101

100

200

300

400

500

600

650

201

301

401

501

601

667855899998876467765678888887578758999986467 88 8888888888888988888899988888889888877767888777888757

2222454345655744642554435764462443558468643653433344333424435334233333332333332333354335333344334354

LDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITH

9999997797689998787758999999999859996577754788888888875333467888868987688888888888877888855675799987

9585847535375857543447459666955563844544585374435443365845846434457567533353232334334444433337484944

REVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDAT

5888757887577654788887766654677776555468877888765455546887766555688888877777777777776556788888767766

5344456484742545656645594364384534437445444342323455453535435463434323333343434333333334333335433333

EFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESI

5666776654456777888888888888866889999999998848888876677778999999998776686899998589875579999987486899

4635343574354322343434533333343548479747945564424647635575478569766456468999999754233454687457844669

VSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDE

9999888589997557877545444345677876335554467888876899986799876447777677888999999998877555778887777778

6778547999996674433343444333333335466456434433487999895353333464346434365468755846444545454565446565

REARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVF

8888888887788877766545557777777666777777777776678877777766578888888888885799998589975788758987589999

4545445564445344433443354555556653665456544454434444444443344334333333446859596743354745795535796898

DFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKE

99997799988779985788754577888765665678898589998589

67974525444795877564635844444476875837446989897366

(b)

Figure 2 FAF1 structure (a) A schematic of functional domains in FAF1 with annotations (adapted from [6ndash8]) There are two ubiquitin-like domains flanking the S181 site The PDB structure 2DZM identifies the N-terminal one while the C-terminal prediction is by sequencesimilarity Whereas the mutation S181G is not expected to disrupt the protein structure per se this amino acid has a high score as a possiblephosphorylation site for a number of kinases involved inDNAdamage repair (b) Secondary structure prediction of FAF1The residues aroundS181 appear unstructured

reflected in STAT3 constitutive phosphorylation The lack ofphosphorylation in this case suggests that the leiomyosar-coma may not depend on the STAT3 pathway

36 Pharmacogenetic Evaluation Predicting the sensitivityto anticancer drugs is a main goal of molecular analysisFor this the over- or underexpression of genes for drugtransport and metabolism is of key importance Analysis ofthe RNASeq data for these groups of gene products identified

a surprisingly large number of deregulations compared tomuscle or bone (Tables 3(a) and 3(b)) Those alterationsmay affect choices for drug treatment For example the highlevels of glutathione S-transferase may render carmustinethioTEPA cisplatin chlorambucil melphalan nitrogenmus-tard phosphoramide mustard acrolein or steroids ineffec-tive The overexpression ofN-acetyltransferase may compro-mise 5-fluorouracil or taxolThemodest upregulation of onlytwo export transporters (ABC-transporters) and specifically

Sarcoma 11

Bone Tumor

Muscle

(a)

Transcription factor Description Reference

E2F1 Transcriptional activation of cell cycle progression is selectively activated in response to specific cues key downstream target of RB1 can promote proliferation or apoptosis when activated [14]

AP-33 interact in a mutually exclusive manner [15]

CCAC CCAC box is an essential element for muscle-specific transcription from the myoglobin promoter [16]

LIII-BP The L-III transcriptional regulatory element (a carbohydrate-response element) is in the pyruvate kinase L gene necessary for cell type-specific expression and transcriptional stimulation by carbohydrates [17]

ADD-1 (SREBP-1) activates transcription from sterol-regulated elements and from an E-box sequence present in the promoter of fatty acid synthase [18]

PAX6 Member of the paired box gene family transcriptional regulator involved in developmental processes [19]

48kD transcription factor activator protein-3 binds to the core element and recognizes the SV40 enhancer and AP-2 and AP-

(b)

Figure 3 Transcription factor activation Nuclear extracts from tumor muscle and bone were tested for DNA binding activity on a protein-DNA array (a) Transcription factor binding that was high in all 3 tissues and is therefore considered constitutively active is circled in yellow(left to right top to bottom AhRAmt GATA1 GATA2 GATA12 HIF1 and HOXD8910) Transcription factors that show high binding inthe cancer but not in muscle or bone are circled in red (left to right top to bottom E2F1 AP3 LIII-BP PAX6 ADD-1 and CCAC) (b) Thefunctions of transcription factors that display high DNA binding in the cancer but not in muscle or bone are described

the lack of ABCB1 overexpression is favorable for avoidingdrug resistance

37 Population Analysis The above-described results indi-cated that a FAF1 mutation which replaces serine in position181 thus preventing FAF1 phosphorylation and activationmay be a driver for leiomyosarcomagenesis A custom Taq-Man assay confirmed the presence of the somatic mutationin the patient To assess whether this single nucleotidereplacement is common in this type of cancer we analyzedDNA from 29 leiomyosarcomas and 1 blood sample from aleiomyosarcoma patient For comparison to nonsarcomatoustumors 34 breast cancers served as a reference None of themdisplayed amutation in the same locus By contrast there wasa distribution across all leiomyosarcomas in the osteopontin

promoter position minus443 (used as a reference) with 12 CC 10TC and 9 TT

4 Discussion

TheFAF1mutation identified as the likely cause for the cancerunder study gives room for an explanation of the sarcomatoustransformation (Figure 5) DNA damage to mesenchymalcells occurs persistently in an oxidizing environment at 37∘CThese insults are rarely transforming and such an occurrencewould trigger the initiation of programmed cell death inapoptosis-competent cells Intact FAF1 associates with FASand enhances apoptosis mediated through this receptor [12]A loss of function in FAF1 could lead to transformation viaantiapoptosis Whereas the mutation S181G is not expected

12 Sarcoma

Muscle

Bone

Tumor

220

94

6043

29

14

220

94

60

43

29

14

220

94

60

43

29

14

1

7

6

23

24

21

22

25

26

89 1011 12

1516 1719

pH 4 8 pH 4 8

8pH 4

(a)

SwissProt or NCBISpot number Protein identified accession Description

(Structural)6 Transgelin Q01995 Smooth muscle-specific cross-links actin24 Transgelin-2 P37802 Smooth muscle-specific cross-links actin

Vimentin P08670 Intermediate filament in mesenchymal tissue1 Filamin-A P21333 Actin-binding protein regulates the cytoskeleton 21 P06709 Cytoskeleton

(Calcium homeostasis)8 9 Calumenin O43852 Calcium-binding protein in the ER and golgi apparatus26 Protein S100-A11 P31949 Calcium-binding EF hand protein10 Reticulocalbin-3 Q96D15 Calcium-binding protein located in the ER12 P11021 Heat shock 70-kD protein 5 ER lumenal calcium-binding

(Protein processing)25 40S ribosomal protein S12 P25398 Core component of the ribosomal accuracy center7 Glycine-tRNA ligase P41250 Catalyzes the attachment of glycine to tRNA19 P01009 Protease inhibitor23 P01009 Protease inhibitor21 Proteasome activator complex subunit 2 Q9UL46 Proteasome activation10 P07900 Chaperone

(Other)7 Serum albumin P02768 Carrier protein in blood22 Protein disulfide isomerase (C-terminal fragment) P07237 Prolyl hydroxylation in collagen microsomal triglyceride transfer

120573 actin (C-terminal fragment)

78kD glucose-regulated protein

120572-1-antitrypsin120572-1-antitrypsin (C-terminal fragment)

Heat shock protein HSP 90120572 (N-terminal fragment)

11 15ndash17

(b)

Figure 4 Continued

Sarcoma 13

Tumor Muscle

OPN

CD44

STAT3

P-STAT3

Tubulin

75

75

50

15010075

10075

50

(c)

Tumor bone Tumor muscleGene ID Symbol Name Fold change Expression Fold change Expression6876 TAGLN Transgelin 3274 2455 3087 15088407 TAGLN2 Transgelin-2 2164 7338 6975 13037431 VIM Vimentin 1653 295010 4061 696042316 FLNA Filamin A alpha 1304 7791 9005 065160 ACTB Actin beta 0656 973187 5491 67368813 CALU Calumenin 7601 19328 12063 70596282 S100A11 S100 calcium binding protein A11 0931 158914 5071 1686357333 RCN3 Reticulocalbin 3 EF-hand calcium binding domain 2439 0604 6412 01223309 HSPA5 1068 92754 2607 220356696 SPP1 Secreted phosphoprotein 1 7572 46508 547929 0355960 CD44 CD44 molecule (Indian blood group) 2053 26708 15436 20566774 STAT3 Signal transducer and activator of transcription 3 0617 46534 0832 200165925 RB1 Retinoblastoma 1 0736 14772 0442 14268

Heat shock 70kDa protein 5 (glucose-regulated protein 78kDa)

(d)

Figure 4 Protein overexpression (a) 2D protein gel electrophoresis of lysates from muscle (upper left) tumor (upper right) and bone(bottom) in RIPA buffer Red circles indicate the spots that were identified as overexpressed and were further analyzed by mass spectrometry(b) Proteins identified in 2D gel electrophoresis as overexpressed in the tumor in comparison to muscle and bone were analyzed for theiridentity by mass spectrometry The left column indicates the spot number corresponding to the 2D gel The next column contains theprotein name followed by the accession number and a description of the protein functionThe protein functions are grouped into structuralcalcium homeostasis and various others (c) Western blot 10 120583g lysates of tumor muscle and bone in RIPA buffer were loaded per laneand electrophoresed on 10 SDS-polyacrylamide minigels with reducing denaturing sample buffer After transfer to PVDF membranesthey were probed for markers of cancer progression including osteopontin CD44 STAT3 and phospho-STAT3 Antitubulin served as aloading control (d) RNA levels corresponding to the proteins found to be affected in the sarcoma With four exceptions in the tumor-bonecomparison (gray font) upregulated proteins are associated with increased RNA levels STAT3 is not increased on the protein or RNA levelThe reduced level of RB1 expression is consistent with the elevated DNA binding activity of E2F1

to disrupt the structure of the protein this site does scorehigh as a possible phosphorylation site for a number ofkinases involved in DNA damage repair supporting thehypothesis that the cancer cells containing thismutation havelost their ability to respond to transforming DNA damagewith programmed cell death FAF1 antagonizes WNT signal-ing by promoting 120573-catenin degradation in the proteasome[21] a function that may be lost after the point mutationThe elevated DNA binding activity of the protooncogenictranscription factor E2F1 (see Figure 3) could be caused byits interaction with LEF-1 [20] consecutive to persistent

WNT signaling The RNA level of IGF2BP1 (IMP-1) a stress-responsive regulator of mRNA stability is highly upregulatedin the tumor compared to muscle as well as bone (seeTable 2(c)) IGF2BP1 is a transcriptional target of the WNTpathway that regulates NF-120581B activity (see Table 2(e)) andis antiapoptotic [22 23] IGF2BP1 may be upregulated asa consequence of mutated FAF1 not being able to suppressWNT signaling in this specific cancerOf noteWNTpathwayoveractivity may not be required for transformation ratherthe persistence of a WNT pathway signal due to the lack of aFAF1-mediated termination signal may suffice

14 Sarcoma

WNTFrizzled

Axin Dvl

igf2bp1

FAF1

P65 P50

IKK

FAS

120573-Catenin

LEF + E2F1

I120581B

Figure 5 Possible transforming pathway The point mutation inFAF1 is believed to cause a loss of function (crossed out in red)This may lead to an overactivity of the WNT signaling pathwayConsistently E2F1 (activated by LEF1) and igf2bp1 (a transcriptionaltarget of the WNT pathway) have been identified to be upregulatedin the molecular analysis In contrast to the negative regulator FAF1IGF2BP1 is a positive regulator of NF-120581B activityThe overactivity ofthe NF-120581B pathway and the reduced efficacy of FAS signaling can betransforming

The role ofWNT signaling in sarcoma has been subject todebate (eg [24]) In metastatic leiomyosarcoma 120573-Cateninmay accumulate in the nucleus despite a relatively weakexpression of WNT [25] This may be due to WNT signalactivation via noncanonical ligands [26] or to 120573-Cateninbinding the nuclear receptor NR4A2 and releasing it from thecorepressor protein LEF-1 [27] Of note in the cancer understudy here the mRNA level of NR4A2 was overexpressed10-fold compared to bone and 3-fold compared to muscleThe results from this study are consistent with the possibilitythat a lack of termination in the WNT signal rather than itsoveractivation could contribute to sarcomatous transforma-tion Such a mechanism may be reflected in an upregulationof downstream targets even though overexpression of WNTpathway components is not detectable

Other mutations beside FAF1 are less likely to becausative for the cancer EPHA3 was revealed as mutated inthis cancer by DNA exome sequencing and RNASeq EPHA3is a receptor tyrosine kinase that is frequentlymutated in lungcancer Tumor-suppressive effects of wild-type EPHA3 can beoverridden by dominant negative EPHA3 somatic mutations[28]Thismechanism is unlikely to play a role in this sarcomaas the detected mutation is located far N-terminally on theextracellular Ephrin binding domain not on the intracellularkinase domain

FAF1 has been described to act as a tumor suppressor gene[29] Its depletion due to chromosome breakage can affectprognosis in glioblastoma patients [30 31] Single nucleotidepolymorphisms in FAF1 are associated with a risk for gastriccancer [32] While numerous FAF1 mutations are associatedwith various cancers none of these genetic changes in theTCGA database affects the amino acid position 181 (Table 4)

The major upregulations identified in this tumor com-prise muscle-specific gene products (transcription factors

CAAC binding proteomics transgelin transgelin-2 RNAanoctamin-4 and immunohistochemistry smooth mus-cle actin) and calcium-regulating molecules (proteomicscalumenin S100-A11 reticulocalbin-3 and 78 kD glucose-regulated protein) The muscle-specific gene products con-firm this recurrent sarcoma as a leiomyosarcoma (the firsttumor distal to the site of the recurring one had beendiagnosed as an osteosarcoma) Calcium is one of the majorsecond messengers in smooth muscle cells Its uptake isregulated by potential-sensitive ion channels in the cellmembrane and by the activities of various receptors Calciumis stored in the sarcoplasmic reticulum from where it canbe released to facilitate actin-myosin interaction and tensiongeneration Phosphorylation of the myosin light chain by acalmodulin-regulated enzyme is important for contractionThe upregulation of gene products associated with migrationand invasion (osteopontin MMP1 vimentin filamin-A and120573-actin) and gene products for extracellularmatrixmoleculesand their modulators (fibronectin collagen ITGBL1 andMXRA5) reflects the invasive nature of this cancer

The recurrence of a sarcoma after 14 years has twoprobable explanations either it is due to a cancer pre-disposition syndrome based on a germ-line mutation (theage of the patient weakens this hypothesis) or the secondtumor is a metastatic colony of the first that was reactivatedafter dormancy The location of the sarcoma in the sameextremity and proximal to a preceding mesenchymal cancer(and therefore in its natural path of dissemination) impliedthe probability that this was a relapse in a metastatic siteThe different histologic assessment as osteosarcoma in thefirst occurrence and leiomyosarcoma as the second cancerdoes not necessarily negate that Mixed histology [33 34]and transdifferentiation [35ndash37] have been described forsarcomatous tumors Of note however in this scenarioosteosarcoma seems to more commonly follow leiomyosar-coma than precede it Material from the first cancer of thispatient was not accessible to us It is very plausible that thiscould have been a mineralized leiomyosarcoma In thosetumors the differential diagnosis from osteosarcoma can bedifficult [38] The extracompartmental location of the firsttumor supports this interpretation

On the molecular pathology level sarcomas fall intotwo groups comprising tumors with simple karyotypes(with pathogenetic translocations or specific genetic muta-tions) and tumors with very complex karyotypes (overtchromosome and genomic instability with numerous gainsand losses) [39] Some molecular alterations that lead tocarcinogenesis can be defined in absolute termsThey includegain-of-function mutations or chromosome translocationsthat transformprotooncogenes to oncogenes However otherchanges are relative to the normal tissue of origin suchas pathway overactivity or overexpression on the proteinor RNA levels We have combined the analysis of absolutechanges (DNA exome sequence RNA sequence) with theanalysis of relative changes using skeletal muscle and bone asreference organs (protein-DNA array 2D gel electrophoresisand RNA expression levels) This choice was determined inpart by tissue availability after surgery andwas intended to aidin the distinction of osteosarcoma from myosarcoma While

Sarcoma 15

Table 3 Deregulation of genes for drug disposition Analysis of RNASeq for at least twofold overexpression (italic font) or underexpression(bold font) of genes for (a) transport and (b) metabolism in tumor compared to muscle and bone For the export transporters (ABCtransporters) only overexpression is considered relevant for drug resistance Not shown in the table are the underexpressed genes (ABCA7ABCA8 ABCA13 ABCB6 ABCB10 ABCC6 ABCC6P1 ABCC6P2 ABCC8 ABCC11 ABCD2 ABCG2 and ABCG5)

(a) Transport

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

650655 ABCA17P ATP-binding cassette subfamily A (ABC1) member 17 pseudogene 1360 211624 ABCA4 ATP-binding cassette subfamily A (ABC1) member 4 2038 2802

6555 SLC10A2 Solute carrier family 10 (sodiumbile acid cotransporter family)member 2 minus1962 minus2112

345274 SLC10A6 Solute carrier family 10 (sodiumbile acid cotransporter family)member 6 2623 3240

6563 SLC14A1 Solute carrier family 14 (urea transporter) member 1 (Kidd bloodgroup) minus3074 minus3152

6565 SLC15A2 Solute carrier family 15 (H+peptide transporter) member 2 minus2027 minus2152

6566 SLC16A1 Solute carrier family 16 member 1 (monocarboxylic acid transporter1) 1401 2170

117247 SLC16A10 Solute carrier family 16 member 10 (aromatic amino acid transporter) 2113 2848

6567 SLC16A2 Solute carrier family 16 member 2 (monocarboxylic acid transporter8) 3242 3848

10786 SLC17A3 Solute carrier family 17 (sodium phosphate) member 3 minus2868 minus29596571 SLC18A2 Solute carrier family 18 (vesicular monoamine) member 2 minus2087 minus21946573 SLC19A1 Solute carrier family 19 (folate transporter) member 1 minus2099 minus220310560 SLC19A2 Solute carrier family 19 (thiamine transporter) member 2 1820 254880704 SLC19A3 Solute carrier family 19 member 3 minus3331 minus3478387775 SLC22A10 Solute carrier family 22 member 10 5711 60859390 SLC22A13 Solute carrier family 22 (organic anion transporter) member 13 2623 3217

85413 SLC22A16 Solute carrier family 22 (organic cationcarnitine transporter)member 16 minus8101 minus7299

51310 SLC22A17 Solute carrier family 22 member 17 1475 21755002 SLC22A18 Solute carrier family 22 member 18 2038 27226582 SLC22A2 Solute carrier family 22 (organic cation transporter) member 2 4793 5216

6581 SLC22A3 Solute carrier family 22 (extraneuronal monoamine transporter)member 3 2554 3182

6583 SLC22A4 Solute carrier family 22 (organic cationergothioneine transporter)member 4 minus3603 minus3776

151295 SLC23A3 Solute carrier family 23 (nucleobase transporters) member 3 2109 284810478 SLC25A17 Solute carrier family 25 (mitochondrial carrier) member 17 1311 207783733 SLC25A18 Solute carrier family 25 (mitochondrial carrier) member 18 1846 2570

788 SLC25A20 Solute carrier family 25 (carnitineacylcarnitine translocase) member20 minus2105 minus2207

89874 SLC25A21 Solute carrier family 25 (mitochondrial oxodicarboxylate carrier)member 21 minus2962 minus3105

51312 SLC25A37 Solute carrier family 25 member 37 minus4633 minus486651629 SLC25A39 Solute carrier family 25 member 39 minus2585 minus2737203427 SLC25A43 Solute carrier family 25 member 43 1325 208865012 SLC26A10 Solute carrier family 26 member 10 3896 4472115111 SLC26A7 Solute carrier family 26 member 7 3431 4018116369 SLC26A8 Solute carrier family 26 member 8 minus5354 minus5536115019 SLC26A9 Solute carrier family 26 member 9 1623 241911001 SLC27A2 Solute carrier family 27 (fatty acid transporter) member 2 minus4278 minus4487

16 Sarcoma

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

64078 SLC28A3 Solute carrier family 28 (sodium-coupled nucleoside transporter)member 3 minus4543 minus4812

222962 SLC29A4 Solute carrier family 29 (nucleoside transporters) member 4 minus1962 minus204581031 SLC2A10 Solute carrier family 2 (facilitated glucose transporter) member 10 2532 3170

6518 SLC2A5 Solute carrier family 2 (facilitated glucosefructose transporter)member 5 minus3647 minus3821

55532 SLC30A10 Solute carrier family 30 member 10 minus3547 minus37257782 SLC30A4 Solute carrier family 30 (zinc transporter) member 4 2832 33726569 SLC34A1 Solute carrier family 34 (sodium phosphate) member 1 minus4716 minus4941340146 SLC35D3 Solute carrier family 35 member D3 minus5662 minus590654733 SLC35F2 Solute carrier family 35 member F2 1433 2170206358 SLC36A1 Solute carrier family 36 (protonamino acid symporter) member 1 minus2265 minus2345285641 SLC36A3 Solute carrier family 36 (protonamino acid symporter) member 3 minus2284 minus239354020 SLC37A1 Solute carrier family 37 (glycerol-3-phosphate transporter) member 1 minus2112 minus22122542 SLC37A4 Solute carrier family 37 (glucose-6-phosphate transporter) member 4 minus2117 minus2219151258 SLC38A11 Solute carrier family 38 member 11 2410 308555089 SLC38A4 Solute carrier family 38 member 4 1837 254892745 SLC38A5 Solute carrier family 38 member 5 minus1994 minus215291252 SLC39A13 Solute carrier family 39 (zinc transporter) member 13 1301 207023516 SLC39A14 Solute carrier family 39 (zinc transporter) member 14 2569 318829985 SLC39A3 Solute carrier family 39 (zinc transporter) member 3 minus2032 minus2152283375 SLC39A5 Solute carrier family 39 (metal ion transporter) member 5 1623 23707922 SLC39A7 Solute carrier family 39 (zinc transporter) member 7 1273 205830061 SLC40A1 Solute carrier family 40 (iron-regulated transporter) member 1 minus3612 minus379684102 SLC41A2 Solute carrier family 41 member 2 1293 20708501 SLC43A1 Solute carrier family 43 member 1 minus2013 minus215257153 SLC44A2 Solute carrier family 44 member 2 minus2047 minus215250651 SLC45A1 Solute carrier family 45 member 1 2038 2722146802 SLC47A2 Solute carrier family 47 member 2 minus1962 minus20266521 SLC4A1 Solute carrier family 4 anion exchanger member 1 minus6513 minus645357282 SLC4A10 Solute carrier family 4 sodium bicarbonate transporter member 10 minus2640 minus275383959 SLC4A11 Solute carrier family 4 sodium borate transporter member 11 1846 25696508 SLC4A3 Solute carrier family 4 anion exchanger member 3 1261 20458671 SLC4A4 Solute carrier family 4 sodium bicarbonate cotransporter member 4 2445 31139497 SLC4A7 Solute carrier family 4 sodium bicarbonate cotransporter member 7 2717 33076523 SLC5A1 Solute carrier family 5 (sodiumglucose cotransporter) member 1 minus2547 minus2705159963 SLC5A12 Solute carrier family 5 (sodiumglucose cotransporter) member 12 4753 52066527 SLC5A4 Solute carrier family 5 (low affinity glucose cotransporter) member 4 minus3969 minus4249

6540 SLC6A13 Solute carrier family 6 (neurotransmitter transporter GABA)member 13 1328 2092

55117 SLC6A15 Solute carrier family 6 (neutral amino acid transporter) member 15 1623 2333388662 SLC6A17 Solute carrier family 6 member 17 2038 272854716 SLC6A20 Solute carrier family 6 (proline IMINO transporter) member 20 1697 2433

6532 SLC6A4 Solute carrier family 6 (neurotransmitter transporter serotonin)member 4 minus2512 minus2611

6534 SLC6A7 Solute carrier family 6 (neurotransmitter transporter L-proline)member 7 2623 3228

56301 SLC7A10 Solute carrier family 7 (neutral amino acid transporter y+ system)member 10 minus3421 minus3603

Sarcoma 17

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

6547 SLC8A3 Solute carrier family 8 (sodiumcalcium exchanger) member 3 minus2204 minus2309285335 SLC9A10 Solute carrier family 9 member 10 1208 20006549 SLC9A2 Solute carrier family 9 (sodiumhydrogen exchanger) member 2 2038 2706

9368 SLC9A3R1 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 1 minus2760 minus2846

9351 SLC9A3R2 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 2 1889 2619

84679 SLC9A7 Solute carrier family 9 (sodiumhydrogen exchanger) member 7 3347 393510599 SLCO1B1 Solute carrier organic anion transporter family member 1B1 3360 397728234 SLCO1B3 Solute carrier organic anion transporter family member 1B3 9760 95696578 SLCO2A1 Solute carrier organic anion transporter family member 2A1 2432 309628232 SLCO3A1 Solute carrier organic anion transporter family member 3A1 minus2032 minus2152353189 SLCO4C1 Solute carrier organic anion transporter family member 4C1 minus6850 minus6611

(b) Metabolism

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold Cchange

1583 CYP11A1 Cytochrome P450 family 11 subfamily A polypeptide 1 1623 23961589 CYP21A2 Cytochrome P450 family 21 subfamily A polypeptide 2 minus1962 minus20361591 CYP24A1 Cytochrome P450 family 24 subfamily A polypeptide 1 5208 55771592 CYP26A1 Cytochrome P450 family 26 subfamily A polypeptide 1 2623 32571594 CYP27B1 Cytochrome P450 family 27 subfamily B polypeptide 1 1846 2585339761 CYP27C1 Cytochrome P450 family 27 subfamily C polypeptide 1 3585 41781553 CYP2A13 Cytochrome P450 family 2 subfamily A polypeptide 13 minus1962 minus20351580 CYP4B1 Cytochrome P450 family 4 subfamily B polypeptide 1 minus4820 minus506566002 CYP4F12 Cytochrome P450 family 4 subfamily F polypeptide 12 minus6070 minus61958529 CYP4F2 Cytochrome P450 family 4 subfamily F polypeptide 2 minus7769 minus70604051 CYP4F3 Cytochrome P450 family 4 subfamily F polypeptide 3 minus8244 minus749111283 CYP4F8 Cytochrome P450 family 4 subfamily F polypeptide 8 minus5006 minus5270260293 CYP4X1 Cytochrome P450 family 4 subfamily X polypeptide 1 minus2476 minus25809420 CYP7B1 Cytochrome P450 family 7 subfamily B polypeptide 1 2502 31702326 FMO1 Flavin containing monooxygenase 1 4360 48592327 FMO2 Flavin containing monooxygenase 2 (nonfunctional) minus4074 minus43372328 FMO3 Flavin containing monooxygenase 3 minus4036 minus4309388714 FMO6P Flavin containing monooxygenase 6 pseudogene minus2569 minus2737493869 GPX8 Glutathione peroxidase 8 (putative) 2814 33712938 GSTA1 Glutathione S-transferase alpha 1 1623 23632939 GSTA2 Glutathione S-transferase alpha 2 2038 27412941 GSTA4 Glutathione S-transferase alpha 4 1492 21882953 GSTT2 Glutathione S-transferase theta 2 4682 5170653689 GSTT2B Glutathione S-transferase theta 2B (genepseudogene) 3739 430784779 NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439 79969027 NAT8 N-acetyltransferase 8 (GCN5-related putative) minus2769 minus29207358 UGDH UDP-glucose 6-dehydrogenase 2333 301855757 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 1604 227110720 UGT2B11 UDP glucuronosyltransferase 2 family polypeptide B11 minus3586 minus37687367 UGT2B17 UDP glucuronosyltransferase 2 family polypeptide B17 minus2836 minus295954490 UGT2B28 UDP glucuronosyltransferase 2 family polypeptide B28 minus3132 minus3284167127 UGT3A2 UDP glycosyltransferase 3 family polypeptide A2 minus4716 minus4907

18 Sarcoma

Table 4 FAF1 mutations in various cancers FAF1 mutations listed in the TCGA data base were identified without restriction to any type ofcancer For the location of the affected domains on the protein compare Figure 2

AA Mutation Cancer DomainN4S Missense Cutaneous melanomaI10S Missense Stomach adenocarcinoma UBAE21lowast Nonsense Cutaneous melanoma UBAE25K Missense Uterine endometrioid carcinoma UBAV38 splice Splice Stomach adenocarcinoma UBAP86fs FS del Colorectal adenocarcinomaG123 splice Splice Uterine endometrioid carcinoma UB1P136H Missense Colorectal adenocarcinoma UB1T147M Missense Brain lower grade glioma UB1D149Y Missense Uterine endometrioid carcinoma UB1L159V Missense Lung adenocarcinoma UB1K163N Missense Uterine endometrioid carcinoma UB1L170F Missense Cutaneous melanomaG184 splice Splice Colorectal cancerQ187H Missense Stomach adenocarcinomaS214N Missense Colorectal adenocarcinoma UB2R222I Missense Uterine endometrioid carcinoma UB2E238D Missense Lung adenocarcinoma UB2P241S Missense Uterine endometrioid carcinoma UB2T245A Missense Renal clear cell carcinoma UB2M249V Missense Uterine endometrioid carcinoma UB2E280K Missense Brain lower grade gliomaG293lowast Nonsense Colorectal cancerT300I Missense Colorectal adenocarcinomaD305H Missense Lung adenocarcinomaE308Q Missense Lung adenocarcinomaA316V Missense Stomach adenocarcinomaK319fs FS ins Head and neck squamous cell carcinomaR344G Missense Stomach adenocarcinoma UASF353I Missense Cutaneous melanoma UASL379V Missense Breast invasive carcinoma UASC396F Missense Cutaneous melanoma UASS459lowast Nonsense Uterine endometrioid carcinoma UASG469 splice Splice Glioblastoma multiforme UASR509G Missense Lung squamous cell carcinomaE510fs FS del Cutaneous melanomaR516C Missense Uterine endometrioid carcinomaA534V Missense Stomach adenocarcinomaF537L Missense Stomach adenocarcinomaE551lowast Nonsense Lung adenocarcinomaR554W Missense Colorectal cancerS582I Missense Lung adenocarcinoma UBXF585L Missense Uterine endometrioid carcinoma UBXE587lowast Nonsense Lung adenocarcinoma UBXA592V Missense Stomach adenocarcinoma UBXW610lowast Nonsense Breast invasive carcinoma UBXD611Y Missense Lung adenocarcinoma UBXE635fs FS del Brain lower grade glioma UBXP640fs FS del Cutaneous melanoma UBX

Sarcoma 19

a more accurate reference point for a leiomyosarcoma wouldhave been smooth muscle we believe that the comparison tostriated muscle is sufficient to allow the assessment of tumorspecific changes We have measured RNA DNA and proteinwith various assays Similar assessments in the future shouldalso include chromosome analysis for possible translocations

In the first-line defense against cancer the gradualreplacement of conventional chemotherapy with molecularlytargeted agents has opened the possibility to tailor drug treat-ments to particular tumors This transition necessitates thecharacterization of the molecular lesions that are causativefor the transformation of healthy cells into cancerous cellsbecause drugs need to be matched with the underlying carci-nogenic defect to be effective An additional caveat causedby unique genetic changes in the primary tumor can affectdrug transport and metabolism and needs to be taken intoconsideration In this case the RNA levels for genes that regu-late transport andmetabolismwere extensively skewed in thetumor tissue as compared to muscle and bone (see Table 3)implying potential challenges to chemotherapy An advancedmolecular treatment strategy for cancer will rely on themolecular definition of drug target drug transport and drugmetabolism pharmacogenetics in the primary tumor Con-secutive to cancer dissemination it will also require adjust-ments to account for the genetic changes in the metastases

The cost of health care has been under increasing scrutinyThe treatment of cancer patients is expensive in particularwhen hospitalization is required and further in cases ofend-of-life care Avoidable expenditures are generated bysuboptimal treatment decisions that result in low efficacy orhigh toxicity of anticancer regimens Molecular medicine hasthe potential to preempt those problems and reduce wastefulspending Yet it requires the upfront cost of molecular cancerexamination The analysis performed in this study requiredabout $11000- in nonsalary expenses to perform While ithas not identified a drug target it has specified possibleconfines for drug treatment The costs for molecular analysisneed to be weighed against the societal cost derived fromlost productivity in the workforce disrupted lives of familiesand premature deaths While currently limited drug optionsconstitute the major constraint to the approach taken herethe foreseeable future will bring an increasing spectrum ofmolecularly targeted drugs along with faster and cheapertechnologies for the molecular assessment of cancers Theywill facilitate the clinical translation of our approach

Consent

The patient provided informed consent

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by DOD Grant PR094070under University of Cincinnati IRB protocol 04-01-29-01The

author is grateful to Dr Rhett Kovall for helping with thestructural analysis of FAF1

References

[1] G F Weber Molecular Mechanisms of Cancer Springer Dor-drecht The Netherlands 2007

[2] N G Sanerkin ldquoPrimary leiomyosarcoma of the bone and itscomparison with fibrosarcomardquoCancer vol 44 no 4 pp 1375ndash1387 1979

[3] J M Weaver and J A Abraham ldquoLeiomyosarcoma of the Boneand Soft Tissue A Reviewrdquo httpsarcomahelporglearningcenterleiomyosarcomahtml

[4] M A Depristo E Banks R Poplin et al ldquoA framework forvariation discovery and genotyping using next-generationDNAsequencing datardquo Nature Genetics vol 43 no 5 pp 491ndash5012011

[5] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[6] C W Menges D A Altomare and J R Testa ldquoFAS-associatedfactor 1 (FAF1) diverse functions and implications for oncoge-nesisrdquo Cell Cycle vol 8 no 16 pp 2528ndash2534 2009

[7] M Kim J H Lee S Y Lee E Kim and J Chung ldquoCaspar asuppressor of antibacterial immunity in Drosophilardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 44 pp 16358ndash16363 2006

[8] J Song K P Joon J-J Lee et al ldquoStructure and interaction ofubiquitin-associated domain of human Fas-associated factor 1rdquoProtein Science vol 18 no 11 pp 2265ndash2276 2009

[9] P H OrsquoFarrell ldquoHigh resolution two dimensional electrophore-sis of proteinsrdquo Journal of Biological Chemistry vol 250 no 10pp 4007ndash4021 1975

[10] A Burgess-Cassler J J Johansen D A Santek J R Ideand N C Kendrick ldquoComputerized quantitative analysis ofCoomassie-Blue-stained serum proteins separated by two-dimensional electrophoresisrdquo Clinical Chemistry vol 35 no 12pp 2297ndash2304 1989

[11] B R Oakley D R Kirsch and N RMorris ldquoA simplified ultra-sensitive silver stain for detecting proteins in polyacrylamidegelsrdquo Analytical Biochemistry vol 105 no 2 pp 361ndash363 1980

[12] K Chu X Niu and L T Williams ldquoA Fas-associated proteinfactor FAF1 potentiates Fas-mediated apoptosisrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 25 pp 11894ndash11898 1995

[13] B Rikhof S de Jong A J H Suurmeijer C Meijer and W TA van der Graaf ldquoThe insulin-like growth factor system andsarcomasrdquoThe Journal of Pathology vol 217 no 4 pp 469ndash4822009

[14] RGirling J F Partridge L R Bandara et al ldquoAnew componentof the transcription factor DRTF1E2Frdquo Nature vol 362 no6415 pp 83ndash87 1993

[15] F Mercurio and M Karin ldquoTranscription factors AP-3 andAP-2 interact with the SV40 enhancer in a mutually exclusivemannerrdquo EMBO Journal vol 8 no 5 pp 1455ndash1460 1989

[16] R Bassel-Duby M D Hernandez M A Gonzalez J KKrueger and R S Williams ldquoA 40-kilodalton protein bindsspecifically to an upstream sequence element essential for mus-cle-specific transcription of the human myoglobin promoterrdquoMolecular and Cellular Biology vol 12 no 11 pp 5024ndash50321992

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 3: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

Sarcoma 3

(a)

(b) (c)

Figure 1 Osteosarcoma (a)Whole body scan shows abnormally intense uptake of the radionuclide (Tc) within the middiaphysis of the rightfemur No increased radionuclide uptake is seen anywhere else in the bony skeleton (b) MRI scan displays an extensively abnormal signal inthe diaphysis of the right femur It is surrounded by soft tissue involvement (c) Bone lesion displayed postoperatively

27 Polymorphism Analysis We obtained 22 formalin-fixedparaffin-embedded leiomyosarcoma specimens (stroma andparaffin had been removed from unstained slides undermicroscopic examination) through the Department ofPathology University of Cincinnati DNA was extracted withthe AllPrep DNARNA FFPE kit (Qiagen) We purchased7 frozen leiomyosarcoma tissues from Creative Bioarrayand extracted DNA with the AllPrep DNARNA Mini Kit(Qiagen) One blood sample from a leiomyosarcoma patientwas received from the University of Cincinnati TissueBank Polymorphisms in FAF1 were analyzed in the DNAusing a custom TaqMan assay with the probe ACACCA-GATTTGCCACCACCTTCATCATCT [AG] GTCAT-GCTGGGTAAGTTGTTTATATTTCCTG A TaqMan assaywith an existing probe for position minus443 in the osteopontinpromoter served as a reference assay 34 breast cancerDNA samples served as nonsarcoma control The assay wasperformed by the CCHMC DNA core

3 Results

31 Patient History In 1998 the patient was diagnosedwith high grade stage IIb osteogenic sarcoma of the right

femur which was extracompartmental Upon resection thelesion had a size of 95 times 3 times 4 cm (Figure 1) The tumorwas assessed as stage T2NxMx grade III characterized ashypercellular it showed marked cytologic atypia and a highmitotic rate Histologic features included anaplasia pleomor-phism numerous abnormal mitoses numerous giant cellsand osteoid production with focal calcifications

In 2012 a CT scan done because of hip pain revealed a43 times 34 times 31 cm lytic mass in the superior right acetabulumgrossly stable in size and configuration There was diffuseosteopenia involving the right femoral head and neck withdiffuse atrophy of the right pelvic girdle musculature Perios-titis and cortical interruptionwere associatedwith this lesion

The postsurgical pathology report identified the 57 times55 times 49 cm mass as a high grade leiomyosarcoma stagepT2bpNx Whereas a bone scan revealed intense activityin the left seventh rib a follow-up chest CT providedno indication of pulmonary masses mediastinal or hilarlymphadenopathy enlargement of axillary nodes or pleuraleffusion implying stage M0 The mitotic rate was 70 with60 necrosis The tumor caused extensive bone destructionand involvement of adjacent tissue Histologically the tumorcells stained positively for smooth muscle actin They were

4 Sarcoma

also positive for CD68 and displayed diffuse positive stainingfor vimentin but were negative for CD117 pancreatin S-100and CD34

Seven months after the surgery the patient received aPET-MRI scan for pain which revealed sixmetastatic lesionsincluding both lungs and multiple ribs She was put on three21-day cycles of Gemzar (days 1 and 8) Taxotere (day 8) andNeulasta (beginning on day 9) but was unable to continuepast the first cycle due to hospitalizations for continued andproblematic wound infections at the surgical lung biopsysites

32 DNA Exome Sequence Exome sequencing of thegenomic DNAs for tumor muscle and bone identified65546 potential sequence variants Filtering yielded 46 likelysomatic mutations in the tumor (Table 1) of which 7 (affect-ing EIF4A1 EPHA3 FAF1 IPO8 KIAA1377 LIMCH1 andNIPBL) were confirmed in the RNASeq results FAF1 asso-ciates with FAS and enhances apoptosis mediated throughthis receptor [12] The point mutation S181G (Figure 2) couldcause a loss of function in FAF1 and lead to transformationvia antiapoptosis

33 RNA Analysis Expectedly the gene expression patternsaccording to RNASeq were very different among tumormuscle and bone The cancer contained several gene prod-ucts that were overexpressed compared to both muscle andbone (Tables 2(a) and 2(b)) Among the top 30 changesin the tumormuscle and tumorbone comparisons 13 wereidentical (Table 2(c)) It is implied that these gene productsare quite unique for the tumor and likely contribute to itspathogenesis This notion is supported by the upregulationof the smooth muscle gene Ano4 which corroborates theleiomyosarcomatous nature of the cancer When limiting theanalysis to genes expressed at least at the level of 1 unit the17 genes overexpressed in the tumormuscle and tumorbonecomparisons contain several extracellular matrix proteinsimplying an active remodeling of the tumor microenviron-ment (Table 2(d)) By contrast none of the underexpressedgene products in the tumormuscle comparison matched thetumorbone comparison (not shown)

Of note the RNA level of IGF2BP1 (IMP-1 CRD-BPand ZBP-1) is highly upregulated in the tumor compared tomuscle as well as bone IGF2BP1 is a RNA-binding factorthat affects mRNA nuclear export localization stability andtranslation It regulates mRNA stability during the inte-grated cellular stress response in stress granules IGF2BP1 isa transcriptional target of the WNT pathway which is nega-tively regulated by intact FAF1 and may be unregulated byFAF1S181GThe IGF systemhas been linked to sarcoma patho-genesis [13] and may play a role in this specific cancer OtherIGF family members with increased RNA message levels inthis tumor (compared to muscle and bone) include IGFBPL1(5-6-log

2-fold) IGFL3 (6-7-log

2-fold) and IGF2BP3 (2-6-

log2-fold)The identified point mutation in FAF1 may be patho-

genetic for this cancer FAF1 is a regulator of NF-120581B activa-tion It directly binds to RelA (P65) retaining it in the cyto-plasm It can also interact with IKK120573 thus allowing for the

I120581B-mediated degradation of the transcription factors P65and P50 [6] Consistently the expression of regulators of theNF-120581B activation pathway is skewed in the tumor comparedto muscle or bone (Table 2(e))

34 Transcription Factor Binding ProteinDNA arrays mea-sure the binding activity of transcription factors Theycomprise three basic steps A set of biotin-labeled DNAbinding oligonucleotides are preincubated with a nuclearextract of interestThe proteinDNA complexes are separatedfrom the free probes The probes in the complexes arethen extracted and hybridized to prespotted membranesfollowed by HRP-based chemiluminescence detection Wemade nuclear extracts from tumor bone and muscle andtested them for DNA binding activity Binding that wasinduced in cancer but not in the normal tissues was dis-played by the transcription factors E2F1 AP3 LIII-BP PAX6ADD-1 and CCAC [14ndash19] The CCAC binding activity isconsistent with a muscle-derived tumor E2F1 may associatewith the WNT pathway-induced transcription factor LEF1resulting in transcriptional derepression of E2F1 [20] Likelyconstitutive transcription factors that are active in all 3 tissuescomprise AhRAmt GATA1 GATA2 GATA12 HIF1 andHOXD8910 (Figure 3)

35 Protein Analysis 2D gel electrophoresis of the RIPAlysates from tumor muscle and bone showed very divergentpatterns (Figure 4(a)) Two experienced analysts comparedthe protein pattern from the tumor with the protein patternfrom either bone or muscle Polypeptide spots that wereunique to the gels from the tumor were outlined (spotsunique to or relatively darker in the bone or muscle werenot indicated)The labeled proteins were extracted for identi-fication with mass spectrometry This yielded several struc-tural proteins which may reflect modification of the cellulararchitecture under rapid growth Transgelin-1 and transgelin-2 were abundant and corroborated the identity of thetumor as a leiomyosarcoma Four calcium-binding proteinswere highly expressed in the cancer In addition regulatorsof protein synthesis (40S ribosomal protein S12 glycine-tRNA ligase) protein modification (N-terminal fragmentof heat shock protein HSP 90120572 C-terminal fragment ofprotein disulfide isomerase) and protein degradation (1205721-antitrypsin proteasome activator complex subunit 2) wereidentified (Figure 4(b)) Of interest may be the C-terminalfragment of protein disulfide isomerase which not onlyhydroxylates prolines in preprocollagen but also contributesto microsomal triglyceride transfer It could be reflectiveof a skewed tumor metabolism The protein analysis wascorroborated by the mRNA levels (Figure 4(d))

Cancer markers were tested according to Western blot(Figure 4(c)) The tumor but not normal muscle expressedthe metastasis protein osteopontin and a single small form(lt75 kD) of CD44 that is likely the not alternatively splicedstandard form Unexpectedly while both tumor and muscleexpressed comparably abundant amounts of STAT3 phos-phorylation (reflective of activation) was present in themuscle but not in the tumorThe STAT3 pathway is associatedwith progression in several human cancers and this is often

Sarcoma 5

Table1DNAexom

eSNPsTh

eDNAexom

esfortum

orm

uscle

and

bone

weres

equencedTh

eresultswerefi

lteredin

thefollowingorder(1)d

ifferentgenotypeintumor

from

muscle

and

bonew

ithmuscle

andbo

nebeingidentic

alto

each

other(2)d

eletem

utations

with

lowconfi

dence(

valueo

f20or

lower)inall3

tissues(3)

deleteun

identifi

edgenes(4)d

eletem

utations

thatareh

omozygou

sreference

inthetum

or(5)

deletemutations

thathave

aMAFin

dbSN

Pgt10(6)

deletelowim

pactandmod

ifier

mutationsTh

egenen

ames

arep

arto

fthe

keyin

the

leftcolumnIn

thiscolumnresults

onbo

ldfont

representSNPs

thatwerec

onfirmed

ontheR

NAlevelbyRN

ASeqTh

edatafi

lesh

aveb

eensubm

itted

totheN

CBIsho

rtread

archive(SR

A)

underthe

accessionnu

mberS

RP052797

(biosamples

SAMN03316820SAMN03316821SAMN03316822)

Key

Chromosom

ePo

sition

Reference

Alternate

dbSN

PID

dbSN

PMAF

ESPMAF

(All)

ESPMAF

(EA)

ESPMAF

(AA)

Con

sensus

impact

Bone

muscle

geno

type

Bone

overall

depth

Bone

allele

depths

Bone

quality

Muscle

overall

depth

Muscle

allele

depths

Muscle

quality

Tumor

geno

type

Tumor

overall

depth

Tumor

allele

depths

Tumor

quality

177479

998T

EIF4

A1

177479998

CT

mdash

mdashmdash

mdashMod

erate

00

175

1840

99103

1080

9901

133

7968

99389

2592

00T

EPHA3

389259200

CT

mdash

mdashmdash

mdashMod

erate

00

43450

99117

1230

9901

723048

9915120

4545

CFA

F11

51204545

TC

mdash

mdashmdash

mdashMod

erate

00

87910

99108

1130

9901

8566

27

9912

3083

4620

AIPO8

1230834620

CA

mdash

mdashmdash

mdashMod

erate

00

68712

99120

1260

9901

807018

9911

101834

425A

KIA

A1377

11101834425

GA

mdash

mdashmdash

mdashMod

erate

00

36371

9067

700

9901

863163

9944168

2102

CLIMCH

14

41682102

GC

mdash

000

0077

0000

0227

Mod

erate

00

71740

9996

1010

9901

153

12049

99536

985326

GNIPBL

536985326

AG

mdash

mdashmdash

mdashMod

erate

00

660

1835

360

8101

201012

99377623789

ARO

BO2

377623789

AGA

mdash

mdashmdash

mdashHigh

00

22NA

5429

NA

8701

37NA

99

2217300

095A

SMARC

AL1

2217300

095

ATTG

CATC

A-AC

GTC

GTG

GA

mdash

mdashmdash

mdashHigh

00

95NA

99122

NA

9901

99NA

99

2152236045TTA

ATN

FAIP6

2152236045

TATTA

Ars3506

0021

mdashmdash

mdashmdash

High

02

19NA

124

NA

5701

17NA

383520206

69TAC

Y13

520206

69G

T

mdashmdash

mdashmdash

Mod

erate

00

130

1360

9986

900

9901

804543

999117

130734

GAKN

A9

117130734

CG

mdash

mdashmdash

mdashMod

erate

00

27280

7830

310

9001

503024

9912

6030354A

ANO2

126030354

CA

mdash

mdashmdash

mdashMod

erate

00

101

1060

99114

1200

9901

135

10445

9918

10487667

AAPC

DD1

1810487667

GA

mdash

mdashmdash

mdashMod

erate

00

43450

9938

400

9901

503816

99734118

560C

BMPE

R7

34118

560

GC

mdash

mdashmdash

mdashMod

erate

00

69720

99101

1060

9901

716314

9915

24921273

TC1

5orf2

1524921273

GT

mdash

mdashmdash

mdashMod

erate

00

12120

3638

390

9901

26226

9919

54483249

CCA

CNG8

1954483249

TC

mdash

mdashmdash

mdashMod

erate

00

147

1540

99103

1080

9901

152

13632

9910

16893265

CCU

BN10

16893265

GC

mdash

mdashmdash

mdashMod

erate

00

57600

9990

940

9901

403410

997148489854C

CUL1

7148489854

AC

mdash

mdashmdash

mdashMod

erate

00

73760

9965

680

9901

574221

9917

41566894

CDHX8

1741566894

GC

mdash

mdashmdash

mdashMod

erate

00

106

1110

9988

920

9901

124

9442

9920

61512358

TDID

O1

2061512358

GT

rs73304513

00417

0045932

000

0838

0135456

Mod

erate

00

600

182

00

601

400

2116

23703526

AER

N2

1623703526

GA

mdash

mdashmdash

mdashMod

erate

00

81850

9998

1030

9901

916041

993197880164G

FAM157A

3197880164

GCA

GCA

GCA

AG

mdash

mdashmdash

mdashMod

erate

00

21NA

2525

NA

101

31NA

614

4564

4287

GFA

NCM

144564

4287

AG

000

09mdash

mdashmdash

Mod

erate

00

13130

3331

320

8701

12102

14189637524

CGBP

71

89637524

GC

mdash

mdashmdash

mdashMod

erate

00

171

1790

99201

2110

9901

206

1616

799

742003933

CGLI3

742003933

GC

mdash

mdashmdash

mdashMod

erate

00

87910

9981

850

9901

856132

9917

4837170TGP1BA

174837170

CT

mdash

mdashmdash

mdashMod

erate

00

16160

459

9015

01

11101

114

24635161

GIRF9

1424635161

TG

mdash

mdashmdash

mdashMod

erate

00

66690

9943

450

9901

362513

9915

42133096

AJM

JD7-

PLA2G

4B15

42133096

GA

mdash

mdashmdash

mdashMod

erate

00

111

1160

9975

780

9901

807414

92

1740271678

AKA

T2A

1740271678

GA

mdash

mdashmdash

mdashMod

erate

00

65680

9958

610

9901

563032

99873848894

GKC

NB2

873848894

AG

mdash

mdashmdash

mdashMod

erate

00

15150

3324

250

6601

301517

9919

50827053

TKC

NC3

1950827053

CT

mdash

mdashmdash

mdashMod

erate

00

220

2310

99136

1430

9901

160

13046

9917

21319069

AKC

NJ12

1721319069

GA

rs76265595

mdashmdash

mdashmdash

Mod

erate

00

23241

6343

434

4001

28255

6012

53011932

CKR

T73

1253011932

TC

mdash

mdashmdash

mdashMod

erate

00

146

1530

99157

1650

9901

159

11758

99X

7500

4584

AMAG

EE2

X7500

4584

CA

mdash

mdashmdash

mdashMod

erate

00

29300

8148

500

9901

382616

995140182157A

PCDHA3

5140182157

GA

mdash

mdashmdash

mdashMod

erate

00

180

1890

99153

1610

9901

150

13234

9915

42133096

APL

A2G

4B15

42133096

GA

mdash

mdashmdash

mdashMod

erate

00

111116

099

75780

9901

807414

9210

96005840

CPL

CE1

1096005840

TC

mdash

mdashmdash

mdashMod

erate

00

81851

9973

762

9901

875540

991166816805G

POGK

1166816805

CG

mdash

mdashmdash

mdashMod

erate

00

84880

9982

860

9901

634920

99

6 Sarcoma

Table1Con

tinued

Key

Chromosom

ePo

sition

Reference

Alternate

dbSN

PID

dbSN

PMAF

ESPMAF

(All)

ESPMAF

(EA)

ESPMAF

(AA)

Con

sensus

impact

Bone

muscle

geno

type

Bone

overall

depth

Bone

allele

depths

Bone

quality

Muscle

overall

depth

Muscle

allele

depths

Muscle

quality

Tumor

geno

type

Tumor

overall

depth

Tumor

allele

depths

Tumor

quality

12111

020739

TPP

TC7

12111

020739

TCGC

Trs71083132

mdashmdash

mdashmdash

Mod

erate

00

18NA

3014

NA

1801

19NA

1019

804293

APT

BP1

19804293

CA

mdash

mdashmdash

mdashMod

erate

00

120

1261

9971

741

9901

755330

9971564

51175C

RNF32

71564

51175

GC

mdash

mdashmdash

mdashMod

erate

00

55570

9967

700

9901

594223

993520206

69TRP

11-155D

1811

3520206

69G

T

mdashmdash

mdashmdash

Mod

erate

00

130

1360

9986

900

9901

804543

9921

43838624

TUBA

SH3A

2143838624

GT

mdash

mdashmdash

mdashMod

erate

00

70730

9945

470

9901

897621

9919

58773857

AZN

F544

1958773857

GA

mdash

mdashmdash

mdashMod

erate

00

46480

9943

450

9901

715126

99516465723

CZN

F622

516465723

TC

mdash

mdashmdash

mdashMod

erate

00

17170

3914

140

3301

271415

99

Sarcoma 7

Table 2 RNA induced in the tumor (a) Transcripts overexpressed in the tumor compared to muscle but not bone (b) Transcriptsoverexpressed in the tumor compared to bone but not muscle (c) Transcripts most abundantly overexpressed in the tumor compared tomuscle and bone (d) as (a) but limited to genes expressed at least at the level of 1 unit in the reference tissue (muscle or bone) (e) Alteredexpression of genes associated with NF-120581B activity Analysis of RNASeq for the transcription factor NF-120581B and gene products that regulate itsactivity RNAmessages that are selectively associated with the TNF pathway to NF-120581B activation are marked with bold font log2-fold changeindicates the alteration in the tumor compared to muscle or bone (the respective columns indicate the expression level for each organ)

(a)

log2-fold changeMuscle

Tumor over muscleNRG1 Neuregulin 1 9909KSR2 Kinase suppressor of ras 2 9675MMP13 Matrix metallopeptidase 13 (collagenase 3) 9528SPP1 Secreted phosphoprotein 1 9098INHBA Inhibin beta A 8570COL11A1 Collagen type XI alpha 1 8564MMP9 Matrix metallopeptidase 9 (gelatinase B 92 kda gelatinase 92 kda type IV collagenase) 8552FBN2 Fibrillin 2 8495LRRC15 Leucine rich repeat containing 15 8429MMP11 Matrix metallopeptidase 11 (stromelysin 3) 8336E2F7 E2F transcription factor 7 8314PRAME Preferentially expressed antigen in melanoma 8179PTK7 PTK7 protein tyrosine kinase 7 7996DSCAM Down syndrome cell adhesion molecule 7761RGS4 Regulator of G-protein signaling 4 7633CNIH3 Cornichon homolog 3 (Drosophila) 7632OR10V1 Olfactory receptor family 10 subfamily V member 1 7610CEP55 Centrosomal protein 55 kda 7583WNT5B Wingless-type MMTV integration site family member 5B 7569CA12 Carbonic anhydrase XII 7520

GALNT5 UDP-N-acetyl-alpha-D-galactosaminepolypeptide N-acetylgalactosaminyltransferase 5(GalNAc-T5) 7517

(b)

log2-fold changeBone

Tumor over boneROS1 c-ros oncogene 1 receptor tyrosine kinase 10987GREM1 Gremlin 1 10604NPTX1 Neuronal pentraxin I 8813GJB2 Gap junction protein beta 2 26 kDa 8597CREB3L1 cAMP responsive element binding protein 3-like 1 8506KRT14 Keratin 14 8351SERPINE1 Serpin peptidase inhibitor clade E (nexin plasminogen activator inhibitor type 1) member 1 8288HOXD10 Homeobox D10 8105ALPK2 Alpha-Kinase 2 7783POU3F2 POU class 3 homeobox 2 7530POSTN Periostin osteoblast specific factor 7497NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439STC2 Stanniocalcin 2 7434WNT5A Wingless-type MMTV integration site family member 5A 7412IGFN1 Immunoglobulin-like and fibronectin type III domain containing 1 7387

8 Sarcoma

(b) Continued

log2-fold changeBone

STC1 Stanniocalcin 1 7385FAM180A Family with sequence similarity 180 member A 7315KRT17 Keratin 17 7305BBOX1 Butyrobetaine (gamma) 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 7277MAGEA1 Melanoma antigen family A 1 (directs expression of antigen MZ2-E) 7267

(c)

log2-fold changeMuscle Bone

Tumor over bonemuscle

ANO4 Anoctamin 4 (TMEM16D) transmembrane calcium-activated chloride channelfacilitates smooth muscle contraction 9937 8542

SLCO1B3 (OATP1B3) Solute carrier organic anion transporter family member 1B3 9569 9760

MARCH4 Membrane-associated ring finger (C3HC4) 4 E3 ubiquitin ligase located predominantlyto the endoplasmic reticulum 9538 7406

IGF2BP1 Insulin-like growth factor 2 mRNA binding protein 1 binds to and stabilizes mRNA 9440 9631ADAMTS16 ADAMmetallopeptidase with thrombospondin type 1 motif 16 zinc-dependent protease 9260 8450SOX11 SRY (sex determining region Y)-box 11 important in brain development 9178 9954

HAPLN1 Hyaluronan and proteoglycan link protein 1 stabilizes aggregates of aggrecan andhyaluronan giving cartilage its tensile strength and elasticity 8951 8142

MUC15 Mucin 15 cell surface associated 8801 7992HOXB9 Homeobox B9 8773 7378MAGEC2 Melanoma antigen family C 2 8693 8884

ST6GALNAC5 ST6 (alpha-N-acetyl-neuraminyl-23-beta-galactosyl-13)-N-acetylgalactosaminidealpha-26-sialyltransferase 5 7848 8038

C11orf41 Chromosome 11 open reading frame 41 7781 8141MMP1 Matrix metallopeptidase 1 (interstitial collagenase) 7455 7646

(d)

Symbol Name log2-fold change log2-fold changeMuscle Bone

Tumor over bonemuscleFN1 Fibronectin 1 7328 6293 6457 19860COL1A1 Collagen type I alpha 1 6768 3706 5868 11934CCND1 Cyclin D1 5595 3958 5098 9637RGS1 Regulator of G-protein signaling 1 5118 1056 4653 2533ITGBL1 Integrin beta-like 1 (with EGF-like repeat domains) 5071 3177 3895 12415COL1A2 Collagen type I alpha 2 4852 8756 4910 14503MXRA5 Matrix-remodelling associated 5 4834 1352 4631 2685POSTN Periostin osteoblast specific factor 4513 5870 7497 1267PLOD2 Procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 4285 1801 4023 3730COL5A2 Collagen type V alpha 2 4275 1297 4833 1517COL3A1 Collagen type III alpha 1 4003 13118 5801 6500

SEMA3C Sema domain immunoglobulin domain (Ig) shortbasic domain secreted 3954 1164 4215 1675

FBN1 Fibrillin 1 3912 6957 4018 11148AEBP1 AE binding protein 1 3808 3212 4002 4843SIK1 Salt-inducible kinase 1 3805 1219 3566 2484

Sarcoma 9

(d) Continued

Symbol Name log2-fold change log2-fold changeMuscle Bone

SERPINH1 Serpin peptidase inhibitor clade H (heat shock protein47) member 1 3706 1652 4145 2098

ANTXR1 Anthrax toxin receptor 1 3670 3789 3690 6445

(e)

Symbol Name log2-fold change log2-fold changeMuscle Bone

HELLS Helicase lymphoid-specific 5315602 0155898 minus082713 202489

TNFRSF11A Tumor necrosis factor receptor superfamily member 11a andNFKB activator 3017922 003091 1400993 0202453

NFKBID Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor delta 2655352 0 minus147615 0504341

TNFRSF25 Tumor necrosis factor receptor superfamily member 25 2432959 0046388 0163954 0490795

NFKBIE Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor epsilon 2121015 0062395 0311441 043382

TNF Tumor necrosis factor 1847997 0 minus142101 003733

TNFRSF10D Tumor necrosis factor receptor superfamily member 10d decoywith truncated death domain 1754888 0172968 minus01757 1183343

TNFRSF1B Tumor necrosis factor receptor superfamily member 1B 1473438 0709902 minus097011 6729401TNFRSF10A Tumor necrosis factor receptor superfamily member 10a 1411898 0828219 0357701 3004386

NFKBIB Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor beta 1193993 1209816 046055 3484692

TNFRSF10B Tumor necrosis factor receptor superfamily member 10b 1094157 1908597 0425446 5242006TNFRSF21 Tumor necrosis factor receptor superfamily member 21 1055762 3882038 0745815 8305711TNFRSF1A Tumor necrosis factor receptor superfamily member 1A 0875167 3790938 minus011707 130344

NFKBIZ Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor zeta 0575974 3698951 0344657 7493136

RIPK1 Receptor (TNFRSF)-interacting serine-threonine kinase 1 0494467 311749 minus108854 161392NKRF NFKB repressing factor 0475722 2156087 minus086397 9434166NFKB1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 0432959 2054532 minus065865 7568586CHUK Conserved helix-loop-helix ubiquitous kinase 0176126 2961281 minus120204 1330333RELA V-rel reticuloendotheliosis viral oncogene homolog A (avian) 0013848 1263837 0287167 1803044

NFKB2 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 2(p49p100) minus008641 0312993 0485882 0360442

NKAPP1 NFKB activating protein pseudogene 1 minus010692 0825177 minus099449 2655392

TNFRSF10C Tumor necrosis factor receptor superfamily member 10c decoywithout an intracellular domain minus0152 0064676 minus556891 6321515

NKIRAS2 NFKB inhibitor interacting Ras-like 2 minus060768 1095135 minus088758 2299946

NFKBIL1 Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor-like 1 minus08719 0141933 minus008357 0140418

NKIRAS1 NFKB inhibitor interacting Ras-like 1 minus087428 6296399 1467735 2122983TANK TRAF family member-associated NFKB activator minus0983 6777124 minus151908 1695872NKAP NFKB activating protein minus135735 1422458 minus078243 1646287

NFKBIA Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor alpha minus185483 4032743 minus031802 2395275

NKAPL NFKB activating protein-like minus557347 5875743 minus140215 0534643

10 Sarcoma

FAS associating region

DED interacting region

UB12 DZM

UB2domain

UASdomain

UBXdomain

UBAdomain

S289 S291

Aurora ACK II

S181

AKT1ATMGSK3

AuroraPKG

PLK1RSK

STK4CHK1

650566481336260205169110475

(a)

MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRM

6

1

101

100

200

300

400

500

600

650

201

301

401

501

601

667855899998876467765678888887578758999986467 88 8888888888888988888899988888889888877767888777888757

2222454345655744642554435764462443558468643653433344333424435334233333332333332333354335333344334354

LDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITH

9999997797689998787758999999999859996577754788888888875333467888868987688888888888877888855675799987

9585847535375857543447459666955563844544585374435443365845846434457567533353232334334444433337484944

REVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDAT

5888757887577654788887766654677776555468877888765455546887766555688888877777777777776556788888767766

5344456484742545656645594364384534437445444342323455453535435463434323333343434333333334333335433333

EFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESI

5666776654456777888888888888866889999999998848888876677778999999998776686899998589875579999987486899

4635343574354322343434533333343548479747945564424647635575478569766456468999999754233454687457844669

VSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDE

9999888589997557877545444345677876335554467888876899986799876447777677888999999998877555778887777778

6778547999996674433343444333333335466456434433487999895353333464346434365468755846444545454565446565

REARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVF

8888888887788877766545557777777666777777777776678877777766578888888888885799998589975788758987589999

4545445564445344433443354555556653665456544454434444444443344334333333446859596743354745795535796898

DFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKE

99997799988779985788754577888765665678898589998589

67974525444795877564635844444476875837446989897366

(b)

Figure 2 FAF1 structure (a) A schematic of functional domains in FAF1 with annotations (adapted from [6ndash8]) There are two ubiquitin-like domains flanking the S181 site The PDB structure 2DZM identifies the N-terminal one while the C-terminal prediction is by sequencesimilarity Whereas the mutation S181G is not expected to disrupt the protein structure per se this amino acid has a high score as a possiblephosphorylation site for a number of kinases involved inDNAdamage repair (b) Secondary structure prediction of FAF1The residues aroundS181 appear unstructured

reflected in STAT3 constitutive phosphorylation The lack ofphosphorylation in this case suggests that the leiomyosar-coma may not depend on the STAT3 pathway

36 Pharmacogenetic Evaluation Predicting the sensitivityto anticancer drugs is a main goal of molecular analysisFor this the over- or underexpression of genes for drugtransport and metabolism is of key importance Analysis ofthe RNASeq data for these groups of gene products identified

a surprisingly large number of deregulations compared tomuscle or bone (Tables 3(a) and 3(b)) Those alterationsmay affect choices for drug treatment For example the highlevels of glutathione S-transferase may render carmustinethioTEPA cisplatin chlorambucil melphalan nitrogenmus-tard phosphoramide mustard acrolein or steroids ineffec-tive The overexpression ofN-acetyltransferase may compro-mise 5-fluorouracil or taxolThemodest upregulation of onlytwo export transporters (ABC-transporters) and specifically

Sarcoma 11

Bone Tumor

Muscle

(a)

Transcription factor Description Reference

E2F1 Transcriptional activation of cell cycle progression is selectively activated in response to specific cues key downstream target of RB1 can promote proliferation or apoptosis when activated [14]

AP-33 interact in a mutually exclusive manner [15]

CCAC CCAC box is an essential element for muscle-specific transcription from the myoglobin promoter [16]

LIII-BP The L-III transcriptional regulatory element (a carbohydrate-response element) is in the pyruvate kinase L gene necessary for cell type-specific expression and transcriptional stimulation by carbohydrates [17]

ADD-1 (SREBP-1) activates transcription from sterol-regulated elements and from an E-box sequence present in the promoter of fatty acid synthase [18]

PAX6 Member of the paired box gene family transcriptional regulator involved in developmental processes [19]

48kD transcription factor activator protein-3 binds to the core element and recognizes the SV40 enhancer and AP-2 and AP-

(b)

Figure 3 Transcription factor activation Nuclear extracts from tumor muscle and bone were tested for DNA binding activity on a protein-DNA array (a) Transcription factor binding that was high in all 3 tissues and is therefore considered constitutively active is circled in yellow(left to right top to bottom AhRAmt GATA1 GATA2 GATA12 HIF1 and HOXD8910) Transcription factors that show high binding inthe cancer but not in muscle or bone are circled in red (left to right top to bottom E2F1 AP3 LIII-BP PAX6 ADD-1 and CCAC) (b) Thefunctions of transcription factors that display high DNA binding in the cancer but not in muscle or bone are described

the lack of ABCB1 overexpression is favorable for avoidingdrug resistance

37 Population Analysis The above-described results indi-cated that a FAF1 mutation which replaces serine in position181 thus preventing FAF1 phosphorylation and activationmay be a driver for leiomyosarcomagenesis A custom Taq-Man assay confirmed the presence of the somatic mutationin the patient To assess whether this single nucleotidereplacement is common in this type of cancer we analyzedDNA from 29 leiomyosarcomas and 1 blood sample from aleiomyosarcoma patient For comparison to nonsarcomatoustumors 34 breast cancers served as a reference None of themdisplayed amutation in the same locus By contrast there wasa distribution across all leiomyosarcomas in the osteopontin

promoter position minus443 (used as a reference) with 12 CC 10TC and 9 TT

4 Discussion

TheFAF1mutation identified as the likely cause for the cancerunder study gives room for an explanation of the sarcomatoustransformation (Figure 5) DNA damage to mesenchymalcells occurs persistently in an oxidizing environment at 37∘CThese insults are rarely transforming and such an occurrencewould trigger the initiation of programmed cell death inapoptosis-competent cells Intact FAF1 associates with FASand enhances apoptosis mediated through this receptor [12]A loss of function in FAF1 could lead to transformation viaantiapoptosis Whereas the mutation S181G is not expected

12 Sarcoma

Muscle

Bone

Tumor

220

94

6043

29

14

220

94

60

43

29

14

220

94

60

43

29

14

1

7

6

23

24

21

22

25

26

89 1011 12

1516 1719

pH 4 8 pH 4 8

8pH 4

(a)

SwissProt or NCBISpot number Protein identified accession Description

(Structural)6 Transgelin Q01995 Smooth muscle-specific cross-links actin24 Transgelin-2 P37802 Smooth muscle-specific cross-links actin

Vimentin P08670 Intermediate filament in mesenchymal tissue1 Filamin-A P21333 Actin-binding protein regulates the cytoskeleton 21 P06709 Cytoskeleton

(Calcium homeostasis)8 9 Calumenin O43852 Calcium-binding protein in the ER and golgi apparatus26 Protein S100-A11 P31949 Calcium-binding EF hand protein10 Reticulocalbin-3 Q96D15 Calcium-binding protein located in the ER12 P11021 Heat shock 70-kD protein 5 ER lumenal calcium-binding

(Protein processing)25 40S ribosomal protein S12 P25398 Core component of the ribosomal accuracy center7 Glycine-tRNA ligase P41250 Catalyzes the attachment of glycine to tRNA19 P01009 Protease inhibitor23 P01009 Protease inhibitor21 Proteasome activator complex subunit 2 Q9UL46 Proteasome activation10 P07900 Chaperone

(Other)7 Serum albumin P02768 Carrier protein in blood22 Protein disulfide isomerase (C-terminal fragment) P07237 Prolyl hydroxylation in collagen microsomal triglyceride transfer

120573 actin (C-terminal fragment)

78kD glucose-regulated protein

120572-1-antitrypsin120572-1-antitrypsin (C-terminal fragment)

Heat shock protein HSP 90120572 (N-terminal fragment)

11 15ndash17

(b)

Figure 4 Continued

Sarcoma 13

Tumor Muscle

OPN

CD44

STAT3

P-STAT3

Tubulin

75

75

50

15010075

10075

50

(c)

Tumor bone Tumor muscleGene ID Symbol Name Fold change Expression Fold change Expression6876 TAGLN Transgelin 3274 2455 3087 15088407 TAGLN2 Transgelin-2 2164 7338 6975 13037431 VIM Vimentin 1653 295010 4061 696042316 FLNA Filamin A alpha 1304 7791 9005 065160 ACTB Actin beta 0656 973187 5491 67368813 CALU Calumenin 7601 19328 12063 70596282 S100A11 S100 calcium binding protein A11 0931 158914 5071 1686357333 RCN3 Reticulocalbin 3 EF-hand calcium binding domain 2439 0604 6412 01223309 HSPA5 1068 92754 2607 220356696 SPP1 Secreted phosphoprotein 1 7572 46508 547929 0355960 CD44 CD44 molecule (Indian blood group) 2053 26708 15436 20566774 STAT3 Signal transducer and activator of transcription 3 0617 46534 0832 200165925 RB1 Retinoblastoma 1 0736 14772 0442 14268

Heat shock 70kDa protein 5 (glucose-regulated protein 78kDa)

(d)

Figure 4 Protein overexpression (a) 2D protein gel electrophoresis of lysates from muscle (upper left) tumor (upper right) and bone(bottom) in RIPA buffer Red circles indicate the spots that were identified as overexpressed and were further analyzed by mass spectrometry(b) Proteins identified in 2D gel electrophoresis as overexpressed in the tumor in comparison to muscle and bone were analyzed for theiridentity by mass spectrometry The left column indicates the spot number corresponding to the 2D gel The next column contains theprotein name followed by the accession number and a description of the protein functionThe protein functions are grouped into structuralcalcium homeostasis and various others (c) Western blot 10 120583g lysates of tumor muscle and bone in RIPA buffer were loaded per laneand electrophoresed on 10 SDS-polyacrylamide minigels with reducing denaturing sample buffer After transfer to PVDF membranesthey were probed for markers of cancer progression including osteopontin CD44 STAT3 and phospho-STAT3 Antitubulin served as aloading control (d) RNA levels corresponding to the proteins found to be affected in the sarcoma With four exceptions in the tumor-bonecomparison (gray font) upregulated proteins are associated with increased RNA levels STAT3 is not increased on the protein or RNA levelThe reduced level of RB1 expression is consistent with the elevated DNA binding activity of E2F1

to disrupt the structure of the protein this site does scorehigh as a possible phosphorylation site for a number ofkinases involved in DNA damage repair supporting thehypothesis that the cancer cells containing thismutation havelost their ability to respond to transforming DNA damagewith programmed cell death FAF1 antagonizes WNT signal-ing by promoting 120573-catenin degradation in the proteasome[21] a function that may be lost after the point mutationThe elevated DNA binding activity of the protooncogenictranscription factor E2F1 (see Figure 3) could be caused byits interaction with LEF-1 [20] consecutive to persistent

WNT signaling The RNA level of IGF2BP1 (IMP-1) a stress-responsive regulator of mRNA stability is highly upregulatedin the tumor compared to muscle as well as bone (seeTable 2(c)) IGF2BP1 is a transcriptional target of the WNTpathway that regulates NF-120581B activity (see Table 2(e)) andis antiapoptotic [22 23] IGF2BP1 may be upregulated asa consequence of mutated FAF1 not being able to suppressWNT signaling in this specific cancerOf noteWNTpathwayoveractivity may not be required for transformation ratherthe persistence of a WNT pathway signal due to the lack of aFAF1-mediated termination signal may suffice

14 Sarcoma

WNTFrizzled

Axin Dvl

igf2bp1

FAF1

P65 P50

IKK

FAS

120573-Catenin

LEF + E2F1

I120581B

Figure 5 Possible transforming pathway The point mutation inFAF1 is believed to cause a loss of function (crossed out in red)This may lead to an overactivity of the WNT signaling pathwayConsistently E2F1 (activated by LEF1) and igf2bp1 (a transcriptionaltarget of the WNT pathway) have been identified to be upregulatedin the molecular analysis In contrast to the negative regulator FAF1IGF2BP1 is a positive regulator of NF-120581B activityThe overactivity ofthe NF-120581B pathway and the reduced efficacy of FAS signaling can betransforming

The role ofWNT signaling in sarcoma has been subject todebate (eg [24]) In metastatic leiomyosarcoma 120573-Cateninmay accumulate in the nucleus despite a relatively weakexpression of WNT [25] This may be due to WNT signalactivation via noncanonical ligands [26] or to 120573-Cateninbinding the nuclear receptor NR4A2 and releasing it from thecorepressor protein LEF-1 [27] Of note in the cancer understudy here the mRNA level of NR4A2 was overexpressed10-fold compared to bone and 3-fold compared to muscleThe results from this study are consistent with the possibilitythat a lack of termination in the WNT signal rather than itsoveractivation could contribute to sarcomatous transforma-tion Such a mechanism may be reflected in an upregulationof downstream targets even though overexpression of WNTpathway components is not detectable

Other mutations beside FAF1 are less likely to becausative for the cancer EPHA3 was revealed as mutated inthis cancer by DNA exome sequencing and RNASeq EPHA3is a receptor tyrosine kinase that is frequentlymutated in lungcancer Tumor-suppressive effects of wild-type EPHA3 can beoverridden by dominant negative EPHA3 somatic mutations[28]Thismechanism is unlikely to play a role in this sarcomaas the detected mutation is located far N-terminally on theextracellular Ephrin binding domain not on the intracellularkinase domain

FAF1 has been described to act as a tumor suppressor gene[29] Its depletion due to chromosome breakage can affectprognosis in glioblastoma patients [30 31] Single nucleotidepolymorphisms in FAF1 are associated with a risk for gastriccancer [32] While numerous FAF1 mutations are associatedwith various cancers none of these genetic changes in theTCGA database affects the amino acid position 181 (Table 4)

The major upregulations identified in this tumor com-prise muscle-specific gene products (transcription factors

CAAC binding proteomics transgelin transgelin-2 RNAanoctamin-4 and immunohistochemistry smooth mus-cle actin) and calcium-regulating molecules (proteomicscalumenin S100-A11 reticulocalbin-3 and 78 kD glucose-regulated protein) The muscle-specific gene products con-firm this recurrent sarcoma as a leiomyosarcoma (the firsttumor distal to the site of the recurring one had beendiagnosed as an osteosarcoma) Calcium is one of the majorsecond messengers in smooth muscle cells Its uptake isregulated by potential-sensitive ion channels in the cellmembrane and by the activities of various receptors Calciumis stored in the sarcoplasmic reticulum from where it canbe released to facilitate actin-myosin interaction and tensiongeneration Phosphorylation of the myosin light chain by acalmodulin-regulated enzyme is important for contractionThe upregulation of gene products associated with migrationand invasion (osteopontin MMP1 vimentin filamin-A and120573-actin) and gene products for extracellularmatrixmoleculesand their modulators (fibronectin collagen ITGBL1 andMXRA5) reflects the invasive nature of this cancer

The recurrence of a sarcoma after 14 years has twoprobable explanations either it is due to a cancer pre-disposition syndrome based on a germ-line mutation (theage of the patient weakens this hypothesis) or the secondtumor is a metastatic colony of the first that was reactivatedafter dormancy The location of the sarcoma in the sameextremity and proximal to a preceding mesenchymal cancer(and therefore in its natural path of dissemination) impliedthe probability that this was a relapse in a metastatic siteThe different histologic assessment as osteosarcoma in thefirst occurrence and leiomyosarcoma as the second cancerdoes not necessarily negate that Mixed histology [33 34]and transdifferentiation [35ndash37] have been described forsarcomatous tumors Of note however in this scenarioosteosarcoma seems to more commonly follow leiomyosar-coma than precede it Material from the first cancer of thispatient was not accessible to us It is very plausible that thiscould have been a mineralized leiomyosarcoma In thosetumors the differential diagnosis from osteosarcoma can bedifficult [38] The extracompartmental location of the firsttumor supports this interpretation

On the molecular pathology level sarcomas fall intotwo groups comprising tumors with simple karyotypes(with pathogenetic translocations or specific genetic muta-tions) and tumors with very complex karyotypes (overtchromosome and genomic instability with numerous gainsand losses) [39] Some molecular alterations that lead tocarcinogenesis can be defined in absolute termsThey includegain-of-function mutations or chromosome translocationsthat transformprotooncogenes to oncogenes However otherchanges are relative to the normal tissue of origin suchas pathway overactivity or overexpression on the proteinor RNA levels We have combined the analysis of absolutechanges (DNA exome sequence RNA sequence) with theanalysis of relative changes using skeletal muscle and bone asreference organs (protein-DNA array 2D gel electrophoresisand RNA expression levels) This choice was determined inpart by tissue availability after surgery andwas intended to aidin the distinction of osteosarcoma from myosarcoma While

Sarcoma 15

Table 3 Deregulation of genes for drug disposition Analysis of RNASeq for at least twofold overexpression (italic font) or underexpression(bold font) of genes for (a) transport and (b) metabolism in tumor compared to muscle and bone For the export transporters (ABCtransporters) only overexpression is considered relevant for drug resistance Not shown in the table are the underexpressed genes (ABCA7ABCA8 ABCA13 ABCB6 ABCB10 ABCC6 ABCC6P1 ABCC6P2 ABCC8 ABCC11 ABCD2 ABCG2 and ABCG5)

(a) Transport

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

650655 ABCA17P ATP-binding cassette subfamily A (ABC1) member 17 pseudogene 1360 211624 ABCA4 ATP-binding cassette subfamily A (ABC1) member 4 2038 2802

6555 SLC10A2 Solute carrier family 10 (sodiumbile acid cotransporter family)member 2 minus1962 minus2112

345274 SLC10A6 Solute carrier family 10 (sodiumbile acid cotransporter family)member 6 2623 3240

6563 SLC14A1 Solute carrier family 14 (urea transporter) member 1 (Kidd bloodgroup) minus3074 minus3152

6565 SLC15A2 Solute carrier family 15 (H+peptide transporter) member 2 minus2027 minus2152

6566 SLC16A1 Solute carrier family 16 member 1 (monocarboxylic acid transporter1) 1401 2170

117247 SLC16A10 Solute carrier family 16 member 10 (aromatic amino acid transporter) 2113 2848

6567 SLC16A2 Solute carrier family 16 member 2 (monocarboxylic acid transporter8) 3242 3848

10786 SLC17A3 Solute carrier family 17 (sodium phosphate) member 3 minus2868 minus29596571 SLC18A2 Solute carrier family 18 (vesicular monoamine) member 2 minus2087 minus21946573 SLC19A1 Solute carrier family 19 (folate transporter) member 1 minus2099 minus220310560 SLC19A2 Solute carrier family 19 (thiamine transporter) member 2 1820 254880704 SLC19A3 Solute carrier family 19 member 3 minus3331 minus3478387775 SLC22A10 Solute carrier family 22 member 10 5711 60859390 SLC22A13 Solute carrier family 22 (organic anion transporter) member 13 2623 3217

85413 SLC22A16 Solute carrier family 22 (organic cationcarnitine transporter)member 16 minus8101 minus7299

51310 SLC22A17 Solute carrier family 22 member 17 1475 21755002 SLC22A18 Solute carrier family 22 member 18 2038 27226582 SLC22A2 Solute carrier family 22 (organic cation transporter) member 2 4793 5216

6581 SLC22A3 Solute carrier family 22 (extraneuronal monoamine transporter)member 3 2554 3182

6583 SLC22A4 Solute carrier family 22 (organic cationergothioneine transporter)member 4 minus3603 minus3776

151295 SLC23A3 Solute carrier family 23 (nucleobase transporters) member 3 2109 284810478 SLC25A17 Solute carrier family 25 (mitochondrial carrier) member 17 1311 207783733 SLC25A18 Solute carrier family 25 (mitochondrial carrier) member 18 1846 2570

788 SLC25A20 Solute carrier family 25 (carnitineacylcarnitine translocase) member20 minus2105 minus2207

89874 SLC25A21 Solute carrier family 25 (mitochondrial oxodicarboxylate carrier)member 21 minus2962 minus3105

51312 SLC25A37 Solute carrier family 25 member 37 minus4633 minus486651629 SLC25A39 Solute carrier family 25 member 39 minus2585 minus2737203427 SLC25A43 Solute carrier family 25 member 43 1325 208865012 SLC26A10 Solute carrier family 26 member 10 3896 4472115111 SLC26A7 Solute carrier family 26 member 7 3431 4018116369 SLC26A8 Solute carrier family 26 member 8 minus5354 minus5536115019 SLC26A9 Solute carrier family 26 member 9 1623 241911001 SLC27A2 Solute carrier family 27 (fatty acid transporter) member 2 minus4278 minus4487

16 Sarcoma

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

64078 SLC28A3 Solute carrier family 28 (sodium-coupled nucleoside transporter)member 3 minus4543 minus4812

222962 SLC29A4 Solute carrier family 29 (nucleoside transporters) member 4 minus1962 minus204581031 SLC2A10 Solute carrier family 2 (facilitated glucose transporter) member 10 2532 3170

6518 SLC2A5 Solute carrier family 2 (facilitated glucosefructose transporter)member 5 minus3647 minus3821

55532 SLC30A10 Solute carrier family 30 member 10 minus3547 minus37257782 SLC30A4 Solute carrier family 30 (zinc transporter) member 4 2832 33726569 SLC34A1 Solute carrier family 34 (sodium phosphate) member 1 minus4716 minus4941340146 SLC35D3 Solute carrier family 35 member D3 minus5662 minus590654733 SLC35F2 Solute carrier family 35 member F2 1433 2170206358 SLC36A1 Solute carrier family 36 (protonamino acid symporter) member 1 minus2265 minus2345285641 SLC36A3 Solute carrier family 36 (protonamino acid symporter) member 3 minus2284 minus239354020 SLC37A1 Solute carrier family 37 (glycerol-3-phosphate transporter) member 1 minus2112 minus22122542 SLC37A4 Solute carrier family 37 (glucose-6-phosphate transporter) member 4 minus2117 minus2219151258 SLC38A11 Solute carrier family 38 member 11 2410 308555089 SLC38A4 Solute carrier family 38 member 4 1837 254892745 SLC38A5 Solute carrier family 38 member 5 minus1994 minus215291252 SLC39A13 Solute carrier family 39 (zinc transporter) member 13 1301 207023516 SLC39A14 Solute carrier family 39 (zinc transporter) member 14 2569 318829985 SLC39A3 Solute carrier family 39 (zinc transporter) member 3 minus2032 minus2152283375 SLC39A5 Solute carrier family 39 (metal ion transporter) member 5 1623 23707922 SLC39A7 Solute carrier family 39 (zinc transporter) member 7 1273 205830061 SLC40A1 Solute carrier family 40 (iron-regulated transporter) member 1 minus3612 minus379684102 SLC41A2 Solute carrier family 41 member 2 1293 20708501 SLC43A1 Solute carrier family 43 member 1 minus2013 minus215257153 SLC44A2 Solute carrier family 44 member 2 minus2047 minus215250651 SLC45A1 Solute carrier family 45 member 1 2038 2722146802 SLC47A2 Solute carrier family 47 member 2 minus1962 minus20266521 SLC4A1 Solute carrier family 4 anion exchanger member 1 minus6513 minus645357282 SLC4A10 Solute carrier family 4 sodium bicarbonate transporter member 10 minus2640 minus275383959 SLC4A11 Solute carrier family 4 sodium borate transporter member 11 1846 25696508 SLC4A3 Solute carrier family 4 anion exchanger member 3 1261 20458671 SLC4A4 Solute carrier family 4 sodium bicarbonate cotransporter member 4 2445 31139497 SLC4A7 Solute carrier family 4 sodium bicarbonate cotransporter member 7 2717 33076523 SLC5A1 Solute carrier family 5 (sodiumglucose cotransporter) member 1 minus2547 minus2705159963 SLC5A12 Solute carrier family 5 (sodiumglucose cotransporter) member 12 4753 52066527 SLC5A4 Solute carrier family 5 (low affinity glucose cotransporter) member 4 minus3969 minus4249

6540 SLC6A13 Solute carrier family 6 (neurotransmitter transporter GABA)member 13 1328 2092

55117 SLC6A15 Solute carrier family 6 (neutral amino acid transporter) member 15 1623 2333388662 SLC6A17 Solute carrier family 6 member 17 2038 272854716 SLC6A20 Solute carrier family 6 (proline IMINO transporter) member 20 1697 2433

6532 SLC6A4 Solute carrier family 6 (neurotransmitter transporter serotonin)member 4 minus2512 minus2611

6534 SLC6A7 Solute carrier family 6 (neurotransmitter transporter L-proline)member 7 2623 3228

56301 SLC7A10 Solute carrier family 7 (neutral amino acid transporter y+ system)member 10 minus3421 minus3603

Sarcoma 17

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

6547 SLC8A3 Solute carrier family 8 (sodiumcalcium exchanger) member 3 minus2204 minus2309285335 SLC9A10 Solute carrier family 9 member 10 1208 20006549 SLC9A2 Solute carrier family 9 (sodiumhydrogen exchanger) member 2 2038 2706

9368 SLC9A3R1 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 1 minus2760 minus2846

9351 SLC9A3R2 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 2 1889 2619

84679 SLC9A7 Solute carrier family 9 (sodiumhydrogen exchanger) member 7 3347 393510599 SLCO1B1 Solute carrier organic anion transporter family member 1B1 3360 397728234 SLCO1B3 Solute carrier organic anion transporter family member 1B3 9760 95696578 SLCO2A1 Solute carrier organic anion transporter family member 2A1 2432 309628232 SLCO3A1 Solute carrier organic anion transporter family member 3A1 minus2032 minus2152353189 SLCO4C1 Solute carrier organic anion transporter family member 4C1 minus6850 minus6611

(b) Metabolism

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold Cchange

1583 CYP11A1 Cytochrome P450 family 11 subfamily A polypeptide 1 1623 23961589 CYP21A2 Cytochrome P450 family 21 subfamily A polypeptide 2 minus1962 minus20361591 CYP24A1 Cytochrome P450 family 24 subfamily A polypeptide 1 5208 55771592 CYP26A1 Cytochrome P450 family 26 subfamily A polypeptide 1 2623 32571594 CYP27B1 Cytochrome P450 family 27 subfamily B polypeptide 1 1846 2585339761 CYP27C1 Cytochrome P450 family 27 subfamily C polypeptide 1 3585 41781553 CYP2A13 Cytochrome P450 family 2 subfamily A polypeptide 13 minus1962 minus20351580 CYP4B1 Cytochrome P450 family 4 subfamily B polypeptide 1 minus4820 minus506566002 CYP4F12 Cytochrome P450 family 4 subfamily F polypeptide 12 minus6070 minus61958529 CYP4F2 Cytochrome P450 family 4 subfamily F polypeptide 2 minus7769 minus70604051 CYP4F3 Cytochrome P450 family 4 subfamily F polypeptide 3 minus8244 minus749111283 CYP4F8 Cytochrome P450 family 4 subfamily F polypeptide 8 minus5006 minus5270260293 CYP4X1 Cytochrome P450 family 4 subfamily X polypeptide 1 minus2476 minus25809420 CYP7B1 Cytochrome P450 family 7 subfamily B polypeptide 1 2502 31702326 FMO1 Flavin containing monooxygenase 1 4360 48592327 FMO2 Flavin containing monooxygenase 2 (nonfunctional) minus4074 minus43372328 FMO3 Flavin containing monooxygenase 3 minus4036 minus4309388714 FMO6P Flavin containing monooxygenase 6 pseudogene minus2569 minus2737493869 GPX8 Glutathione peroxidase 8 (putative) 2814 33712938 GSTA1 Glutathione S-transferase alpha 1 1623 23632939 GSTA2 Glutathione S-transferase alpha 2 2038 27412941 GSTA4 Glutathione S-transferase alpha 4 1492 21882953 GSTT2 Glutathione S-transferase theta 2 4682 5170653689 GSTT2B Glutathione S-transferase theta 2B (genepseudogene) 3739 430784779 NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439 79969027 NAT8 N-acetyltransferase 8 (GCN5-related putative) minus2769 minus29207358 UGDH UDP-glucose 6-dehydrogenase 2333 301855757 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 1604 227110720 UGT2B11 UDP glucuronosyltransferase 2 family polypeptide B11 minus3586 minus37687367 UGT2B17 UDP glucuronosyltransferase 2 family polypeptide B17 minus2836 minus295954490 UGT2B28 UDP glucuronosyltransferase 2 family polypeptide B28 minus3132 minus3284167127 UGT3A2 UDP glycosyltransferase 3 family polypeptide A2 minus4716 minus4907

18 Sarcoma

Table 4 FAF1 mutations in various cancers FAF1 mutations listed in the TCGA data base were identified without restriction to any type ofcancer For the location of the affected domains on the protein compare Figure 2

AA Mutation Cancer DomainN4S Missense Cutaneous melanomaI10S Missense Stomach adenocarcinoma UBAE21lowast Nonsense Cutaneous melanoma UBAE25K Missense Uterine endometrioid carcinoma UBAV38 splice Splice Stomach adenocarcinoma UBAP86fs FS del Colorectal adenocarcinomaG123 splice Splice Uterine endometrioid carcinoma UB1P136H Missense Colorectal adenocarcinoma UB1T147M Missense Brain lower grade glioma UB1D149Y Missense Uterine endometrioid carcinoma UB1L159V Missense Lung adenocarcinoma UB1K163N Missense Uterine endometrioid carcinoma UB1L170F Missense Cutaneous melanomaG184 splice Splice Colorectal cancerQ187H Missense Stomach adenocarcinomaS214N Missense Colorectal adenocarcinoma UB2R222I Missense Uterine endometrioid carcinoma UB2E238D Missense Lung adenocarcinoma UB2P241S Missense Uterine endometrioid carcinoma UB2T245A Missense Renal clear cell carcinoma UB2M249V Missense Uterine endometrioid carcinoma UB2E280K Missense Brain lower grade gliomaG293lowast Nonsense Colorectal cancerT300I Missense Colorectal adenocarcinomaD305H Missense Lung adenocarcinomaE308Q Missense Lung adenocarcinomaA316V Missense Stomach adenocarcinomaK319fs FS ins Head and neck squamous cell carcinomaR344G Missense Stomach adenocarcinoma UASF353I Missense Cutaneous melanoma UASL379V Missense Breast invasive carcinoma UASC396F Missense Cutaneous melanoma UASS459lowast Nonsense Uterine endometrioid carcinoma UASG469 splice Splice Glioblastoma multiforme UASR509G Missense Lung squamous cell carcinomaE510fs FS del Cutaneous melanomaR516C Missense Uterine endometrioid carcinomaA534V Missense Stomach adenocarcinomaF537L Missense Stomach adenocarcinomaE551lowast Nonsense Lung adenocarcinomaR554W Missense Colorectal cancerS582I Missense Lung adenocarcinoma UBXF585L Missense Uterine endometrioid carcinoma UBXE587lowast Nonsense Lung adenocarcinoma UBXA592V Missense Stomach adenocarcinoma UBXW610lowast Nonsense Breast invasive carcinoma UBXD611Y Missense Lung adenocarcinoma UBXE635fs FS del Brain lower grade glioma UBXP640fs FS del Cutaneous melanoma UBX

Sarcoma 19

a more accurate reference point for a leiomyosarcoma wouldhave been smooth muscle we believe that the comparison tostriated muscle is sufficient to allow the assessment of tumorspecific changes We have measured RNA DNA and proteinwith various assays Similar assessments in the future shouldalso include chromosome analysis for possible translocations

In the first-line defense against cancer the gradualreplacement of conventional chemotherapy with molecularlytargeted agents has opened the possibility to tailor drug treat-ments to particular tumors This transition necessitates thecharacterization of the molecular lesions that are causativefor the transformation of healthy cells into cancerous cellsbecause drugs need to be matched with the underlying carci-nogenic defect to be effective An additional caveat causedby unique genetic changes in the primary tumor can affectdrug transport and metabolism and needs to be taken intoconsideration In this case the RNA levels for genes that regu-late transport andmetabolismwere extensively skewed in thetumor tissue as compared to muscle and bone (see Table 3)implying potential challenges to chemotherapy An advancedmolecular treatment strategy for cancer will rely on themolecular definition of drug target drug transport and drugmetabolism pharmacogenetics in the primary tumor Con-secutive to cancer dissemination it will also require adjust-ments to account for the genetic changes in the metastases

The cost of health care has been under increasing scrutinyThe treatment of cancer patients is expensive in particularwhen hospitalization is required and further in cases ofend-of-life care Avoidable expenditures are generated bysuboptimal treatment decisions that result in low efficacy orhigh toxicity of anticancer regimens Molecular medicine hasthe potential to preempt those problems and reduce wastefulspending Yet it requires the upfront cost of molecular cancerexamination The analysis performed in this study requiredabout $11000- in nonsalary expenses to perform While ithas not identified a drug target it has specified possibleconfines for drug treatment The costs for molecular analysisneed to be weighed against the societal cost derived fromlost productivity in the workforce disrupted lives of familiesand premature deaths While currently limited drug optionsconstitute the major constraint to the approach taken herethe foreseeable future will bring an increasing spectrum ofmolecularly targeted drugs along with faster and cheapertechnologies for the molecular assessment of cancers Theywill facilitate the clinical translation of our approach

Consent

The patient provided informed consent

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by DOD Grant PR094070under University of Cincinnati IRB protocol 04-01-29-01The

author is grateful to Dr Rhett Kovall for helping with thestructural analysis of FAF1

References

[1] G F Weber Molecular Mechanisms of Cancer Springer Dor-drecht The Netherlands 2007

[2] N G Sanerkin ldquoPrimary leiomyosarcoma of the bone and itscomparison with fibrosarcomardquoCancer vol 44 no 4 pp 1375ndash1387 1979

[3] J M Weaver and J A Abraham ldquoLeiomyosarcoma of the Boneand Soft Tissue A Reviewrdquo httpsarcomahelporglearningcenterleiomyosarcomahtml

[4] M A Depristo E Banks R Poplin et al ldquoA framework forvariation discovery and genotyping using next-generationDNAsequencing datardquo Nature Genetics vol 43 no 5 pp 491ndash5012011

[5] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[6] C W Menges D A Altomare and J R Testa ldquoFAS-associatedfactor 1 (FAF1) diverse functions and implications for oncoge-nesisrdquo Cell Cycle vol 8 no 16 pp 2528ndash2534 2009

[7] M Kim J H Lee S Y Lee E Kim and J Chung ldquoCaspar asuppressor of antibacterial immunity in Drosophilardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 44 pp 16358ndash16363 2006

[8] J Song K P Joon J-J Lee et al ldquoStructure and interaction ofubiquitin-associated domain of human Fas-associated factor 1rdquoProtein Science vol 18 no 11 pp 2265ndash2276 2009

[9] P H OrsquoFarrell ldquoHigh resolution two dimensional electrophore-sis of proteinsrdquo Journal of Biological Chemistry vol 250 no 10pp 4007ndash4021 1975

[10] A Burgess-Cassler J J Johansen D A Santek J R Ideand N C Kendrick ldquoComputerized quantitative analysis ofCoomassie-Blue-stained serum proteins separated by two-dimensional electrophoresisrdquo Clinical Chemistry vol 35 no 12pp 2297ndash2304 1989

[11] B R Oakley D R Kirsch and N RMorris ldquoA simplified ultra-sensitive silver stain for detecting proteins in polyacrylamidegelsrdquo Analytical Biochemistry vol 105 no 2 pp 361ndash363 1980

[12] K Chu X Niu and L T Williams ldquoA Fas-associated proteinfactor FAF1 potentiates Fas-mediated apoptosisrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 25 pp 11894ndash11898 1995

[13] B Rikhof S de Jong A J H Suurmeijer C Meijer and W TA van der Graaf ldquoThe insulin-like growth factor system andsarcomasrdquoThe Journal of Pathology vol 217 no 4 pp 469ndash4822009

[14] RGirling J F Partridge L R Bandara et al ldquoAnew componentof the transcription factor DRTF1E2Frdquo Nature vol 362 no6415 pp 83ndash87 1993

[15] F Mercurio and M Karin ldquoTranscription factors AP-3 andAP-2 interact with the SV40 enhancer in a mutually exclusivemannerrdquo EMBO Journal vol 8 no 5 pp 1455ndash1460 1989

[16] R Bassel-Duby M D Hernandez M A Gonzalez J KKrueger and R S Williams ldquoA 40-kilodalton protein bindsspecifically to an upstream sequence element essential for mus-cle-specific transcription of the human myoglobin promoterrdquoMolecular and Cellular Biology vol 12 no 11 pp 5024ndash50321992

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Behavioural Neurology

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 4: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

4 Sarcoma

also positive for CD68 and displayed diffuse positive stainingfor vimentin but were negative for CD117 pancreatin S-100and CD34

Seven months after the surgery the patient received aPET-MRI scan for pain which revealed sixmetastatic lesionsincluding both lungs and multiple ribs She was put on three21-day cycles of Gemzar (days 1 and 8) Taxotere (day 8) andNeulasta (beginning on day 9) but was unable to continuepast the first cycle due to hospitalizations for continued andproblematic wound infections at the surgical lung biopsysites

32 DNA Exome Sequence Exome sequencing of thegenomic DNAs for tumor muscle and bone identified65546 potential sequence variants Filtering yielded 46 likelysomatic mutations in the tumor (Table 1) of which 7 (affect-ing EIF4A1 EPHA3 FAF1 IPO8 KIAA1377 LIMCH1 andNIPBL) were confirmed in the RNASeq results FAF1 asso-ciates with FAS and enhances apoptosis mediated throughthis receptor [12] The point mutation S181G (Figure 2) couldcause a loss of function in FAF1 and lead to transformationvia antiapoptosis

33 RNA Analysis Expectedly the gene expression patternsaccording to RNASeq were very different among tumormuscle and bone The cancer contained several gene prod-ucts that were overexpressed compared to both muscle andbone (Tables 2(a) and 2(b)) Among the top 30 changesin the tumormuscle and tumorbone comparisons 13 wereidentical (Table 2(c)) It is implied that these gene productsare quite unique for the tumor and likely contribute to itspathogenesis This notion is supported by the upregulationof the smooth muscle gene Ano4 which corroborates theleiomyosarcomatous nature of the cancer When limiting theanalysis to genes expressed at least at the level of 1 unit the17 genes overexpressed in the tumormuscle and tumorbonecomparisons contain several extracellular matrix proteinsimplying an active remodeling of the tumor microenviron-ment (Table 2(d)) By contrast none of the underexpressedgene products in the tumormuscle comparison matched thetumorbone comparison (not shown)

Of note the RNA level of IGF2BP1 (IMP-1 CRD-BPand ZBP-1) is highly upregulated in the tumor compared tomuscle as well as bone IGF2BP1 is a RNA-binding factorthat affects mRNA nuclear export localization stability andtranslation It regulates mRNA stability during the inte-grated cellular stress response in stress granules IGF2BP1 isa transcriptional target of the WNT pathway which is nega-tively regulated by intact FAF1 and may be unregulated byFAF1S181GThe IGF systemhas been linked to sarcoma patho-genesis [13] and may play a role in this specific cancer OtherIGF family members with increased RNA message levels inthis tumor (compared to muscle and bone) include IGFBPL1(5-6-log

2-fold) IGFL3 (6-7-log

2-fold) and IGF2BP3 (2-6-

log2-fold)The identified point mutation in FAF1 may be patho-

genetic for this cancer FAF1 is a regulator of NF-120581B activa-tion It directly binds to RelA (P65) retaining it in the cyto-plasm It can also interact with IKK120573 thus allowing for the

I120581B-mediated degradation of the transcription factors P65and P50 [6] Consistently the expression of regulators of theNF-120581B activation pathway is skewed in the tumor comparedto muscle or bone (Table 2(e))

34 Transcription Factor Binding ProteinDNA arrays mea-sure the binding activity of transcription factors Theycomprise three basic steps A set of biotin-labeled DNAbinding oligonucleotides are preincubated with a nuclearextract of interestThe proteinDNA complexes are separatedfrom the free probes The probes in the complexes arethen extracted and hybridized to prespotted membranesfollowed by HRP-based chemiluminescence detection Wemade nuclear extracts from tumor bone and muscle andtested them for DNA binding activity Binding that wasinduced in cancer but not in the normal tissues was dis-played by the transcription factors E2F1 AP3 LIII-BP PAX6ADD-1 and CCAC [14ndash19] The CCAC binding activity isconsistent with a muscle-derived tumor E2F1 may associatewith the WNT pathway-induced transcription factor LEF1resulting in transcriptional derepression of E2F1 [20] Likelyconstitutive transcription factors that are active in all 3 tissuescomprise AhRAmt GATA1 GATA2 GATA12 HIF1 andHOXD8910 (Figure 3)

35 Protein Analysis 2D gel electrophoresis of the RIPAlysates from tumor muscle and bone showed very divergentpatterns (Figure 4(a)) Two experienced analysts comparedthe protein pattern from the tumor with the protein patternfrom either bone or muscle Polypeptide spots that wereunique to the gels from the tumor were outlined (spotsunique to or relatively darker in the bone or muscle werenot indicated)The labeled proteins were extracted for identi-fication with mass spectrometry This yielded several struc-tural proteins which may reflect modification of the cellulararchitecture under rapid growth Transgelin-1 and transgelin-2 were abundant and corroborated the identity of thetumor as a leiomyosarcoma Four calcium-binding proteinswere highly expressed in the cancer In addition regulatorsof protein synthesis (40S ribosomal protein S12 glycine-tRNA ligase) protein modification (N-terminal fragmentof heat shock protein HSP 90120572 C-terminal fragment ofprotein disulfide isomerase) and protein degradation (1205721-antitrypsin proteasome activator complex subunit 2) wereidentified (Figure 4(b)) Of interest may be the C-terminalfragment of protein disulfide isomerase which not onlyhydroxylates prolines in preprocollagen but also contributesto microsomal triglyceride transfer It could be reflectiveof a skewed tumor metabolism The protein analysis wascorroborated by the mRNA levels (Figure 4(d))

Cancer markers were tested according to Western blot(Figure 4(c)) The tumor but not normal muscle expressedthe metastasis protein osteopontin and a single small form(lt75 kD) of CD44 that is likely the not alternatively splicedstandard form Unexpectedly while both tumor and muscleexpressed comparably abundant amounts of STAT3 phos-phorylation (reflective of activation) was present in themuscle but not in the tumorThe STAT3 pathway is associatedwith progression in several human cancers and this is often

Sarcoma 5

Table1DNAexom

eSNPsTh

eDNAexom

esfortum

orm

uscle

and

bone

weres

equencedTh

eresultswerefi

lteredin

thefollowingorder(1)d

ifferentgenotypeintumor

from

muscle

and

bonew

ithmuscle

andbo

nebeingidentic

alto

each

other(2)d

eletem

utations

with

lowconfi

dence(

valueo

f20or

lower)inall3

tissues(3)

deleteun

identifi

edgenes(4)d

eletem

utations

thatareh

omozygou

sreference

inthetum

or(5)

deletemutations

thathave

aMAFin

dbSN

Pgt10(6)

deletelowim

pactandmod

ifier

mutationsTh

egenen

ames

arep

arto

fthe

keyin

the

leftcolumnIn

thiscolumnresults

onbo

ldfont

representSNPs

thatwerec

onfirmed

ontheR

NAlevelbyRN

ASeqTh

edatafi

lesh

aveb

eensubm

itted

totheN

CBIsho

rtread

archive(SR

A)

underthe

accessionnu

mberS

RP052797

(biosamples

SAMN03316820SAMN03316821SAMN03316822)

Key

Chromosom

ePo

sition

Reference

Alternate

dbSN

PID

dbSN

PMAF

ESPMAF

(All)

ESPMAF

(EA)

ESPMAF

(AA)

Con

sensus

impact

Bone

muscle

geno

type

Bone

overall

depth

Bone

allele

depths

Bone

quality

Muscle

overall

depth

Muscle

allele

depths

Muscle

quality

Tumor

geno

type

Tumor

overall

depth

Tumor

allele

depths

Tumor

quality

177479

998T

EIF4

A1

177479998

CT

mdash

mdashmdash

mdashMod

erate

00

175

1840

99103

1080

9901

133

7968

99389

2592

00T

EPHA3

389259200

CT

mdash

mdashmdash

mdashMod

erate

00

43450

99117

1230

9901

723048

9915120

4545

CFA

F11

51204545

TC

mdash

mdashmdash

mdashMod

erate

00

87910

99108

1130

9901

8566

27

9912

3083

4620

AIPO8

1230834620

CA

mdash

mdashmdash

mdashMod

erate

00

68712

99120

1260

9901

807018

9911

101834

425A

KIA

A1377

11101834425

GA

mdash

mdashmdash

mdashMod

erate

00

36371

9067

700

9901

863163

9944168

2102

CLIMCH

14

41682102

GC

mdash

000

0077

0000

0227

Mod

erate

00

71740

9996

1010

9901

153

12049

99536

985326

GNIPBL

536985326

AG

mdash

mdashmdash

mdashMod

erate

00

660

1835

360

8101

201012

99377623789

ARO

BO2

377623789

AGA

mdash

mdashmdash

mdashHigh

00

22NA

5429

NA

8701

37NA

99

2217300

095A

SMARC

AL1

2217300

095

ATTG

CATC

A-AC

GTC

GTG

GA

mdash

mdashmdash

mdashHigh

00

95NA

99122

NA

9901

99NA

99

2152236045TTA

ATN

FAIP6

2152236045

TATTA

Ars3506

0021

mdashmdash

mdashmdash

High

02

19NA

124

NA

5701

17NA

383520206

69TAC

Y13

520206

69G

T

mdashmdash

mdashmdash

Mod

erate

00

130

1360

9986

900

9901

804543

999117

130734

GAKN

A9

117130734

CG

mdash

mdashmdash

mdashMod

erate

00

27280

7830

310

9001

503024

9912

6030354A

ANO2

126030354

CA

mdash

mdashmdash

mdashMod

erate

00

101

1060

99114

1200

9901

135

10445

9918

10487667

AAPC

DD1

1810487667

GA

mdash

mdashmdash

mdashMod

erate

00

43450

9938

400

9901

503816

99734118

560C

BMPE

R7

34118

560

GC

mdash

mdashmdash

mdashMod

erate

00

69720

99101

1060

9901

716314

9915

24921273

TC1

5orf2

1524921273

GT

mdash

mdashmdash

mdashMod

erate

00

12120

3638

390

9901

26226

9919

54483249

CCA

CNG8

1954483249

TC

mdash

mdashmdash

mdashMod

erate

00

147

1540

99103

1080

9901

152

13632

9910

16893265

CCU

BN10

16893265

GC

mdash

mdashmdash

mdashMod

erate

00

57600

9990

940

9901

403410

997148489854C

CUL1

7148489854

AC

mdash

mdashmdash

mdashMod

erate

00

73760

9965

680

9901

574221

9917

41566894

CDHX8

1741566894

GC

mdash

mdashmdash

mdashMod

erate

00

106

1110

9988

920

9901

124

9442

9920

61512358

TDID

O1

2061512358

GT

rs73304513

00417

0045932

000

0838

0135456

Mod

erate

00

600

182

00

601

400

2116

23703526

AER

N2

1623703526

GA

mdash

mdashmdash

mdashMod

erate

00

81850

9998

1030

9901

916041

993197880164G

FAM157A

3197880164

GCA

GCA

GCA

AG

mdash

mdashmdash

mdashMod

erate

00

21NA

2525

NA

101

31NA

614

4564

4287

GFA

NCM

144564

4287

AG

000

09mdash

mdashmdash

Mod

erate

00

13130

3331

320

8701

12102

14189637524

CGBP

71

89637524

GC

mdash

mdashmdash

mdashMod

erate

00

171

1790

99201

2110

9901

206

1616

799

742003933

CGLI3

742003933

GC

mdash

mdashmdash

mdashMod

erate

00

87910

9981

850

9901

856132

9917

4837170TGP1BA

174837170

CT

mdash

mdashmdash

mdashMod

erate

00

16160

459

9015

01

11101

114

24635161

GIRF9

1424635161

TG

mdash

mdashmdash

mdashMod

erate

00

66690

9943

450

9901

362513

9915

42133096

AJM

JD7-

PLA2G

4B15

42133096

GA

mdash

mdashmdash

mdashMod

erate

00

111

1160

9975

780

9901

807414

92

1740271678

AKA

T2A

1740271678

GA

mdash

mdashmdash

mdashMod

erate

00

65680

9958

610

9901

563032

99873848894

GKC

NB2

873848894

AG

mdash

mdashmdash

mdashMod

erate

00

15150

3324

250

6601

301517

9919

50827053

TKC

NC3

1950827053

CT

mdash

mdashmdash

mdashMod

erate

00

220

2310

99136

1430

9901

160

13046

9917

21319069

AKC

NJ12

1721319069

GA

rs76265595

mdashmdash

mdashmdash

Mod

erate

00

23241

6343

434

4001

28255

6012

53011932

CKR

T73

1253011932

TC

mdash

mdashmdash

mdashMod

erate

00

146

1530

99157

1650

9901

159

11758

99X

7500

4584

AMAG

EE2

X7500

4584

CA

mdash

mdashmdash

mdashMod

erate

00

29300

8148

500

9901

382616

995140182157A

PCDHA3

5140182157

GA

mdash

mdashmdash

mdashMod

erate

00

180

1890

99153

1610

9901

150

13234

9915

42133096

APL

A2G

4B15

42133096

GA

mdash

mdashmdash

mdashMod

erate

00

111116

099

75780

9901

807414

9210

96005840

CPL

CE1

1096005840

TC

mdash

mdashmdash

mdashMod

erate

00

81851

9973

762

9901

875540

991166816805G

POGK

1166816805

CG

mdash

mdashmdash

mdashMod

erate

00

84880

9982

860

9901

634920

99

6 Sarcoma

Table1Con

tinued

Key

Chromosom

ePo

sition

Reference

Alternate

dbSN

PID

dbSN

PMAF

ESPMAF

(All)

ESPMAF

(EA)

ESPMAF

(AA)

Con

sensus

impact

Bone

muscle

geno

type

Bone

overall

depth

Bone

allele

depths

Bone

quality

Muscle

overall

depth

Muscle

allele

depths

Muscle

quality

Tumor

geno

type

Tumor

overall

depth

Tumor

allele

depths

Tumor

quality

12111

020739

TPP

TC7

12111

020739

TCGC

Trs71083132

mdashmdash

mdashmdash

Mod

erate

00

18NA

3014

NA

1801

19NA

1019

804293

APT

BP1

19804293

CA

mdash

mdashmdash

mdashMod

erate

00

120

1261

9971

741

9901

755330

9971564

51175C

RNF32

71564

51175

GC

mdash

mdashmdash

mdashMod

erate

00

55570

9967

700

9901

594223

993520206

69TRP

11-155D

1811

3520206

69G

T

mdashmdash

mdashmdash

Mod

erate

00

130

1360

9986

900

9901

804543

9921

43838624

TUBA

SH3A

2143838624

GT

mdash

mdashmdash

mdashMod

erate

00

70730

9945

470

9901

897621

9919

58773857

AZN

F544

1958773857

GA

mdash

mdashmdash

mdashMod

erate

00

46480

9943

450

9901

715126

99516465723

CZN

F622

516465723

TC

mdash

mdashmdash

mdashMod

erate

00

17170

3914

140

3301

271415

99

Sarcoma 7

Table 2 RNA induced in the tumor (a) Transcripts overexpressed in the tumor compared to muscle but not bone (b) Transcriptsoverexpressed in the tumor compared to bone but not muscle (c) Transcripts most abundantly overexpressed in the tumor compared tomuscle and bone (d) as (a) but limited to genes expressed at least at the level of 1 unit in the reference tissue (muscle or bone) (e) Alteredexpression of genes associated with NF-120581B activity Analysis of RNASeq for the transcription factor NF-120581B and gene products that regulate itsactivity RNAmessages that are selectively associated with the TNF pathway to NF-120581B activation are marked with bold font log2-fold changeindicates the alteration in the tumor compared to muscle or bone (the respective columns indicate the expression level for each organ)

(a)

log2-fold changeMuscle

Tumor over muscleNRG1 Neuregulin 1 9909KSR2 Kinase suppressor of ras 2 9675MMP13 Matrix metallopeptidase 13 (collagenase 3) 9528SPP1 Secreted phosphoprotein 1 9098INHBA Inhibin beta A 8570COL11A1 Collagen type XI alpha 1 8564MMP9 Matrix metallopeptidase 9 (gelatinase B 92 kda gelatinase 92 kda type IV collagenase) 8552FBN2 Fibrillin 2 8495LRRC15 Leucine rich repeat containing 15 8429MMP11 Matrix metallopeptidase 11 (stromelysin 3) 8336E2F7 E2F transcription factor 7 8314PRAME Preferentially expressed antigen in melanoma 8179PTK7 PTK7 protein tyrosine kinase 7 7996DSCAM Down syndrome cell adhesion molecule 7761RGS4 Regulator of G-protein signaling 4 7633CNIH3 Cornichon homolog 3 (Drosophila) 7632OR10V1 Olfactory receptor family 10 subfamily V member 1 7610CEP55 Centrosomal protein 55 kda 7583WNT5B Wingless-type MMTV integration site family member 5B 7569CA12 Carbonic anhydrase XII 7520

GALNT5 UDP-N-acetyl-alpha-D-galactosaminepolypeptide N-acetylgalactosaminyltransferase 5(GalNAc-T5) 7517

(b)

log2-fold changeBone

Tumor over boneROS1 c-ros oncogene 1 receptor tyrosine kinase 10987GREM1 Gremlin 1 10604NPTX1 Neuronal pentraxin I 8813GJB2 Gap junction protein beta 2 26 kDa 8597CREB3L1 cAMP responsive element binding protein 3-like 1 8506KRT14 Keratin 14 8351SERPINE1 Serpin peptidase inhibitor clade E (nexin plasminogen activator inhibitor type 1) member 1 8288HOXD10 Homeobox D10 8105ALPK2 Alpha-Kinase 2 7783POU3F2 POU class 3 homeobox 2 7530POSTN Periostin osteoblast specific factor 7497NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439STC2 Stanniocalcin 2 7434WNT5A Wingless-type MMTV integration site family member 5A 7412IGFN1 Immunoglobulin-like and fibronectin type III domain containing 1 7387

8 Sarcoma

(b) Continued

log2-fold changeBone

STC1 Stanniocalcin 1 7385FAM180A Family with sequence similarity 180 member A 7315KRT17 Keratin 17 7305BBOX1 Butyrobetaine (gamma) 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 7277MAGEA1 Melanoma antigen family A 1 (directs expression of antigen MZ2-E) 7267

(c)

log2-fold changeMuscle Bone

Tumor over bonemuscle

ANO4 Anoctamin 4 (TMEM16D) transmembrane calcium-activated chloride channelfacilitates smooth muscle contraction 9937 8542

SLCO1B3 (OATP1B3) Solute carrier organic anion transporter family member 1B3 9569 9760

MARCH4 Membrane-associated ring finger (C3HC4) 4 E3 ubiquitin ligase located predominantlyto the endoplasmic reticulum 9538 7406

IGF2BP1 Insulin-like growth factor 2 mRNA binding protein 1 binds to and stabilizes mRNA 9440 9631ADAMTS16 ADAMmetallopeptidase with thrombospondin type 1 motif 16 zinc-dependent protease 9260 8450SOX11 SRY (sex determining region Y)-box 11 important in brain development 9178 9954

HAPLN1 Hyaluronan and proteoglycan link protein 1 stabilizes aggregates of aggrecan andhyaluronan giving cartilage its tensile strength and elasticity 8951 8142

MUC15 Mucin 15 cell surface associated 8801 7992HOXB9 Homeobox B9 8773 7378MAGEC2 Melanoma antigen family C 2 8693 8884

ST6GALNAC5 ST6 (alpha-N-acetyl-neuraminyl-23-beta-galactosyl-13)-N-acetylgalactosaminidealpha-26-sialyltransferase 5 7848 8038

C11orf41 Chromosome 11 open reading frame 41 7781 8141MMP1 Matrix metallopeptidase 1 (interstitial collagenase) 7455 7646

(d)

Symbol Name log2-fold change log2-fold changeMuscle Bone

Tumor over bonemuscleFN1 Fibronectin 1 7328 6293 6457 19860COL1A1 Collagen type I alpha 1 6768 3706 5868 11934CCND1 Cyclin D1 5595 3958 5098 9637RGS1 Regulator of G-protein signaling 1 5118 1056 4653 2533ITGBL1 Integrin beta-like 1 (with EGF-like repeat domains) 5071 3177 3895 12415COL1A2 Collagen type I alpha 2 4852 8756 4910 14503MXRA5 Matrix-remodelling associated 5 4834 1352 4631 2685POSTN Periostin osteoblast specific factor 4513 5870 7497 1267PLOD2 Procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 4285 1801 4023 3730COL5A2 Collagen type V alpha 2 4275 1297 4833 1517COL3A1 Collagen type III alpha 1 4003 13118 5801 6500

SEMA3C Sema domain immunoglobulin domain (Ig) shortbasic domain secreted 3954 1164 4215 1675

FBN1 Fibrillin 1 3912 6957 4018 11148AEBP1 AE binding protein 1 3808 3212 4002 4843SIK1 Salt-inducible kinase 1 3805 1219 3566 2484

Sarcoma 9

(d) Continued

Symbol Name log2-fold change log2-fold changeMuscle Bone

SERPINH1 Serpin peptidase inhibitor clade H (heat shock protein47) member 1 3706 1652 4145 2098

ANTXR1 Anthrax toxin receptor 1 3670 3789 3690 6445

(e)

Symbol Name log2-fold change log2-fold changeMuscle Bone

HELLS Helicase lymphoid-specific 5315602 0155898 minus082713 202489

TNFRSF11A Tumor necrosis factor receptor superfamily member 11a andNFKB activator 3017922 003091 1400993 0202453

NFKBID Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor delta 2655352 0 minus147615 0504341

TNFRSF25 Tumor necrosis factor receptor superfamily member 25 2432959 0046388 0163954 0490795

NFKBIE Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor epsilon 2121015 0062395 0311441 043382

TNF Tumor necrosis factor 1847997 0 minus142101 003733

TNFRSF10D Tumor necrosis factor receptor superfamily member 10d decoywith truncated death domain 1754888 0172968 minus01757 1183343

TNFRSF1B Tumor necrosis factor receptor superfamily member 1B 1473438 0709902 minus097011 6729401TNFRSF10A Tumor necrosis factor receptor superfamily member 10a 1411898 0828219 0357701 3004386

NFKBIB Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor beta 1193993 1209816 046055 3484692

TNFRSF10B Tumor necrosis factor receptor superfamily member 10b 1094157 1908597 0425446 5242006TNFRSF21 Tumor necrosis factor receptor superfamily member 21 1055762 3882038 0745815 8305711TNFRSF1A Tumor necrosis factor receptor superfamily member 1A 0875167 3790938 minus011707 130344

NFKBIZ Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor zeta 0575974 3698951 0344657 7493136

RIPK1 Receptor (TNFRSF)-interacting serine-threonine kinase 1 0494467 311749 minus108854 161392NKRF NFKB repressing factor 0475722 2156087 minus086397 9434166NFKB1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 0432959 2054532 minus065865 7568586CHUK Conserved helix-loop-helix ubiquitous kinase 0176126 2961281 minus120204 1330333RELA V-rel reticuloendotheliosis viral oncogene homolog A (avian) 0013848 1263837 0287167 1803044

NFKB2 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 2(p49p100) minus008641 0312993 0485882 0360442

NKAPP1 NFKB activating protein pseudogene 1 minus010692 0825177 minus099449 2655392

TNFRSF10C Tumor necrosis factor receptor superfamily member 10c decoywithout an intracellular domain minus0152 0064676 minus556891 6321515

NKIRAS2 NFKB inhibitor interacting Ras-like 2 minus060768 1095135 minus088758 2299946

NFKBIL1 Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor-like 1 minus08719 0141933 minus008357 0140418

NKIRAS1 NFKB inhibitor interacting Ras-like 1 minus087428 6296399 1467735 2122983TANK TRAF family member-associated NFKB activator minus0983 6777124 minus151908 1695872NKAP NFKB activating protein minus135735 1422458 minus078243 1646287

NFKBIA Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor alpha minus185483 4032743 minus031802 2395275

NKAPL NFKB activating protein-like minus557347 5875743 minus140215 0534643

10 Sarcoma

FAS associating region

DED interacting region

UB12 DZM

UB2domain

UASdomain

UBXdomain

UBAdomain

S289 S291

Aurora ACK II

S181

AKT1ATMGSK3

AuroraPKG

PLK1RSK

STK4CHK1

650566481336260205169110475

(a)

MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRM

6

1

101

100

200

300

400

500

600

650

201

301

401

501

601

667855899998876467765678888887578758999986467 88 8888888888888988888899988888889888877767888777888757

2222454345655744642554435764462443558468643653433344333424435334233333332333332333354335333344334354

LDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITH

9999997797689998787758999999999859996577754788888888875333467888868987688888888888877888855675799987

9585847535375857543447459666955563844544585374435443365845846434457567533353232334334444433337484944

REVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDAT

5888757887577654788887766654677776555468877888765455546887766555688888877777777777776556788888767766

5344456484742545656645594364384534437445444342323455453535435463434323333343434333333334333335433333

EFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESI

5666776654456777888888888888866889999999998848888876677778999999998776686899998589875579999987486899

4635343574354322343434533333343548479747945564424647635575478569766456468999999754233454687457844669

VSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDE

9999888589997557877545444345677876335554467888876899986799876447777677888999999998877555778887777778

6778547999996674433343444333333335466456434433487999895353333464346434365468755846444545454565446565

REARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVF

8888888887788877766545557777777666777777777776678877777766578888888888885799998589975788758987589999

4545445564445344433443354555556653665456544454434444444443344334333333446859596743354745795535796898

DFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKE

99997799988779985788754577888765665678898589998589

67974525444795877564635844444476875837446989897366

(b)

Figure 2 FAF1 structure (a) A schematic of functional domains in FAF1 with annotations (adapted from [6ndash8]) There are two ubiquitin-like domains flanking the S181 site The PDB structure 2DZM identifies the N-terminal one while the C-terminal prediction is by sequencesimilarity Whereas the mutation S181G is not expected to disrupt the protein structure per se this amino acid has a high score as a possiblephosphorylation site for a number of kinases involved inDNAdamage repair (b) Secondary structure prediction of FAF1The residues aroundS181 appear unstructured

reflected in STAT3 constitutive phosphorylation The lack ofphosphorylation in this case suggests that the leiomyosar-coma may not depend on the STAT3 pathway

36 Pharmacogenetic Evaluation Predicting the sensitivityto anticancer drugs is a main goal of molecular analysisFor this the over- or underexpression of genes for drugtransport and metabolism is of key importance Analysis ofthe RNASeq data for these groups of gene products identified

a surprisingly large number of deregulations compared tomuscle or bone (Tables 3(a) and 3(b)) Those alterationsmay affect choices for drug treatment For example the highlevels of glutathione S-transferase may render carmustinethioTEPA cisplatin chlorambucil melphalan nitrogenmus-tard phosphoramide mustard acrolein or steroids ineffec-tive The overexpression ofN-acetyltransferase may compro-mise 5-fluorouracil or taxolThemodest upregulation of onlytwo export transporters (ABC-transporters) and specifically

Sarcoma 11

Bone Tumor

Muscle

(a)

Transcription factor Description Reference

E2F1 Transcriptional activation of cell cycle progression is selectively activated in response to specific cues key downstream target of RB1 can promote proliferation or apoptosis when activated [14]

AP-33 interact in a mutually exclusive manner [15]

CCAC CCAC box is an essential element for muscle-specific transcription from the myoglobin promoter [16]

LIII-BP The L-III transcriptional regulatory element (a carbohydrate-response element) is in the pyruvate kinase L gene necessary for cell type-specific expression and transcriptional stimulation by carbohydrates [17]

ADD-1 (SREBP-1) activates transcription from sterol-regulated elements and from an E-box sequence present in the promoter of fatty acid synthase [18]

PAX6 Member of the paired box gene family transcriptional regulator involved in developmental processes [19]

48kD transcription factor activator protein-3 binds to the core element and recognizes the SV40 enhancer and AP-2 and AP-

(b)

Figure 3 Transcription factor activation Nuclear extracts from tumor muscle and bone were tested for DNA binding activity on a protein-DNA array (a) Transcription factor binding that was high in all 3 tissues and is therefore considered constitutively active is circled in yellow(left to right top to bottom AhRAmt GATA1 GATA2 GATA12 HIF1 and HOXD8910) Transcription factors that show high binding inthe cancer but not in muscle or bone are circled in red (left to right top to bottom E2F1 AP3 LIII-BP PAX6 ADD-1 and CCAC) (b) Thefunctions of transcription factors that display high DNA binding in the cancer but not in muscle or bone are described

the lack of ABCB1 overexpression is favorable for avoidingdrug resistance

37 Population Analysis The above-described results indi-cated that a FAF1 mutation which replaces serine in position181 thus preventing FAF1 phosphorylation and activationmay be a driver for leiomyosarcomagenesis A custom Taq-Man assay confirmed the presence of the somatic mutationin the patient To assess whether this single nucleotidereplacement is common in this type of cancer we analyzedDNA from 29 leiomyosarcomas and 1 blood sample from aleiomyosarcoma patient For comparison to nonsarcomatoustumors 34 breast cancers served as a reference None of themdisplayed amutation in the same locus By contrast there wasa distribution across all leiomyosarcomas in the osteopontin

promoter position minus443 (used as a reference) with 12 CC 10TC and 9 TT

4 Discussion

TheFAF1mutation identified as the likely cause for the cancerunder study gives room for an explanation of the sarcomatoustransformation (Figure 5) DNA damage to mesenchymalcells occurs persistently in an oxidizing environment at 37∘CThese insults are rarely transforming and such an occurrencewould trigger the initiation of programmed cell death inapoptosis-competent cells Intact FAF1 associates with FASand enhances apoptosis mediated through this receptor [12]A loss of function in FAF1 could lead to transformation viaantiapoptosis Whereas the mutation S181G is not expected

12 Sarcoma

Muscle

Bone

Tumor

220

94

6043

29

14

220

94

60

43

29

14

220

94

60

43

29

14

1

7

6

23

24

21

22

25

26

89 1011 12

1516 1719

pH 4 8 pH 4 8

8pH 4

(a)

SwissProt or NCBISpot number Protein identified accession Description

(Structural)6 Transgelin Q01995 Smooth muscle-specific cross-links actin24 Transgelin-2 P37802 Smooth muscle-specific cross-links actin

Vimentin P08670 Intermediate filament in mesenchymal tissue1 Filamin-A P21333 Actin-binding protein regulates the cytoskeleton 21 P06709 Cytoskeleton

(Calcium homeostasis)8 9 Calumenin O43852 Calcium-binding protein in the ER and golgi apparatus26 Protein S100-A11 P31949 Calcium-binding EF hand protein10 Reticulocalbin-3 Q96D15 Calcium-binding protein located in the ER12 P11021 Heat shock 70-kD protein 5 ER lumenal calcium-binding

(Protein processing)25 40S ribosomal protein S12 P25398 Core component of the ribosomal accuracy center7 Glycine-tRNA ligase P41250 Catalyzes the attachment of glycine to tRNA19 P01009 Protease inhibitor23 P01009 Protease inhibitor21 Proteasome activator complex subunit 2 Q9UL46 Proteasome activation10 P07900 Chaperone

(Other)7 Serum albumin P02768 Carrier protein in blood22 Protein disulfide isomerase (C-terminal fragment) P07237 Prolyl hydroxylation in collagen microsomal triglyceride transfer

120573 actin (C-terminal fragment)

78kD glucose-regulated protein

120572-1-antitrypsin120572-1-antitrypsin (C-terminal fragment)

Heat shock protein HSP 90120572 (N-terminal fragment)

11 15ndash17

(b)

Figure 4 Continued

Sarcoma 13

Tumor Muscle

OPN

CD44

STAT3

P-STAT3

Tubulin

75

75

50

15010075

10075

50

(c)

Tumor bone Tumor muscleGene ID Symbol Name Fold change Expression Fold change Expression6876 TAGLN Transgelin 3274 2455 3087 15088407 TAGLN2 Transgelin-2 2164 7338 6975 13037431 VIM Vimentin 1653 295010 4061 696042316 FLNA Filamin A alpha 1304 7791 9005 065160 ACTB Actin beta 0656 973187 5491 67368813 CALU Calumenin 7601 19328 12063 70596282 S100A11 S100 calcium binding protein A11 0931 158914 5071 1686357333 RCN3 Reticulocalbin 3 EF-hand calcium binding domain 2439 0604 6412 01223309 HSPA5 1068 92754 2607 220356696 SPP1 Secreted phosphoprotein 1 7572 46508 547929 0355960 CD44 CD44 molecule (Indian blood group) 2053 26708 15436 20566774 STAT3 Signal transducer and activator of transcription 3 0617 46534 0832 200165925 RB1 Retinoblastoma 1 0736 14772 0442 14268

Heat shock 70kDa protein 5 (glucose-regulated protein 78kDa)

(d)

Figure 4 Protein overexpression (a) 2D protein gel electrophoresis of lysates from muscle (upper left) tumor (upper right) and bone(bottom) in RIPA buffer Red circles indicate the spots that were identified as overexpressed and were further analyzed by mass spectrometry(b) Proteins identified in 2D gel electrophoresis as overexpressed in the tumor in comparison to muscle and bone were analyzed for theiridentity by mass spectrometry The left column indicates the spot number corresponding to the 2D gel The next column contains theprotein name followed by the accession number and a description of the protein functionThe protein functions are grouped into structuralcalcium homeostasis and various others (c) Western blot 10 120583g lysates of tumor muscle and bone in RIPA buffer were loaded per laneand electrophoresed on 10 SDS-polyacrylamide minigels with reducing denaturing sample buffer After transfer to PVDF membranesthey were probed for markers of cancer progression including osteopontin CD44 STAT3 and phospho-STAT3 Antitubulin served as aloading control (d) RNA levels corresponding to the proteins found to be affected in the sarcoma With four exceptions in the tumor-bonecomparison (gray font) upregulated proteins are associated with increased RNA levels STAT3 is not increased on the protein or RNA levelThe reduced level of RB1 expression is consistent with the elevated DNA binding activity of E2F1

to disrupt the structure of the protein this site does scorehigh as a possible phosphorylation site for a number ofkinases involved in DNA damage repair supporting thehypothesis that the cancer cells containing thismutation havelost their ability to respond to transforming DNA damagewith programmed cell death FAF1 antagonizes WNT signal-ing by promoting 120573-catenin degradation in the proteasome[21] a function that may be lost after the point mutationThe elevated DNA binding activity of the protooncogenictranscription factor E2F1 (see Figure 3) could be caused byits interaction with LEF-1 [20] consecutive to persistent

WNT signaling The RNA level of IGF2BP1 (IMP-1) a stress-responsive regulator of mRNA stability is highly upregulatedin the tumor compared to muscle as well as bone (seeTable 2(c)) IGF2BP1 is a transcriptional target of the WNTpathway that regulates NF-120581B activity (see Table 2(e)) andis antiapoptotic [22 23] IGF2BP1 may be upregulated asa consequence of mutated FAF1 not being able to suppressWNT signaling in this specific cancerOf noteWNTpathwayoveractivity may not be required for transformation ratherthe persistence of a WNT pathway signal due to the lack of aFAF1-mediated termination signal may suffice

14 Sarcoma

WNTFrizzled

Axin Dvl

igf2bp1

FAF1

P65 P50

IKK

FAS

120573-Catenin

LEF + E2F1

I120581B

Figure 5 Possible transforming pathway The point mutation inFAF1 is believed to cause a loss of function (crossed out in red)This may lead to an overactivity of the WNT signaling pathwayConsistently E2F1 (activated by LEF1) and igf2bp1 (a transcriptionaltarget of the WNT pathway) have been identified to be upregulatedin the molecular analysis In contrast to the negative regulator FAF1IGF2BP1 is a positive regulator of NF-120581B activityThe overactivity ofthe NF-120581B pathway and the reduced efficacy of FAS signaling can betransforming

The role ofWNT signaling in sarcoma has been subject todebate (eg [24]) In metastatic leiomyosarcoma 120573-Cateninmay accumulate in the nucleus despite a relatively weakexpression of WNT [25] This may be due to WNT signalactivation via noncanonical ligands [26] or to 120573-Cateninbinding the nuclear receptor NR4A2 and releasing it from thecorepressor protein LEF-1 [27] Of note in the cancer understudy here the mRNA level of NR4A2 was overexpressed10-fold compared to bone and 3-fold compared to muscleThe results from this study are consistent with the possibilitythat a lack of termination in the WNT signal rather than itsoveractivation could contribute to sarcomatous transforma-tion Such a mechanism may be reflected in an upregulationof downstream targets even though overexpression of WNTpathway components is not detectable

Other mutations beside FAF1 are less likely to becausative for the cancer EPHA3 was revealed as mutated inthis cancer by DNA exome sequencing and RNASeq EPHA3is a receptor tyrosine kinase that is frequentlymutated in lungcancer Tumor-suppressive effects of wild-type EPHA3 can beoverridden by dominant negative EPHA3 somatic mutations[28]Thismechanism is unlikely to play a role in this sarcomaas the detected mutation is located far N-terminally on theextracellular Ephrin binding domain not on the intracellularkinase domain

FAF1 has been described to act as a tumor suppressor gene[29] Its depletion due to chromosome breakage can affectprognosis in glioblastoma patients [30 31] Single nucleotidepolymorphisms in FAF1 are associated with a risk for gastriccancer [32] While numerous FAF1 mutations are associatedwith various cancers none of these genetic changes in theTCGA database affects the amino acid position 181 (Table 4)

The major upregulations identified in this tumor com-prise muscle-specific gene products (transcription factors

CAAC binding proteomics transgelin transgelin-2 RNAanoctamin-4 and immunohistochemistry smooth mus-cle actin) and calcium-regulating molecules (proteomicscalumenin S100-A11 reticulocalbin-3 and 78 kD glucose-regulated protein) The muscle-specific gene products con-firm this recurrent sarcoma as a leiomyosarcoma (the firsttumor distal to the site of the recurring one had beendiagnosed as an osteosarcoma) Calcium is one of the majorsecond messengers in smooth muscle cells Its uptake isregulated by potential-sensitive ion channels in the cellmembrane and by the activities of various receptors Calciumis stored in the sarcoplasmic reticulum from where it canbe released to facilitate actin-myosin interaction and tensiongeneration Phosphorylation of the myosin light chain by acalmodulin-regulated enzyme is important for contractionThe upregulation of gene products associated with migrationand invasion (osteopontin MMP1 vimentin filamin-A and120573-actin) and gene products for extracellularmatrixmoleculesand their modulators (fibronectin collagen ITGBL1 andMXRA5) reflects the invasive nature of this cancer

The recurrence of a sarcoma after 14 years has twoprobable explanations either it is due to a cancer pre-disposition syndrome based on a germ-line mutation (theage of the patient weakens this hypothesis) or the secondtumor is a metastatic colony of the first that was reactivatedafter dormancy The location of the sarcoma in the sameextremity and proximal to a preceding mesenchymal cancer(and therefore in its natural path of dissemination) impliedthe probability that this was a relapse in a metastatic siteThe different histologic assessment as osteosarcoma in thefirst occurrence and leiomyosarcoma as the second cancerdoes not necessarily negate that Mixed histology [33 34]and transdifferentiation [35ndash37] have been described forsarcomatous tumors Of note however in this scenarioosteosarcoma seems to more commonly follow leiomyosar-coma than precede it Material from the first cancer of thispatient was not accessible to us It is very plausible that thiscould have been a mineralized leiomyosarcoma In thosetumors the differential diagnosis from osteosarcoma can bedifficult [38] The extracompartmental location of the firsttumor supports this interpretation

On the molecular pathology level sarcomas fall intotwo groups comprising tumors with simple karyotypes(with pathogenetic translocations or specific genetic muta-tions) and tumors with very complex karyotypes (overtchromosome and genomic instability with numerous gainsand losses) [39] Some molecular alterations that lead tocarcinogenesis can be defined in absolute termsThey includegain-of-function mutations or chromosome translocationsthat transformprotooncogenes to oncogenes However otherchanges are relative to the normal tissue of origin suchas pathway overactivity or overexpression on the proteinor RNA levels We have combined the analysis of absolutechanges (DNA exome sequence RNA sequence) with theanalysis of relative changes using skeletal muscle and bone asreference organs (protein-DNA array 2D gel electrophoresisand RNA expression levels) This choice was determined inpart by tissue availability after surgery andwas intended to aidin the distinction of osteosarcoma from myosarcoma While

Sarcoma 15

Table 3 Deregulation of genes for drug disposition Analysis of RNASeq for at least twofold overexpression (italic font) or underexpression(bold font) of genes for (a) transport and (b) metabolism in tumor compared to muscle and bone For the export transporters (ABCtransporters) only overexpression is considered relevant for drug resistance Not shown in the table are the underexpressed genes (ABCA7ABCA8 ABCA13 ABCB6 ABCB10 ABCC6 ABCC6P1 ABCC6P2 ABCC8 ABCC11 ABCD2 ABCG2 and ABCG5)

(a) Transport

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

650655 ABCA17P ATP-binding cassette subfamily A (ABC1) member 17 pseudogene 1360 211624 ABCA4 ATP-binding cassette subfamily A (ABC1) member 4 2038 2802

6555 SLC10A2 Solute carrier family 10 (sodiumbile acid cotransporter family)member 2 minus1962 minus2112

345274 SLC10A6 Solute carrier family 10 (sodiumbile acid cotransporter family)member 6 2623 3240

6563 SLC14A1 Solute carrier family 14 (urea transporter) member 1 (Kidd bloodgroup) minus3074 minus3152

6565 SLC15A2 Solute carrier family 15 (H+peptide transporter) member 2 minus2027 minus2152

6566 SLC16A1 Solute carrier family 16 member 1 (monocarboxylic acid transporter1) 1401 2170

117247 SLC16A10 Solute carrier family 16 member 10 (aromatic amino acid transporter) 2113 2848

6567 SLC16A2 Solute carrier family 16 member 2 (monocarboxylic acid transporter8) 3242 3848

10786 SLC17A3 Solute carrier family 17 (sodium phosphate) member 3 minus2868 minus29596571 SLC18A2 Solute carrier family 18 (vesicular monoamine) member 2 minus2087 minus21946573 SLC19A1 Solute carrier family 19 (folate transporter) member 1 minus2099 minus220310560 SLC19A2 Solute carrier family 19 (thiamine transporter) member 2 1820 254880704 SLC19A3 Solute carrier family 19 member 3 minus3331 minus3478387775 SLC22A10 Solute carrier family 22 member 10 5711 60859390 SLC22A13 Solute carrier family 22 (organic anion transporter) member 13 2623 3217

85413 SLC22A16 Solute carrier family 22 (organic cationcarnitine transporter)member 16 minus8101 minus7299

51310 SLC22A17 Solute carrier family 22 member 17 1475 21755002 SLC22A18 Solute carrier family 22 member 18 2038 27226582 SLC22A2 Solute carrier family 22 (organic cation transporter) member 2 4793 5216

6581 SLC22A3 Solute carrier family 22 (extraneuronal monoamine transporter)member 3 2554 3182

6583 SLC22A4 Solute carrier family 22 (organic cationergothioneine transporter)member 4 minus3603 minus3776

151295 SLC23A3 Solute carrier family 23 (nucleobase transporters) member 3 2109 284810478 SLC25A17 Solute carrier family 25 (mitochondrial carrier) member 17 1311 207783733 SLC25A18 Solute carrier family 25 (mitochondrial carrier) member 18 1846 2570

788 SLC25A20 Solute carrier family 25 (carnitineacylcarnitine translocase) member20 minus2105 minus2207

89874 SLC25A21 Solute carrier family 25 (mitochondrial oxodicarboxylate carrier)member 21 minus2962 minus3105

51312 SLC25A37 Solute carrier family 25 member 37 minus4633 minus486651629 SLC25A39 Solute carrier family 25 member 39 minus2585 minus2737203427 SLC25A43 Solute carrier family 25 member 43 1325 208865012 SLC26A10 Solute carrier family 26 member 10 3896 4472115111 SLC26A7 Solute carrier family 26 member 7 3431 4018116369 SLC26A8 Solute carrier family 26 member 8 minus5354 minus5536115019 SLC26A9 Solute carrier family 26 member 9 1623 241911001 SLC27A2 Solute carrier family 27 (fatty acid transporter) member 2 minus4278 minus4487

16 Sarcoma

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

64078 SLC28A3 Solute carrier family 28 (sodium-coupled nucleoside transporter)member 3 minus4543 minus4812

222962 SLC29A4 Solute carrier family 29 (nucleoside transporters) member 4 minus1962 minus204581031 SLC2A10 Solute carrier family 2 (facilitated glucose transporter) member 10 2532 3170

6518 SLC2A5 Solute carrier family 2 (facilitated glucosefructose transporter)member 5 minus3647 minus3821

55532 SLC30A10 Solute carrier family 30 member 10 minus3547 minus37257782 SLC30A4 Solute carrier family 30 (zinc transporter) member 4 2832 33726569 SLC34A1 Solute carrier family 34 (sodium phosphate) member 1 minus4716 minus4941340146 SLC35D3 Solute carrier family 35 member D3 minus5662 minus590654733 SLC35F2 Solute carrier family 35 member F2 1433 2170206358 SLC36A1 Solute carrier family 36 (protonamino acid symporter) member 1 minus2265 minus2345285641 SLC36A3 Solute carrier family 36 (protonamino acid symporter) member 3 minus2284 minus239354020 SLC37A1 Solute carrier family 37 (glycerol-3-phosphate transporter) member 1 minus2112 minus22122542 SLC37A4 Solute carrier family 37 (glucose-6-phosphate transporter) member 4 minus2117 minus2219151258 SLC38A11 Solute carrier family 38 member 11 2410 308555089 SLC38A4 Solute carrier family 38 member 4 1837 254892745 SLC38A5 Solute carrier family 38 member 5 minus1994 minus215291252 SLC39A13 Solute carrier family 39 (zinc transporter) member 13 1301 207023516 SLC39A14 Solute carrier family 39 (zinc transporter) member 14 2569 318829985 SLC39A3 Solute carrier family 39 (zinc transporter) member 3 minus2032 minus2152283375 SLC39A5 Solute carrier family 39 (metal ion transporter) member 5 1623 23707922 SLC39A7 Solute carrier family 39 (zinc transporter) member 7 1273 205830061 SLC40A1 Solute carrier family 40 (iron-regulated transporter) member 1 minus3612 minus379684102 SLC41A2 Solute carrier family 41 member 2 1293 20708501 SLC43A1 Solute carrier family 43 member 1 minus2013 minus215257153 SLC44A2 Solute carrier family 44 member 2 minus2047 minus215250651 SLC45A1 Solute carrier family 45 member 1 2038 2722146802 SLC47A2 Solute carrier family 47 member 2 minus1962 minus20266521 SLC4A1 Solute carrier family 4 anion exchanger member 1 minus6513 minus645357282 SLC4A10 Solute carrier family 4 sodium bicarbonate transporter member 10 minus2640 minus275383959 SLC4A11 Solute carrier family 4 sodium borate transporter member 11 1846 25696508 SLC4A3 Solute carrier family 4 anion exchanger member 3 1261 20458671 SLC4A4 Solute carrier family 4 sodium bicarbonate cotransporter member 4 2445 31139497 SLC4A7 Solute carrier family 4 sodium bicarbonate cotransporter member 7 2717 33076523 SLC5A1 Solute carrier family 5 (sodiumglucose cotransporter) member 1 minus2547 minus2705159963 SLC5A12 Solute carrier family 5 (sodiumglucose cotransporter) member 12 4753 52066527 SLC5A4 Solute carrier family 5 (low affinity glucose cotransporter) member 4 minus3969 minus4249

6540 SLC6A13 Solute carrier family 6 (neurotransmitter transporter GABA)member 13 1328 2092

55117 SLC6A15 Solute carrier family 6 (neutral amino acid transporter) member 15 1623 2333388662 SLC6A17 Solute carrier family 6 member 17 2038 272854716 SLC6A20 Solute carrier family 6 (proline IMINO transporter) member 20 1697 2433

6532 SLC6A4 Solute carrier family 6 (neurotransmitter transporter serotonin)member 4 minus2512 minus2611

6534 SLC6A7 Solute carrier family 6 (neurotransmitter transporter L-proline)member 7 2623 3228

56301 SLC7A10 Solute carrier family 7 (neutral amino acid transporter y+ system)member 10 minus3421 minus3603

Sarcoma 17

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

6547 SLC8A3 Solute carrier family 8 (sodiumcalcium exchanger) member 3 minus2204 minus2309285335 SLC9A10 Solute carrier family 9 member 10 1208 20006549 SLC9A2 Solute carrier family 9 (sodiumhydrogen exchanger) member 2 2038 2706

9368 SLC9A3R1 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 1 minus2760 minus2846

9351 SLC9A3R2 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 2 1889 2619

84679 SLC9A7 Solute carrier family 9 (sodiumhydrogen exchanger) member 7 3347 393510599 SLCO1B1 Solute carrier organic anion transporter family member 1B1 3360 397728234 SLCO1B3 Solute carrier organic anion transporter family member 1B3 9760 95696578 SLCO2A1 Solute carrier organic anion transporter family member 2A1 2432 309628232 SLCO3A1 Solute carrier organic anion transporter family member 3A1 minus2032 minus2152353189 SLCO4C1 Solute carrier organic anion transporter family member 4C1 minus6850 minus6611

(b) Metabolism

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold Cchange

1583 CYP11A1 Cytochrome P450 family 11 subfamily A polypeptide 1 1623 23961589 CYP21A2 Cytochrome P450 family 21 subfamily A polypeptide 2 minus1962 minus20361591 CYP24A1 Cytochrome P450 family 24 subfamily A polypeptide 1 5208 55771592 CYP26A1 Cytochrome P450 family 26 subfamily A polypeptide 1 2623 32571594 CYP27B1 Cytochrome P450 family 27 subfamily B polypeptide 1 1846 2585339761 CYP27C1 Cytochrome P450 family 27 subfamily C polypeptide 1 3585 41781553 CYP2A13 Cytochrome P450 family 2 subfamily A polypeptide 13 minus1962 minus20351580 CYP4B1 Cytochrome P450 family 4 subfamily B polypeptide 1 minus4820 minus506566002 CYP4F12 Cytochrome P450 family 4 subfamily F polypeptide 12 minus6070 minus61958529 CYP4F2 Cytochrome P450 family 4 subfamily F polypeptide 2 minus7769 minus70604051 CYP4F3 Cytochrome P450 family 4 subfamily F polypeptide 3 minus8244 minus749111283 CYP4F8 Cytochrome P450 family 4 subfamily F polypeptide 8 minus5006 minus5270260293 CYP4X1 Cytochrome P450 family 4 subfamily X polypeptide 1 minus2476 minus25809420 CYP7B1 Cytochrome P450 family 7 subfamily B polypeptide 1 2502 31702326 FMO1 Flavin containing monooxygenase 1 4360 48592327 FMO2 Flavin containing monooxygenase 2 (nonfunctional) minus4074 minus43372328 FMO3 Flavin containing monooxygenase 3 minus4036 minus4309388714 FMO6P Flavin containing monooxygenase 6 pseudogene minus2569 minus2737493869 GPX8 Glutathione peroxidase 8 (putative) 2814 33712938 GSTA1 Glutathione S-transferase alpha 1 1623 23632939 GSTA2 Glutathione S-transferase alpha 2 2038 27412941 GSTA4 Glutathione S-transferase alpha 4 1492 21882953 GSTT2 Glutathione S-transferase theta 2 4682 5170653689 GSTT2B Glutathione S-transferase theta 2B (genepseudogene) 3739 430784779 NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439 79969027 NAT8 N-acetyltransferase 8 (GCN5-related putative) minus2769 minus29207358 UGDH UDP-glucose 6-dehydrogenase 2333 301855757 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 1604 227110720 UGT2B11 UDP glucuronosyltransferase 2 family polypeptide B11 minus3586 minus37687367 UGT2B17 UDP glucuronosyltransferase 2 family polypeptide B17 minus2836 minus295954490 UGT2B28 UDP glucuronosyltransferase 2 family polypeptide B28 minus3132 minus3284167127 UGT3A2 UDP glycosyltransferase 3 family polypeptide A2 minus4716 minus4907

18 Sarcoma

Table 4 FAF1 mutations in various cancers FAF1 mutations listed in the TCGA data base were identified without restriction to any type ofcancer For the location of the affected domains on the protein compare Figure 2

AA Mutation Cancer DomainN4S Missense Cutaneous melanomaI10S Missense Stomach adenocarcinoma UBAE21lowast Nonsense Cutaneous melanoma UBAE25K Missense Uterine endometrioid carcinoma UBAV38 splice Splice Stomach adenocarcinoma UBAP86fs FS del Colorectal adenocarcinomaG123 splice Splice Uterine endometrioid carcinoma UB1P136H Missense Colorectal adenocarcinoma UB1T147M Missense Brain lower grade glioma UB1D149Y Missense Uterine endometrioid carcinoma UB1L159V Missense Lung adenocarcinoma UB1K163N Missense Uterine endometrioid carcinoma UB1L170F Missense Cutaneous melanomaG184 splice Splice Colorectal cancerQ187H Missense Stomach adenocarcinomaS214N Missense Colorectal adenocarcinoma UB2R222I Missense Uterine endometrioid carcinoma UB2E238D Missense Lung adenocarcinoma UB2P241S Missense Uterine endometrioid carcinoma UB2T245A Missense Renal clear cell carcinoma UB2M249V Missense Uterine endometrioid carcinoma UB2E280K Missense Brain lower grade gliomaG293lowast Nonsense Colorectal cancerT300I Missense Colorectal adenocarcinomaD305H Missense Lung adenocarcinomaE308Q Missense Lung adenocarcinomaA316V Missense Stomach adenocarcinomaK319fs FS ins Head and neck squamous cell carcinomaR344G Missense Stomach adenocarcinoma UASF353I Missense Cutaneous melanoma UASL379V Missense Breast invasive carcinoma UASC396F Missense Cutaneous melanoma UASS459lowast Nonsense Uterine endometrioid carcinoma UASG469 splice Splice Glioblastoma multiforme UASR509G Missense Lung squamous cell carcinomaE510fs FS del Cutaneous melanomaR516C Missense Uterine endometrioid carcinomaA534V Missense Stomach adenocarcinomaF537L Missense Stomach adenocarcinomaE551lowast Nonsense Lung adenocarcinomaR554W Missense Colorectal cancerS582I Missense Lung adenocarcinoma UBXF585L Missense Uterine endometrioid carcinoma UBXE587lowast Nonsense Lung adenocarcinoma UBXA592V Missense Stomach adenocarcinoma UBXW610lowast Nonsense Breast invasive carcinoma UBXD611Y Missense Lung adenocarcinoma UBXE635fs FS del Brain lower grade glioma UBXP640fs FS del Cutaneous melanoma UBX

Sarcoma 19

a more accurate reference point for a leiomyosarcoma wouldhave been smooth muscle we believe that the comparison tostriated muscle is sufficient to allow the assessment of tumorspecific changes We have measured RNA DNA and proteinwith various assays Similar assessments in the future shouldalso include chromosome analysis for possible translocations

In the first-line defense against cancer the gradualreplacement of conventional chemotherapy with molecularlytargeted agents has opened the possibility to tailor drug treat-ments to particular tumors This transition necessitates thecharacterization of the molecular lesions that are causativefor the transformation of healthy cells into cancerous cellsbecause drugs need to be matched with the underlying carci-nogenic defect to be effective An additional caveat causedby unique genetic changes in the primary tumor can affectdrug transport and metabolism and needs to be taken intoconsideration In this case the RNA levels for genes that regu-late transport andmetabolismwere extensively skewed in thetumor tissue as compared to muscle and bone (see Table 3)implying potential challenges to chemotherapy An advancedmolecular treatment strategy for cancer will rely on themolecular definition of drug target drug transport and drugmetabolism pharmacogenetics in the primary tumor Con-secutive to cancer dissemination it will also require adjust-ments to account for the genetic changes in the metastases

The cost of health care has been under increasing scrutinyThe treatment of cancer patients is expensive in particularwhen hospitalization is required and further in cases ofend-of-life care Avoidable expenditures are generated bysuboptimal treatment decisions that result in low efficacy orhigh toxicity of anticancer regimens Molecular medicine hasthe potential to preempt those problems and reduce wastefulspending Yet it requires the upfront cost of molecular cancerexamination The analysis performed in this study requiredabout $11000- in nonsalary expenses to perform While ithas not identified a drug target it has specified possibleconfines for drug treatment The costs for molecular analysisneed to be weighed against the societal cost derived fromlost productivity in the workforce disrupted lives of familiesand premature deaths While currently limited drug optionsconstitute the major constraint to the approach taken herethe foreseeable future will bring an increasing spectrum ofmolecularly targeted drugs along with faster and cheapertechnologies for the molecular assessment of cancers Theywill facilitate the clinical translation of our approach

Consent

The patient provided informed consent

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by DOD Grant PR094070under University of Cincinnati IRB protocol 04-01-29-01The

author is grateful to Dr Rhett Kovall for helping with thestructural analysis of FAF1

References

[1] G F Weber Molecular Mechanisms of Cancer Springer Dor-drecht The Netherlands 2007

[2] N G Sanerkin ldquoPrimary leiomyosarcoma of the bone and itscomparison with fibrosarcomardquoCancer vol 44 no 4 pp 1375ndash1387 1979

[3] J M Weaver and J A Abraham ldquoLeiomyosarcoma of the Boneand Soft Tissue A Reviewrdquo httpsarcomahelporglearningcenterleiomyosarcomahtml

[4] M A Depristo E Banks R Poplin et al ldquoA framework forvariation discovery and genotyping using next-generationDNAsequencing datardquo Nature Genetics vol 43 no 5 pp 491ndash5012011

[5] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[6] C W Menges D A Altomare and J R Testa ldquoFAS-associatedfactor 1 (FAF1) diverse functions and implications for oncoge-nesisrdquo Cell Cycle vol 8 no 16 pp 2528ndash2534 2009

[7] M Kim J H Lee S Y Lee E Kim and J Chung ldquoCaspar asuppressor of antibacterial immunity in Drosophilardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 44 pp 16358ndash16363 2006

[8] J Song K P Joon J-J Lee et al ldquoStructure and interaction ofubiquitin-associated domain of human Fas-associated factor 1rdquoProtein Science vol 18 no 11 pp 2265ndash2276 2009

[9] P H OrsquoFarrell ldquoHigh resolution two dimensional electrophore-sis of proteinsrdquo Journal of Biological Chemistry vol 250 no 10pp 4007ndash4021 1975

[10] A Burgess-Cassler J J Johansen D A Santek J R Ideand N C Kendrick ldquoComputerized quantitative analysis ofCoomassie-Blue-stained serum proteins separated by two-dimensional electrophoresisrdquo Clinical Chemistry vol 35 no 12pp 2297ndash2304 1989

[11] B R Oakley D R Kirsch and N RMorris ldquoA simplified ultra-sensitive silver stain for detecting proteins in polyacrylamidegelsrdquo Analytical Biochemistry vol 105 no 2 pp 361ndash363 1980

[12] K Chu X Niu and L T Williams ldquoA Fas-associated proteinfactor FAF1 potentiates Fas-mediated apoptosisrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 25 pp 11894ndash11898 1995

[13] B Rikhof S de Jong A J H Suurmeijer C Meijer and W TA van der Graaf ldquoThe insulin-like growth factor system andsarcomasrdquoThe Journal of Pathology vol 217 no 4 pp 469ndash4822009

[14] RGirling J F Partridge L R Bandara et al ldquoAnew componentof the transcription factor DRTF1E2Frdquo Nature vol 362 no6415 pp 83ndash87 1993

[15] F Mercurio and M Karin ldquoTranscription factors AP-3 andAP-2 interact with the SV40 enhancer in a mutually exclusivemannerrdquo EMBO Journal vol 8 no 5 pp 1455ndash1460 1989

[16] R Bassel-Duby M D Hernandez M A Gonzalez J KKrueger and R S Williams ldquoA 40-kilodalton protein bindsspecifically to an upstream sequence element essential for mus-cle-specific transcription of the human myoglobin promoterrdquoMolecular and Cellular Biology vol 12 no 11 pp 5024ndash50321992

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 5: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

Sarcoma 5

Table1DNAexom

eSNPsTh

eDNAexom

esfortum

orm

uscle

and

bone

weres

equencedTh

eresultswerefi

lteredin

thefollowingorder(1)d

ifferentgenotypeintumor

from

muscle

and

bonew

ithmuscle

andbo

nebeingidentic

alto

each

other(2)d

eletem

utations

with

lowconfi

dence(

valueo

f20or

lower)inall3

tissues(3)

deleteun

identifi

edgenes(4)d

eletem

utations

thatareh

omozygou

sreference

inthetum

or(5)

deletemutations

thathave

aMAFin

dbSN

Pgt10(6)

deletelowim

pactandmod

ifier

mutationsTh

egenen

ames

arep

arto

fthe

keyin

the

leftcolumnIn

thiscolumnresults

onbo

ldfont

representSNPs

thatwerec

onfirmed

ontheR

NAlevelbyRN

ASeqTh

edatafi

lesh

aveb

eensubm

itted

totheN

CBIsho

rtread

archive(SR

A)

underthe

accessionnu

mberS

RP052797

(biosamples

SAMN03316820SAMN03316821SAMN03316822)

Key

Chromosom

ePo

sition

Reference

Alternate

dbSN

PID

dbSN

PMAF

ESPMAF

(All)

ESPMAF

(EA)

ESPMAF

(AA)

Con

sensus

impact

Bone

muscle

geno

type

Bone

overall

depth

Bone

allele

depths

Bone

quality

Muscle

overall

depth

Muscle

allele

depths

Muscle

quality

Tumor

geno

type

Tumor

overall

depth

Tumor

allele

depths

Tumor

quality

177479

998T

EIF4

A1

177479998

CT

mdash

mdashmdash

mdashMod

erate

00

175

1840

99103

1080

9901

133

7968

99389

2592

00T

EPHA3

389259200

CT

mdash

mdashmdash

mdashMod

erate

00

43450

99117

1230

9901

723048

9915120

4545

CFA

F11

51204545

TC

mdash

mdashmdash

mdashMod

erate

00

87910

99108

1130

9901

8566

27

9912

3083

4620

AIPO8

1230834620

CA

mdash

mdashmdash

mdashMod

erate

00

68712

99120

1260

9901

807018

9911

101834

425A

KIA

A1377

11101834425

GA

mdash

mdashmdash

mdashMod

erate

00

36371

9067

700

9901

863163

9944168

2102

CLIMCH

14

41682102

GC

mdash

000

0077

0000

0227

Mod

erate

00

71740

9996

1010

9901

153

12049

99536

985326

GNIPBL

536985326

AG

mdash

mdashmdash

mdashMod

erate

00

660

1835

360

8101

201012

99377623789

ARO

BO2

377623789

AGA

mdash

mdashmdash

mdashHigh

00

22NA

5429

NA

8701

37NA

99

2217300

095A

SMARC

AL1

2217300

095

ATTG

CATC

A-AC

GTC

GTG

GA

mdash

mdashmdash

mdashHigh

00

95NA

99122

NA

9901

99NA

99

2152236045TTA

ATN

FAIP6

2152236045

TATTA

Ars3506

0021

mdashmdash

mdashmdash

High

02

19NA

124

NA

5701

17NA

383520206

69TAC

Y13

520206

69G

T

mdashmdash

mdashmdash

Mod

erate

00

130

1360

9986

900

9901

804543

999117

130734

GAKN

A9

117130734

CG

mdash

mdashmdash

mdashMod

erate

00

27280

7830

310

9001

503024

9912

6030354A

ANO2

126030354

CA

mdash

mdashmdash

mdashMod

erate

00

101

1060

99114

1200

9901

135

10445

9918

10487667

AAPC

DD1

1810487667

GA

mdash

mdashmdash

mdashMod

erate

00

43450

9938

400

9901

503816

99734118

560C

BMPE

R7

34118

560

GC

mdash

mdashmdash

mdashMod

erate

00

69720

99101

1060

9901

716314

9915

24921273

TC1

5orf2

1524921273

GT

mdash

mdashmdash

mdashMod

erate

00

12120

3638

390

9901

26226

9919

54483249

CCA

CNG8

1954483249

TC

mdash

mdashmdash

mdashMod

erate

00

147

1540

99103

1080

9901

152

13632

9910

16893265

CCU

BN10

16893265

GC

mdash

mdashmdash

mdashMod

erate

00

57600

9990

940

9901

403410

997148489854C

CUL1

7148489854

AC

mdash

mdashmdash

mdashMod

erate

00

73760

9965

680

9901

574221

9917

41566894

CDHX8

1741566894

GC

mdash

mdashmdash

mdashMod

erate

00

106

1110

9988

920

9901

124

9442

9920

61512358

TDID

O1

2061512358

GT

rs73304513

00417

0045932

000

0838

0135456

Mod

erate

00

600

182

00

601

400

2116

23703526

AER

N2

1623703526

GA

mdash

mdashmdash

mdashMod

erate

00

81850

9998

1030

9901

916041

993197880164G

FAM157A

3197880164

GCA

GCA

GCA

AG

mdash

mdashmdash

mdashMod

erate

00

21NA

2525

NA

101

31NA

614

4564

4287

GFA

NCM

144564

4287

AG

000

09mdash

mdashmdash

Mod

erate

00

13130

3331

320

8701

12102

14189637524

CGBP

71

89637524

GC

mdash

mdashmdash

mdashMod

erate

00

171

1790

99201

2110

9901

206

1616

799

742003933

CGLI3

742003933

GC

mdash

mdashmdash

mdashMod

erate

00

87910

9981

850

9901

856132

9917

4837170TGP1BA

174837170

CT

mdash

mdashmdash

mdashMod

erate

00

16160

459

9015

01

11101

114

24635161

GIRF9

1424635161

TG

mdash

mdashmdash

mdashMod

erate

00

66690

9943

450

9901

362513

9915

42133096

AJM

JD7-

PLA2G

4B15

42133096

GA

mdash

mdashmdash

mdashMod

erate

00

111

1160

9975

780

9901

807414

92

1740271678

AKA

T2A

1740271678

GA

mdash

mdashmdash

mdashMod

erate

00

65680

9958

610

9901

563032

99873848894

GKC

NB2

873848894

AG

mdash

mdashmdash

mdashMod

erate

00

15150

3324

250

6601

301517

9919

50827053

TKC

NC3

1950827053

CT

mdash

mdashmdash

mdashMod

erate

00

220

2310

99136

1430

9901

160

13046

9917

21319069

AKC

NJ12

1721319069

GA

rs76265595

mdashmdash

mdashmdash

Mod

erate

00

23241

6343

434

4001

28255

6012

53011932

CKR

T73

1253011932

TC

mdash

mdashmdash

mdashMod

erate

00

146

1530

99157

1650

9901

159

11758

99X

7500

4584

AMAG

EE2

X7500

4584

CA

mdash

mdashmdash

mdashMod

erate

00

29300

8148

500

9901

382616

995140182157A

PCDHA3

5140182157

GA

mdash

mdashmdash

mdashMod

erate

00

180

1890

99153

1610

9901

150

13234

9915

42133096

APL

A2G

4B15

42133096

GA

mdash

mdashmdash

mdashMod

erate

00

111116

099

75780

9901

807414

9210

96005840

CPL

CE1

1096005840

TC

mdash

mdashmdash

mdashMod

erate

00

81851

9973

762

9901

875540

991166816805G

POGK

1166816805

CG

mdash

mdashmdash

mdashMod

erate

00

84880

9982

860

9901

634920

99

6 Sarcoma

Table1Con

tinued

Key

Chromosom

ePo

sition

Reference

Alternate

dbSN

PID

dbSN

PMAF

ESPMAF

(All)

ESPMAF

(EA)

ESPMAF

(AA)

Con

sensus

impact

Bone

muscle

geno

type

Bone

overall

depth

Bone

allele

depths

Bone

quality

Muscle

overall

depth

Muscle

allele

depths

Muscle

quality

Tumor

geno

type

Tumor

overall

depth

Tumor

allele

depths

Tumor

quality

12111

020739

TPP

TC7

12111

020739

TCGC

Trs71083132

mdashmdash

mdashmdash

Mod

erate

00

18NA

3014

NA

1801

19NA

1019

804293

APT

BP1

19804293

CA

mdash

mdashmdash

mdashMod

erate

00

120

1261

9971

741

9901

755330

9971564

51175C

RNF32

71564

51175

GC

mdash

mdashmdash

mdashMod

erate

00

55570

9967

700

9901

594223

993520206

69TRP

11-155D

1811

3520206

69G

T

mdashmdash

mdashmdash

Mod

erate

00

130

1360

9986

900

9901

804543

9921

43838624

TUBA

SH3A

2143838624

GT

mdash

mdashmdash

mdashMod

erate

00

70730

9945

470

9901

897621

9919

58773857

AZN

F544

1958773857

GA

mdash

mdashmdash

mdashMod

erate

00

46480

9943

450

9901

715126

99516465723

CZN

F622

516465723

TC

mdash

mdashmdash

mdashMod

erate

00

17170

3914

140

3301

271415

99

Sarcoma 7

Table 2 RNA induced in the tumor (a) Transcripts overexpressed in the tumor compared to muscle but not bone (b) Transcriptsoverexpressed in the tumor compared to bone but not muscle (c) Transcripts most abundantly overexpressed in the tumor compared tomuscle and bone (d) as (a) but limited to genes expressed at least at the level of 1 unit in the reference tissue (muscle or bone) (e) Alteredexpression of genes associated with NF-120581B activity Analysis of RNASeq for the transcription factor NF-120581B and gene products that regulate itsactivity RNAmessages that are selectively associated with the TNF pathway to NF-120581B activation are marked with bold font log2-fold changeindicates the alteration in the tumor compared to muscle or bone (the respective columns indicate the expression level for each organ)

(a)

log2-fold changeMuscle

Tumor over muscleNRG1 Neuregulin 1 9909KSR2 Kinase suppressor of ras 2 9675MMP13 Matrix metallopeptidase 13 (collagenase 3) 9528SPP1 Secreted phosphoprotein 1 9098INHBA Inhibin beta A 8570COL11A1 Collagen type XI alpha 1 8564MMP9 Matrix metallopeptidase 9 (gelatinase B 92 kda gelatinase 92 kda type IV collagenase) 8552FBN2 Fibrillin 2 8495LRRC15 Leucine rich repeat containing 15 8429MMP11 Matrix metallopeptidase 11 (stromelysin 3) 8336E2F7 E2F transcription factor 7 8314PRAME Preferentially expressed antigen in melanoma 8179PTK7 PTK7 protein tyrosine kinase 7 7996DSCAM Down syndrome cell adhesion molecule 7761RGS4 Regulator of G-protein signaling 4 7633CNIH3 Cornichon homolog 3 (Drosophila) 7632OR10V1 Olfactory receptor family 10 subfamily V member 1 7610CEP55 Centrosomal protein 55 kda 7583WNT5B Wingless-type MMTV integration site family member 5B 7569CA12 Carbonic anhydrase XII 7520

GALNT5 UDP-N-acetyl-alpha-D-galactosaminepolypeptide N-acetylgalactosaminyltransferase 5(GalNAc-T5) 7517

(b)

log2-fold changeBone

Tumor over boneROS1 c-ros oncogene 1 receptor tyrosine kinase 10987GREM1 Gremlin 1 10604NPTX1 Neuronal pentraxin I 8813GJB2 Gap junction protein beta 2 26 kDa 8597CREB3L1 cAMP responsive element binding protein 3-like 1 8506KRT14 Keratin 14 8351SERPINE1 Serpin peptidase inhibitor clade E (nexin plasminogen activator inhibitor type 1) member 1 8288HOXD10 Homeobox D10 8105ALPK2 Alpha-Kinase 2 7783POU3F2 POU class 3 homeobox 2 7530POSTN Periostin osteoblast specific factor 7497NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439STC2 Stanniocalcin 2 7434WNT5A Wingless-type MMTV integration site family member 5A 7412IGFN1 Immunoglobulin-like and fibronectin type III domain containing 1 7387

8 Sarcoma

(b) Continued

log2-fold changeBone

STC1 Stanniocalcin 1 7385FAM180A Family with sequence similarity 180 member A 7315KRT17 Keratin 17 7305BBOX1 Butyrobetaine (gamma) 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 7277MAGEA1 Melanoma antigen family A 1 (directs expression of antigen MZ2-E) 7267

(c)

log2-fold changeMuscle Bone

Tumor over bonemuscle

ANO4 Anoctamin 4 (TMEM16D) transmembrane calcium-activated chloride channelfacilitates smooth muscle contraction 9937 8542

SLCO1B3 (OATP1B3) Solute carrier organic anion transporter family member 1B3 9569 9760

MARCH4 Membrane-associated ring finger (C3HC4) 4 E3 ubiquitin ligase located predominantlyto the endoplasmic reticulum 9538 7406

IGF2BP1 Insulin-like growth factor 2 mRNA binding protein 1 binds to and stabilizes mRNA 9440 9631ADAMTS16 ADAMmetallopeptidase with thrombospondin type 1 motif 16 zinc-dependent protease 9260 8450SOX11 SRY (sex determining region Y)-box 11 important in brain development 9178 9954

HAPLN1 Hyaluronan and proteoglycan link protein 1 stabilizes aggregates of aggrecan andhyaluronan giving cartilage its tensile strength and elasticity 8951 8142

MUC15 Mucin 15 cell surface associated 8801 7992HOXB9 Homeobox B9 8773 7378MAGEC2 Melanoma antigen family C 2 8693 8884

ST6GALNAC5 ST6 (alpha-N-acetyl-neuraminyl-23-beta-galactosyl-13)-N-acetylgalactosaminidealpha-26-sialyltransferase 5 7848 8038

C11orf41 Chromosome 11 open reading frame 41 7781 8141MMP1 Matrix metallopeptidase 1 (interstitial collagenase) 7455 7646

(d)

Symbol Name log2-fold change log2-fold changeMuscle Bone

Tumor over bonemuscleFN1 Fibronectin 1 7328 6293 6457 19860COL1A1 Collagen type I alpha 1 6768 3706 5868 11934CCND1 Cyclin D1 5595 3958 5098 9637RGS1 Regulator of G-protein signaling 1 5118 1056 4653 2533ITGBL1 Integrin beta-like 1 (with EGF-like repeat domains) 5071 3177 3895 12415COL1A2 Collagen type I alpha 2 4852 8756 4910 14503MXRA5 Matrix-remodelling associated 5 4834 1352 4631 2685POSTN Periostin osteoblast specific factor 4513 5870 7497 1267PLOD2 Procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 4285 1801 4023 3730COL5A2 Collagen type V alpha 2 4275 1297 4833 1517COL3A1 Collagen type III alpha 1 4003 13118 5801 6500

SEMA3C Sema domain immunoglobulin domain (Ig) shortbasic domain secreted 3954 1164 4215 1675

FBN1 Fibrillin 1 3912 6957 4018 11148AEBP1 AE binding protein 1 3808 3212 4002 4843SIK1 Salt-inducible kinase 1 3805 1219 3566 2484

Sarcoma 9

(d) Continued

Symbol Name log2-fold change log2-fold changeMuscle Bone

SERPINH1 Serpin peptidase inhibitor clade H (heat shock protein47) member 1 3706 1652 4145 2098

ANTXR1 Anthrax toxin receptor 1 3670 3789 3690 6445

(e)

Symbol Name log2-fold change log2-fold changeMuscle Bone

HELLS Helicase lymphoid-specific 5315602 0155898 minus082713 202489

TNFRSF11A Tumor necrosis factor receptor superfamily member 11a andNFKB activator 3017922 003091 1400993 0202453

NFKBID Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor delta 2655352 0 minus147615 0504341

TNFRSF25 Tumor necrosis factor receptor superfamily member 25 2432959 0046388 0163954 0490795

NFKBIE Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor epsilon 2121015 0062395 0311441 043382

TNF Tumor necrosis factor 1847997 0 minus142101 003733

TNFRSF10D Tumor necrosis factor receptor superfamily member 10d decoywith truncated death domain 1754888 0172968 minus01757 1183343

TNFRSF1B Tumor necrosis factor receptor superfamily member 1B 1473438 0709902 minus097011 6729401TNFRSF10A Tumor necrosis factor receptor superfamily member 10a 1411898 0828219 0357701 3004386

NFKBIB Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor beta 1193993 1209816 046055 3484692

TNFRSF10B Tumor necrosis factor receptor superfamily member 10b 1094157 1908597 0425446 5242006TNFRSF21 Tumor necrosis factor receptor superfamily member 21 1055762 3882038 0745815 8305711TNFRSF1A Tumor necrosis factor receptor superfamily member 1A 0875167 3790938 minus011707 130344

NFKBIZ Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor zeta 0575974 3698951 0344657 7493136

RIPK1 Receptor (TNFRSF)-interacting serine-threonine kinase 1 0494467 311749 minus108854 161392NKRF NFKB repressing factor 0475722 2156087 minus086397 9434166NFKB1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 0432959 2054532 minus065865 7568586CHUK Conserved helix-loop-helix ubiquitous kinase 0176126 2961281 minus120204 1330333RELA V-rel reticuloendotheliosis viral oncogene homolog A (avian) 0013848 1263837 0287167 1803044

NFKB2 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 2(p49p100) minus008641 0312993 0485882 0360442

NKAPP1 NFKB activating protein pseudogene 1 minus010692 0825177 minus099449 2655392

TNFRSF10C Tumor necrosis factor receptor superfamily member 10c decoywithout an intracellular domain minus0152 0064676 minus556891 6321515

NKIRAS2 NFKB inhibitor interacting Ras-like 2 minus060768 1095135 minus088758 2299946

NFKBIL1 Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor-like 1 minus08719 0141933 minus008357 0140418

NKIRAS1 NFKB inhibitor interacting Ras-like 1 minus087428 6296399 1467735 2122983TANK TRAF family member-associated NFKB activator minus0983 6777124 minus151908 1695872NKAP NFKB activating protein minus135735 1422458 minus078243 1646287

NFKBIA Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor alpha minus185483 4032743 minus031802 2395275

NKAPL NFKB activating protein-like minus557347 5875743 minus140215 0534643

10 Sarcoma

FAS associating region

DED interacting region

UB12 DZM

UB2domain

UASdomain

UBXdomain

UBAdomain

S289 S291

Aurora ACK II

S181

AKT1ATMGSK3

AuroraPKG

PLK1RSK

STK4CHK1

650566481336260205169110475

(a)

MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRM

6

1

101

100

200

300

400

500

600

650

201

301

401

501

601

667855899998876467765678888887578758999986467 88 8888888888888988888899988888889888877767888777888757

2222454345655744642554435764462443558468643653433344333424435334233333332333332333354335333344334354

LDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITH

9999997797689998787758999999999859996577754788888888875333467888868987688888888888877888855675799987

9585847535375857543447459666955563844544585374435443365845846434457567533353232334334444433337484944

REVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDAT

5888757887577654788887766654677776555468877888765455546887766555688888877777777777776556788888767766

5344456484742545656645594364384534437445444342323455453535435463434323333343434333333334333335433333

EFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESI

5666776654456777888888888888866889999999998848888876677778999999998776686899998589875579999987486899

4635343574354322343434533333343548479747945564424647635575478569766456468999999754233454687457844669

VSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDE

9999888589997557877545444345677876335554467888876899986799876447777677888999999998877555778887777778

6778547999996674433343444333333335466456434433487999895353333464346434365468755846444545454565446565

REARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVF

8888888887788877766545557777777666777777777776678877777766578888888888885799998589975788758987589999

4545445564445344433443354555556653665456544454434444444443344334333333446859596743354745795535796898

DFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKE

99997799988779985788754577888765665678898589998589

67974525444795877564635844444476875837446989897366

(b)

Figure 2 FAF1 structure (a) A schematic of functional domains in FAF1 with annotations (adapted from [6ndash8]) There are two ubiquitin-like domains flanking the S181 site The PDB structure 2DZM identifies the N-terminal one while the C-terminal prediction is by sequencesimilarity Whereas the mutation S181G is not expected to disrupt the protein structure per se this amino acid has a high score as a possiblephosphorylation site for a number of kinases involved inDNAdamage repair (b) Secondary structure prediction of FAF1The residues aroundS181 appear unstructured

reflected in STAT3 constitutive phosphorylation The lack ofphosphorylation in this case suggests that the leiomyosar-coma may not depend on the STAT3 pathway

36 Pharmacogenetic Evaluation Predicting the sensitivityto anticancer drugs is a main goal of molecular analysisFor this the over- or underexpression of genes for drugtransport and metabolism is of key importance Analysis ofthe RNASeq data for these groups of gene products identified

a surprisingly large number of deregulations compared tomuscle or bone (Tables 3(a) and 3(b)) Those alterationsmay affect choices for drug treatment For example the highlevels of glutathione S-transferase may render carmustinethioTEPA cisplatin chlorambucil melphalan nitrogenmus-tard phosphoramide mustard acrolein or steroids ineffec-tive The overexpression ofN-acetyltransferase may compro-mise 5-fluorouracil or taxolThemodest upregulation of onlytwo export transporters (ABC-transporters) and specifically

Sarcoma 11

Bone Tumor

Muscle

(a)

Transcription factor Description Reference

E2F1 Transcriptional activation of cell cycle progression is selectively activated in response to specific cues key downstream target of RB1 can promote proliferation or apoptosis when activated [14]

AP-33 interact in a mutually exclusive manner [15]

CCAC CCAC box is an essential element for muscle-specific transcription from the myoglobin promoter [16]

LIII-BP The L-III transcriptional regulatory element (a carbohydrate-response element) is in the pyruvate kinase L gene necessary for cell type-specific expression and transcriptional stimulation by carbohydrates [17]

ADD-1 (SREBP-1) activates transcription from sterol-regulated elements and from an E-box sequence present in the promoter of fatty acid synthase [18]

PAX6 Member of the paired box gene family transcriptional regulator involved in developmental processes [19]

48kD transcription factor activator protein-3 binds to the core element and recognizes the SV40 enhancer and AP-2 and AP-

(b)

Figure 3 Transcription factor activation Nuclear extracts from tumor muscle and bone were tested for DNA binding activity on a protein-DNA array (a) Transcription factor binding that was high in all 3 tissues and is therefore considered constitutively active is circled in yellow(left to right top to bottom AhRAmt GATA1 GATA2 GATA12 HIF1 and HOXD8910) Transcription factors that show high binding inthe cancer but not in muscle or bone are circled in red (left to right top to bottom E2F1 AP3 LIII-BP PAX6 ADD-1 and CCAC) (b) Thefunctions of transcription factors that display high DNA binding in the cancer but not in muscle or bone are described

the lack of ABCB1 overexpression is favorable for avoidingdrug resistance

37 Population Analysis The above-described results indi-cated that a FAF1 mutation which replaces serine in position181 thus preventing FAF1 phosphorylation and activationmay be a driver for leiomyosarcomagenesis A custom Taq-Man assay confirmed the presence of the somatic mutationin the patient To assess whether this single nucleotidereplacement is common in this type of cancer we analyzedDNA from 29 leiomyosarcomas and 1 blood sample from aleiomyosarcoma patient For comparison to nonsarcomatoustumors 34 breast cancers served as a reference None of themdisplayed amutation in the same locus By contrast there wasa distribution across all leiomyosarcomas in the osteopontin

promoter position minus443 (used as a reference) with 12 CC 10TC and 9 TT

4 Discussion

TheFAF1mutation identified as the likely cause for the cancerunder study gives room for an explanation of the sarcomatoustransformation (Figure 5) DNA damage to mesenchymalcells occurs persistently in an oxidizing environment at 37∘CThese insults are rarely transforming and such an occurrencewould trigger the initiation of programmed cell death inapoptosis-competent cells Intact FAF1 associates with FASand enhances apoptosis mediated through this receptor [12]A loss of function in FAF1 could lead to transformation viaantiapoptosis Whereas the mutation S181G is not expected

12 Sarcoma

Muscle

Bone

Tumor

220

94

6043

29

14

220

94

60

43

29

14

220

94

60

43

29

14

1

7

6

23

24

21

22

25

26

89 1011 12

1516 1719

pH 4 8 pH 4 8

8pH 4

(a)

SwissProt or NCBISpot number Protein identified accession Description

(Structural)6 Transgelin Q01995 Smooth muscle-specific cross-links actin24 Transgelin-2 P37802 Smooth muscle-specific cross-links actin

Vimentin P08670 Intermediate filament in mesenchymal tissue1 Filamin-A P21333 Actin-binding protein regulates the cytoskeleton 21 P06709 Cytoskeleton

(Calcium homeostasis)8 9 Calumenin O43852 Calcium-binding protein in the ER and golgi apparatus26 Protein S100-A11 P31949 Calcium-binding EF hand protein10 Reticulocalbin-3 Q96D15 Calcium-binding protein located in the ER12 P11021 Heat shock 70-kD protein 5 ER lumenal calcium-binding

(Protein processing)25 40S ribosomal protein S12 P25398 Core component of the ribosomal accuracy center7 Glycine-tRNA ligase P41250 Catalyzes the attachment of glycine to tRNA19 P01009 Protease inhibitor23 P01009 Protease inhibitor21 Proteasome activator complex subunit 2 Q9UL46 Proteasome activation10 P07900 Chaperone

(Other)7 Serum albumin P02768 Carrier protein in blood22 Protein disulfide isomerase (C-terminal fragment) P07237 Prolyl hydroxylation in collagen microsomal triglyceride transfer

120573 actin (C-terminal fragment)

78kD glucose-regulated protein

120572-1-antitrypsin120572-1-antitrypsin (C-terminal fragment)

Heat shock protein HSP 90120572 (N-terminal fragment)

11 15ndash17

(b)

Figure 4 Continued

Sarcoma 13

Tumor Muscle

OPN

CD44

STAT3

P-STAT3

Tubulin

75

75

50

15010075

10075

50

(c)

Tumor bone Tumor muscleGene ID Symbol Name Fold change Expression Fold change Expression6876 TAGLN Transgelin 3274 2455 3087 15088407 TAGLN2 Transgelin-2 2164 7338 6975 13037431 VIM Vimentin 1653 295010 4061 696042316 FLNA Filamin A alpha 1304 7791 9005 065160 ACTB Actin beta 0656 973187 5491 67368813 CALU Calumenin 7601 19328 12063 70596282 S100A11 S100 calcium binding protein A11 0931 158914 5071 1686357333 RCN3 Reticulocalbin 3 EF-hand calcium binding domain 2439 0604 6412 01223309 HSPA5 1068 92754 2607 220356696 SPP1 Secreted phosphoprotein 1 7572 46508 547929 0355960 CD44 CD44 molecule (Indian blood group) 2053 26708 15436 20566774 STAT3 Signal transducer and activator of transcription 3 0617 46534 0832 200165925 RB1 Retinoblastoma 1 0736 14772 0442 14268

Heat shock 70kDa protein 5 (glucose-regulated protein 78kDa)

(d)

Figure 4 Protein overexpression (a) 2D protein gel electrophoresis of lysates from muscle (upper left) tumor (upper right) and bone(bottom) in RIPA buffer Red circles indicate the spots that were identified as overexpressed and were further analyzed by mass spectrometry(b) Proteins identified in 2D gel electrophoresis as overexpressed in the tumor in comparison to muscle and bone were analyzed for theiridentity by mass spectrometry The left column indicates the spot number corresponding to the 2D gel The next column contains theprotein name followed by the accession number and a description of the protein functionThe protein functions are grouped into structuralcalcium homeostasis and various others (c) Western blot 10 120583g lysates of tumor muscle and bone in RIPA buffer were loaded per laneand electrophoresed on 10 SDS-polyacrylamide minigels with reducing denaturing sample buffer After transfer to PVDF membranesthey were probed for markers of cancer progression including osteopontin CD44 STAT3 and phospho-STAT3 Antitubulin served as aloading control (d) RNA levels corresponding to the proteins found to be affected in the sarcoma With four exceptions in the tumor-bonecomparison (gray font) upregulated proteins are associated with increased RNA levels STAT3 is not increased on the protein or RNA levelThe reduced level of RB1 expression is consistent with the elevated DNA binding activity of E2F1

to disrupt the structure of the protein this site does scorehigh as a possible phosphorylation site for a number ofkinases involved in DNA damage repair supporting thehypothesis that the cancer cells containing thismutation havelost their ability to respond to transforming DNA damagewith programmed cell death FAF1 antagonizes WNT signal-ing by promoting 120573-catenin degradation in the proteasome[21] a function that may be lost after the point mutationThe elevated DNA binding activity of the protooncogenictranscription factor E2F1 (see Figure 3) could be caused byits interaction with LEF-1 [20] consecutive to persistent

WNT signaling The RNA level of IGF2BP1 (IMP-1) a stress-responsive regulator of mRNA stability is highly upregulatedin the tumor compared to muscle as well as bone (seeTable 2(c)) IGF2BP1 is a transcriptional target of the WNTpathway that regulates NF-120581B activity (see Table 2(e)) andis antiapoptotic [22 23] IGF2BP1 may be upregulated asa consequence of mutated FAF1 not being able to suppressWNT signaling in this specific cancerOf noteWNTpathwayoveractivity may not be required for transformation ratherthe persistence of a WNT pathway signal due to the lack of aFAF1-mediated termination signal may suffice

14 Sarcoma

WNTFrizzled

Axin Dvl

igf2bp1

FAF1

P65 P50

IKK

FAS

120573-Catenin

LEF + E2F1

I120581B

Figure 5 Possible transforming pathway The point mutation inFAF1 is believed to cause a loss of function (crossed out in red)This may lead to an overactivity of the WNT signaling pathwayConsistently E2F1 (activated by LEF1) and igf2bp1 (a transcriptionaltarget of the WNT pathway) have been identified to be upregulatedin the molecular analysis In contrast to the negative regulator FAF1IGF2BP1 is a positive regulator of NF-120581B activityThe overactivity ofthe NF-120581B pathway and the reduced efficacy of FAS signaling can betransforming

The role ofWNT signaling in sarcoma has been subject todebate (eg [24]) In metastatic leiomyosarcoma 120573-Cateninmay accumulate in the nucleus despite a relatively weakexpression of WNT [25] This may be due to WNT signalactivation via noncanonical ligands [26] or to 120573-Cateninbinding the nuclear receptor NR4A2 and releasing it from thecorepressor protein LEF-1 [27] Of note in the cancer understudy here the mRNA level of NR4A2 was overexpressed10-fold compared to bone and 3-fold compared to muscleThe results from this study are consistent with the possibilitythat a lack of termination in the WNT signal rather than itsoveractivation could contribute to sarcomatous transforma-tion Such a mechanism may be reflected in an upregulationof downstream targets even though overexpression of WNTpathway components is not detectable

Other mutations beside FAF1 are less likely to becausative for the cancer EPHA3 was revealed as mutated inthis cancer by DNA exome sequencing and RNASeq EPHA3is a receptor tyrosine kinase that is frequentlymutated in lungcancer Tumor-suppressive effects of wild-type EPHA3 can beoverridden by dominant negative EPHA3 somatic mutations[28]Thismechanism is unlikely to play a role in this sarcomaas the detected mutation is located far N-terminally on theextracellular Ephrin binding domain not on the intracellularkinase domain

FAF1 has been described to act as a tumor suppressor gene[29] Its depletion due to chromosome breakage can affectprognosis in glioblastoma patients [30 31] Single nucleotidepolymorphisms in FAF1 are associated with a risk for gastriccancer [32] While numerous FAF1 mutations are associatedwith various cancers none of these genetic changes in theTCGA database affects the amino acid position 181 (Table 4)

The major upregulations identified in this tumor com-prise muscle-specific gene products (transcription factors

CAAC binding proteomics transgelin transgelin-2 RNAanoctamin-4 and immunohistochemistry smooth mus-cle actin) and calcium-regulating molecules (proteomicscalumenin S100-A11 reticulocalbin-3 and 78 kD glucose-regulated protein) The muscle-specific gene products con-firm this recurrent sarcoma as a leiomyosarcoma (the firsttumor distal to the site of the recurring one had beendiagnosed as an osteosarcoma) Calcium is one of the majorsecond messengers in smooth muscle cells Its uptake isregulated by potential-sensitive ion channels in the cellmembrane and by the activities of various receptors Calciumis stored in the sarcoplasmic reticulum from where it canbe released to facilitate actin-myosin interaction and tensiongeneration Phosphorylation of the myosin light chain by acalmodulin-regulated enzyme is important for contractionThe upregulation of gene products associated with migrationand invasion (osteopontin MMP1 vimentin filamin-A and120573-actin) and gene products for extracellularmatrixmoleculesand their modulators (fibronectin collagen ITGBL1 andMXRA5) reflects the invasive nature of this cancer

The recurrence of a sarcoma after 14 years has twoprobable explanations either it is due to a cancer pre-disposition syndrome based on a germ-line mutation (theage of the patient weakens this hypothesis) or the secondtumor is a metastatic colony of the first that was reactivatedafter dormancy The location of the sarcoma in the sameextremity and proximal to a preceding mesenchymal cancer(and therefore in its natural path of dissemination) impliedthe probability that this was a relapse in a metastatic siteThe different histologic assessment as osteosarcoma in thefirst occurrence and leiomyosarcoma as the second cancerdoes not necessarily negate that Mixed histology [33 34]and transdifferentiation [35ndash37] have been described forsarcomatous tumors Of note however in this scenarioosteosarcoma seems to more commonly follow leiomyosar-coma than precede it Material from the first cancer of thispatient was not accessible to us It is very plausible that thiscould have been a mineralized leiomyosarcoma In thosetumors the differential diagnosis from osteosarcoma can bedifficult [38] The extracompartmental location of the firsttumor supports this interpretation

On the molecular pathology level sarcomas fall intotwo groups comprising tumors with simple karyotypes(with pathogenetic translocations or specific genetic muta-tions) and tumors with very complex karyotypes (overtchromosome and genomic instability with numerous gainsand losses) [39] Some molecular alterations that lead tocarcinogenesis can be defined in absolute termsThey includegain-of-function mutations or chromosome translocationsthat transformprotooncogenes to oncogenes However otherchanges are relative to the normal tissue of origin suchas pathway overactivity or overexpression on the proteinor RNA levels We have combined the analysis of absolutechanges (DNA exome sequence RNA sequence) with theanalysis of relative changes using skeletal muscle and bone asreference organs (protein-DNA array 2D gel electrophoresisand RNA expression levels) This choice was determined inpart by tissue availability after surgery andwas intended to aidin the distinction of osteosarcoma from myosarcoma While

Sarcoma 15

Table 3 Deregulation of genes for drug disposition Analysis of RNASeq for at least twofold overexpression (italic font) or underexpression(bold font) of genes for (a) transport and (b) metabolism in tumor compared to muscle and bone For the export transporters (ABCtransporters) only overexpression is considered relevant for drug resistance Not shown in the table are the underexpressed genes (ABCA7ABCA8 ABCA13 ABCB6 ABCB10 ABCC6 ABCC6P1 ABCC6P2 ABCC8 ABCC11 ABCD2 ABCG2 and ABCG5)

(a) Transport

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

650655 ABCA17P ATP-binding cassette subfamily A (ABC1) member 17 pseudogene 1360 211624 ABCA4 ATP-binding cassette subfamily A (ABC1) member 4 2038 2802

6555 SLC10A2 Solute carrier family 10 (sodiumbile acid cotransporter family)member 2 minus1962 minus2112

345274 SLC10A6 Solute carrier family 10 (sodiumbile acid cotransporter family)member 6 2623 3240

6563 SLC14A1 Solute carrier family 14 (urea transporter) member 1 (Kidd bloodgroup) minus3074 minus3152

6565 SLC15A2 Solute carrier family 15 (H+peptide transporter) member 2 minus2027 minus2152

6566 SLC16A1 Solute carrier family 16 member 1 (monocarboxylic acid transporter1) 1401 2170

117247 SLC16A10 Solute carrier family 16 member 10 (aromatic amino acid transporter) 2113 2848

6567 SLC16A2 Solute carrier family 16 member 2 (monocarboxylic acid transporter8) 3242 3848

10786 SLC17A3 Solute carrier family 17 (sodium phosphate) member 3 minus2868 minus29596571 SLC18A2 Solute carrier family 18 (vesicular monoamine) member 2 minus2087 minus21946573 SLC19A1 Solute carrier family 19 (folate transporter) member 1 minus2099 minus220310560 SLC19A2 Solute carrier family 19 (thiamine transporter) member 2 1820 254880704 SLC19A3 Solute carrier family 19 member 3 minus3331 minus3478387775 SLC22A10 Solute carrier family 22 member 10 5711 60859390 SLC22A13 Solute carrier family 22 (organic anion transporter) member 13 2623 3217

85413 SLC22A16 Solute carrier family 22 (organic cationcarnitine transporter)member 16 minus8101 minus7299

51310 SLC22A17 Solute carrier family 22 member 17 1475 21755002 SLC22A18 Solute carrier family 22 member 18 2038 27226582 SLC22A2 Solute carrier family 22 (organic cation transporter) member 2 4793 5216

6581 SLC22A3 Solute carrier family 22 (extraneuronal monoamine transporter)member 3 2554 3182

6583 SLC22A4 Solute carrier family 22 (organic cationergothioneine transporter)member 4 minus3603 minus3776

151295 SLC23A3 Solute carrier family 23 (nucleobase transporters) member 3 2109 284810478 SLC25A17 Solute carrier family 25 (mitochondrial carrier) member 17 1311 207783733 SLC25A18 Solute carrier family 25 (mitochondrial carrier) member 18 1846 2570

788 SLC25A20 Solute carrier family 25 (carnitineacylcarnitine translocase) member20 minus2105 minus2207

89874 SLC25A21 Solute carrier family 25 (mitochondrial oxodicarboxylate carrier)member 21 minus2962 minus3105

51312 SLC25A37 Solute carrier family 25 member 37 minus4633 minus486651629 SLC25A39 Solute carrier family 25 member 39 minus2585 minus2737203427 SLC25A43 Solute carrier family 25 member 43 1325 208865012 SLC26A10 Solute carrier family 26 member 10 3896 4472115111 SLC26A7 Solute carrier family 26 member 7 3431 4018116369 SLC26A8 Solute carrier family 26 member 8 minus5354 minus5536115019 SLC26A9 Solute carrier family 26 member 9 1623 241911001 SLC27A2 Solute carrier family 27 (fatty acid transporter) member 2 minus4278 minus4487

16 Sarcoma

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

64078 SLC28A3 Solute carrier family 28 (sodium-coupled nucleoside transporter)member 3 minus4543 minus4812

222962 SLC29A4 Solute carrier family 29 (nucleoside transporters) member 4 minus1962 minus204581031 SLC2A10 Solute carrier family 2 (facilitated glucose transporter) member 10 2532 3170

6518 SLC2A5 Solute carrier family 2 (facilitated glucosefructose transporter)member 5 minus3647 minus3821

55532 SLC30A10 Solute carrier family 30 member 10 minus3547 minus37257782 SLC30A4 Solute carrier family 30 (zinc transporter) member 4 2832 33726569 SLC34A1 Solute carrier family 34 (sodium phosphate) member 1 minus4716 minus4941340146 SLC35D3 Solute carrier family 35 member D3 minus5662 minus590654733 SLC35F2 Solute carrier family 35 member F2 1433 2170206358 SLC36A1 Solute carrier family 36 (protonamino acid symporter) member 1 minus2265 minus2345285641 SLC36A3 Solute carrier family 36 (protonamino acid symporter) member 3 minus2284 minus239354020 SLC37A1 Solute carrier family 37 (glycerol-3-phosphate transporter) member 1 minus2112 minus22122542 SLC37A4 Solute carrier family 37 (glucose-6-phosphate transporter) member 4 minus2117 minus2219151258 SLC38A11 Solute carrier family 38 member 11 2410 308555089 SLC38A4 Solute carrier family 38 member 4 1837 254892745 SLC38A5 Solute carrier family 38 member 5 minus1994 minus215291252 SLC39A13 Solute carrier family 39 (zinc transporter) member 13 1301 207023516 SLC39A14 Solute carrier family 39 (zinc transporter) member 14 2569 318829985 SLC39A3 Solute carrier family 39 (zinc transporter) member 3 minus2032 minus2152283375 SLC39A5 Solute carrier family 39 (metal ion transporter) member 5 1623 23707922 SLC39A7 Solute carrier family 39 (zinc transporter) member 7 1273 205830061 SLC40A1 Solute carrier family 40 (iron-regulated transporter) member 1 minus3612 minus379684102 SLC41A2 Solute carrier family 41 member 2 1293 20708501 SLC43A1 Solute carrier family 43 member 1 minus2013 minus215257153 SLC44A2 Solute carrier family 44 member 2 minus2047 minus215250651 SLC45A1 Solute carrier family 45 member 1 2038 2722146802 SLC47A2 Solute carrier family 47 member 2 minus1962 minus20266521 SLC4A1 Solute carrier family 4 anion exchanger member 1 minus6513 minus645357282 SLC4A10 Solute carrier family 4 sodium bicarbonate transporter member 10 minus2640 minus275383959 SLC4A11 Solute carrier family 4 sodium borate transporter member 11 1846 25696508 SLC4A3 Solute carrier family 4 anion exchanger member 3 1261 20458671 SLC4A4 Solute carrier family 4 sodium bicarbonate cotransporter member 4 2445 31139497 SLC4A7 Solute carrier family 4 sodium bicarbonate cotransporter member 7 2717 33076523 SLC5A1 Solute carrier family 5 (sodiumglucose cotransporter) member 1 minus2547 minus2705159963 SLC5A12 Solute carrier family 5 (sodiumglucose cotransporter) member 12 4753 52066527 SLC5A4 Solute carrier family 5 (low affinity glucose cotransporter) member 4 minus3969 minus4249

6540 SLC6A13 Solute carrier family 6 (neurotransmitter transporter GABA)member 13 1328 2092

55117 SLC6A15 Solute carrier family 6 (neutral amino acid transporter) member 15 1623 2333388662 SLC6A17 Solute carrier family 6 member 17 2038 272854716 SLC6A20 Solute carrier family 6 (proline IMINO transporter) member 20 1697 2433

6532 SLC6A4 Solute carrier family 6 (neurotransmitter transporter serotonin)member 4 minus2512 minus2611

6534 SLC6A7 Solute carrier family 6 (neurotransmitter transporter L-proline)member 7 2623 3228

56301 SLC7A10 Solute carrier family 7 (neutral amino acid transporter y+ system)member 10 minus3421 minus3603

Sarcoma 17

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

6547 SLC8A3 Solute carrier family 8 (sodiumcalcium exchanger) member 3 minus2204 minus2309285335 SLC9A10 Solute carrier family 9 member 10 1208 20006549 SLC9A2 Solute carrier family 9 (sodiumhydrogen exchanger) member 2 2038 2706

9368 SLC9A3R1 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 1 minus2760 minus2846

9351 SLC9A3R2 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 2 1889 2619

84679 SLC9A7 Solute carrier family 9 (sodiumhydrogen exchanger) member 7 3347 393510599 SLCO1B1 Solute carrier organic anion transporter family member 1B1 3360 397728234 SLCO1B3 Solute carrier organic anion transporter family member 1B3 9760 95696578 SLCO2A1 Solute carrier organic anion transporter family member 2A1 2432 309628232 SLCO3A1 Solute carrier organic anion transporter family member 3A1 minus2032 minus2152353189 SLCO4C1 Solute carrier organic anion transporter family member 4C1 minus6850 minus6611

(b) Metabolism

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold Cchange

1583 CYP11A1 Cytochrome P450 family 11 subfamily A polypeptide 1 1623 23961589 CYP21A2 Cytochrome P450 family 21 subfamily A polypeptide 2 minus1962 minus20361591 CYP24A1 Cytochrome P450 family 24 subfamily A polypeptide 1 5208 55771592 CYP26A1 Cytochrome P450 family 26 subfamily A polypeptide 1 2623 32571594 CYP27B1 Cytochrome P450 family 27 subfamily B polypeptide 1 1846 2585339761 CYP27C1 Cytochrome P450 family 27 subfamily C polypeptide 1 3585 41781553 CYP2A13 Cytochrome P450 family 2 subfamily A polypeptide 13 minus1962 minus20351580 CYP4B1 Cytochrome P450 family 4 subfamily B polypeptide 1 minus4820 minus506566002 CYP4F12 Cytochrome P450 family 4 subfamily F polypeptide 12 minus6070 minus61958529 CYP4F2 Cytochrome P450 family 4 subfamily F polypeptide 2 minus7769 minus70604051 CYP4F3 Cytochrome P450 family 4 subfamily F polypeptide 3 minus8244 minus749111283 CYP4F8 Cytochrome P450 family 4 subfamily F polypeptide 8 minus5006 minus5270260293 CYP4X1 Cytochrome P450 family 4 subfamily X polypeptide 1 minus2476 minus25809420 CYP7B1 Cytochrome P450 family 7 subfamily B polypeptide 1 2502 31702326 FMO1 Flavin containing monooxygenase 1 4360 48592327 FMO2 Flavin containing monooxygenase 2 (nonfunctional) minus4074 minus43372328 FMO3 Flavin containing monooxygenase 3 minus4036 minus4309388714 FMO6P Flavin containing monooxygenase 6 pseudogene minus2569 minus2737493869 GPX8 Glutathione peroxidase 8 (putative) 2814 33712938 GSTA1 Glutathione S-transferase alpha 1 1623 23632939 GSTA2 Glutathione S-transferase alpha 2 2038 27412941 GSTA4 Glutathione S-transferase alpha 4 1492 21882953 GSTT2 Glutathione S-transferase theta 2 4682 5170653689 GSTT2B Glutathione S-transferase theta 2B (genepseudogene) 3739 430784779 NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439 79969027 NAT8 N-acetyltransferase 8 (GCN5-related putative) minus2769 minus29207358 UGDH UDP-glucose 6-dehydrogenase 2333 301855757 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 1604 227110720 UGT2B11 UDP glucuronosyltransferase 2 family polypeptide B11 minus3586 minus37687367 UGT2B17 UDP glucuronosyltransferase 2 family polypeptide B17 minus2836 minus295954490 UGT2B28 UDP glucuronosyltransferase 2 family polypeptide B28 minus3132 minus3284167127 UGT3A2 UDP glycosyltransferase 3 family polypeptide A2 minus4716 minus4907

18 Sarcoma

Table 4 FAF1 mutations in various cancers FAF1 mutations listed in the TCGA data base were identified without restriction to any type ofcancer For the location of the affected domains on the protein compare Figure 2

AA Mutation Cancer DomainN4S Missense Cutaneous melanomaI10S Missense Stomach adenocarcinoma UBAE21lowast Nonsense Cutaneous melanoma UBAE25K Missense Uterine endometrioid carcinoma UBAV38 splice Splice Stomach adenocarcinoma UBAP86fs FS del Colorectal adenocarcinomaG123 splice Splice Uterine endometrioid carcinoma UB1P136H Missense Colorectal adenocarcinoma UB1T147M Missense Brain lower grade glioma UB1D149Y Missense Uterine endometrioid carcinoma UB1L159V Missense Lung adenocarcinoma UB1K163N Missense Uterine endometrioid carcinoma UB1L170F Missense Cutaneous melanomaG184 splice Splice Colorectal cancerQ187H Missense Stomach adenocarcinomaS214N Missense Colorectal adenocarcinoma UB2R222I Missense Uterine endometrioid carcinoma UB2E238D Missense Lung adenocarcinoma UB2P241S Missense Uterine endometrioid carcinoma UB2T245A Missense Renal clear cell carcinoma UB2M249V Missense Uterine endometrioid carcinoma UB2E280K Missense Brain lower grade gliomaG293lowast Nonsense Colorectal cancerT300I Missense Colorectal adenocarcinomaD305H Missense Lung adenocarcinomaE308Q Missense Lung adenocarcinomaA316V Missense Stomach adenocarcinomaK319fs FS ins Head and neck squamous cell carcinomaR344G Missense Stomach adenocarcinoma UASF353I Missense Cutaneous melanoma UASL379V Missense Breast invasive carcinoma UASC396F Missense Cutaneous melanoma UASS459lowast Nonsense Uterine endometrioid carcinoma UASG469 splice Splice Glioblastoma multiforme UASR509G Missense Lung squamous cell carcinomaE510fs FS del Cutaneous melanomaR516C Missense Uterine endometrioid carcinomaA534V Missense Stomach adenocarcinomaF537L Missense Stomach adenocarcinomaE551lowast Nonsense Lung adenocarcinomaR554W Missense Colorectal cancerS582I Missense Lung adenocarcinoma UBXF585L Missense Uterine endometrioid carcinoma UBXE587lowast Nonsense Lung adenocarcinoma UBXA592V Missense Stomach adenocarcinoma UBXW610lowast Nonsense Breast invasive carcinoma UBXD611Y Missense Lung adenocarcinoma UBXE635fs FS del Brain lower grade glioma UBXP640fs FS del Cutaneous melanoma UBX

Sarcoma 19

a more accurate reference point for a leiomyosarcoma wouldhave been smooth muscle we believe that the comparison tostriated muscle is sufficient to allow the assessment of tumorspecific changes We have measured RNA DNA and proteinwith various assays Similar assessments in the future shouldalso include chromosome analysis for possible translocations

In the first-line defense against cancer the gradualreplacement of conventional chemotherapy with molecularlytargeted agents has opened the possibility to tailor drug treat-ments to particular tumors This transition necessitates thecharacterization of the molecular lesions that are causativefor the transformation of healthy cells into cancerous cellsbecause drugs need to be matched with the underlying carci-nogenic defect to be effective An additional caveat causedby unique genetic changes in the primary tumor can affectdrug transport and metabolism and needs to be taken intoconsideration In this case the RNA levels for genes that regu-late transport andmetabolismwere extensively skewed in thetumor tissue as compared to muscle and bone (see Table 3)implying potential challenges to chemotherapy An advancedmolecular treatment strategy for cancer will rely on themolecular definition of drug target drug transport and drugmetabolism pharmacogenetics in the primary tumor Con-secutive to cancer dissemination it will also require adjust-ments to account for the genetic changes in the metastases

The cost of health care has been under increasing scrutinyThe treatment of cancer patients is expensive in particularwhen hospitalization is required and further in cases ofend-of-life care Avoidable expenditures are generated bysuboptimal treatment decisions that result in low efficacy orhigh toxicity of anticancer regimens Molecular medicine hasthe potential to preempt those problems and reduce wastefulspending Yet it requires the upfront cost of molecular cancerexamination The analysis performed in this study requiredabout $11000- in nonsalary expenses to perform While ithas not identified a drug target it has specified possibleconfines for drug treatment The costs for molecular analysisneed to be weighed against the societal cost derived fromlost productivity in the workforce disrupted lives of familiesand premature deaths While currently limited drug optionsconstitute the major constraint to the approach taken herethe foreseeable future will bring an increasing spectrum ofmolecularly targeted drugs along with faster and cheapertechnologies for the molecular assessment of cancers Theywill facilitate the clinical translation of our approach

Consent

The patient provided informed consent

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by DOD Grant PR094070under University of Cincinnati IRB protocol 04-01-29-01The

author is grateful to Dr Rhett Kovall for helping with thestructural analysis of FAF1

References

[1] G F Weber Molecular Mechanisms of Cancer Springer Dor-drecht The Netherlands 2007

[2] N G Sanerkin ldquoPrimary leiomyosarcoma of the bone and itscomparison with fibrosarcomardquoCancer vol 44 no 4 pp 1375ndash1387 1979

[3] J M Weaver and J A Abraham ldquoLeiomyosarcoma of the Boneand Soft Tissue A Reviewrdquo httpsarcomahelporglearningcenterleiomyosarcomahtml

[4] M A Depristo E Banks R Poplin et al ldquoA framework forvariation discovery and genotyping using next-generationDNAsequencing datardquo Nature Genetics vol 43 no 5 pp 491ndash5012011

[5] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[6] C W Menges D A Altomare and J R Testa ldquoFAS-associatedfactor 1 (FAF1) diverse functions and implications for oncoge-nesisrdquo Cell Cycle vol 8 no 16 pp 2528ndash2534 2009

[7] M Kim J H Lee S Y Lee E Kim and J Chung ldquoCaspar asuppressor of antibacterial immunity in Drosophilardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 44 pp 16358ndash16363 2006

[8] J Song K P Joon J-J Lee et al ldquoStructure and interaction ofubiquitin-associated domain of human Fas-associated factor 1rdquoProtein Science vol 18 no 11 pp 2265ndash2276 2009

[9] P H OrsquoFarrell ldquoHigh resolution two dimensional electrophore-sis of proteinsrdquo Journal of Biological Chemistry vol 250 no 10pp 4007ndash4021 1975

[10] A Burgess-Cassler J J Johansen D A Santek J R Ideand N C Kendrick ldquoComputerized quantitative analysis ofCoomassie-Blue-stained serum proteins separated by two-dimensional electrophoresisrdquo Clinical Chemistry vol 35 no 12pp 2297ndash2304 1989

[11] B R Oakley D R Kirsch and N RMorris ldquoA simplified ultra-sensitive silver stain for detecting proteins in polyacrylamidegelsrdquo Analytical Biochemistry vol 105 no 2 pp 361ndash363 1980

[12] K Chu X Niu and L T Williams ldquoA Fas-associated proteinfactor FAF1 potentiates Fas-mediated apoptosisrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 25 pp 11894ndash11898 1995

[13] B Rikhof S de Jong A J H Suurmeijer C Meijer and W TA van der Graaf ldquoThe insulin-like growth factor system andsarcomasrdquoThe Journal of Pathology vol 217 no 4 pp 469ndash4822009

[14] RGirling J F Partridge L R Bandara et al ldquoAnew componentof the transcription factor DRTF1E2Frdquo Nature vol 362 no6415 pp 83ndash87 1993

[15] F Mercurio and M Karin ldquoTranscription factors AP-3 andAP-2 interact with the SV40 enhancer in a mutually exclusivemannerrdquo EMBO Journal vol 8 no 5 pp 1455ndash1460 1989

[16] R Bassel-Duby M D Hernandez M A Gonzalez J KKrueger and R S Williams ldquoA 40-kilodalton protein bindsspecifically to an upstream sequence element essential for mus-cle-specific transcription of the human myoglobin promoterrdquoMolecular and Cellular Biology vol 12 no 11 pp 5024ndash50321992

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 6: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

6 Sarcoma

Table1Con

tinued

Key

Chromosom

ePo

sition

Reference

Alternate

dbSN

PID

dbSN

PMAF

ESPMAF

(All)

ESPMAF

(EA)

ESPMAF

(AA)

Con

sensus

impact

Bone

muscle

geno

type

Bone

overall

depth

Bone

allele

depths

Bone

quality

Muscle

overall

depth

Muscle

allele

depths

Muscle

quality

Tumor

geno

type

Tumor

overall

depth

Tumor

allele

depths

Tumor

quality

12111

020739

TPP

TC7

12111

020739

TCGC

Trs71083132

mdashmdash

mdashmdash

Mod

erate

00

18NA

3014

NA

1801

19NA

1019

804293

APT

BP1

19804293

CA

mdash

mdashmdash

mdashMod

erate

00

120

1261

9971

741

9901

755330

9971564

51175C

RNF32

71564

51175

GC

mdash

mdashmdash

mdashMod

erate

00

55570

9967

700

9901

594223

993520206

69TRP

11-155D

1811

3520206

69G

T

mdashmdash

mdashmdash

Mod

erate

00

130

1360

9986

900

9901

804543

9921

43838624

TUBA

SH3A

2143838624

GT

mdash

mdashmdash

mdashMod

erate

00

70730

9945

470

9901

897621

9919

58773857

AZN

F544

1958773857

GA

mdash

mdashmdash

mdashMod

erate

00

46480

9943

450

9901

715126

99516465723

CZN

F622

516465723

TC

mdash

mdashmdash

mdashMod

erate

00

17170

3914

140

3301

271415

99

Sarcoma 7

Table 2 RNA induced in the tumor (a) Transcripts overexpressed in the tumor compared to muscle but not bone (b) Transcriptsoverexpressed in the tumor compared to bone but not muscle (c) Transcripts most abundantly overexpressed in the tumor compared tomuscle and bone (d) as (a) but limited to genes expressed at least at the level of 1 unit in the reference tissue (muscle or bone) (e) Alteredexpression of genes associated with NF-120581B activity Analysis of RNASeq for the transcription factor NF-120581B and gene products that regulate itsactivity RNAmessages that are selectively associated with the TNF pathway to NF-120581B activation are marked with bold font log2-fold changeindicates the alteration in the tumor compared to muscle or bone (the respective columns indicate the expression level for each organ)

(a)

log2-fold changeMuscle

Tumor over muscleNRG1 Neuregulin 1 9909KSR2 Kinase suppressor of ras 2 9675MMP13 Matrix metallopeptidase 13 (collagenase 3) 9528SPP1 Secreted phosphoprotein 1 9098INHBA Inhibin beta A 8570COL11A1 Collagen type XI alpha 1 8564MMP9 Matrix metallopeptidase 9 (gelatinase B 92 kda gelatinase 92 kda type IV collagenase) 8552FBN2 Fibrillin 2 8495LRRC15 Leucine rich repeat containing 15 8429MMP11 Matrix metallopeptidase 11 (stromelysin 3) 8336E2F7 E2F transcription factor 7 8314PRAME Preferentially expressed antigen in melanoma 8179PTK7 PTK7 protein tyrosine kinase 7 7996DSCAM Down syndrome cell adhesion molecule 7761RGS4 Regulator of G-protein signaling 4 7633CNIH3 Cornichon homolog 3 (Drosophila) 7632OR10V1 Olfactory receptor family 10 subfamily V member 1 7610CEP55 Centrosomal protein 55 kda 7583WNT5B Wingless-type MMTV integration site family member 5B 7569CA12 Carbonic anhydrase XII 7520

GALNT5 UDP-N-acetyl-alpha-D-galactosaminepolypeptide N-acetylgalactosaminyltransferase 5(GalNAc-T5) 7517

(b)

log2-fold changeBone

Tumor over boneROS1 c-ros oncogene 1 receptor tyrosine kinase 10987GREM1 Gremlin 1 10604NPTX1 Neuronal pentraxin I 8813GJB2 Gap junction protein beta 2 26 kDa 8597CREB3L1 cAMP responsive element binding protein 3-like 1 8506KRT14 Keratin 14 8351SERPINE1 Serpin peptidase inhibitor clade E (nexin plasminogen activator inhibitor type 1) member 1 8288HOXD10 Homeobox D10 8105ALPK2 Alpha-Kinase 2 7783POU3F2 POU class 3 homeobox 2 7530POSTN Periostin osteoblast specific factor 7497NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439STC2 Stanniocalcin 2 7434WNT5A Wingless-type MMTV integration site family member 5A 7412IGFN1 Immunoglobulin-like and fibronectin type III domain containing 1 7387

8 Sarcoma

(b) Continued

log2-fold changeBone

STC1 Stanniocalcin 1 7385FAM180A Family with sequence similarity 180 member A 7315KRT17 Keratin 17 7305BBOX1 Butyrobetaine (gamma) 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 7277MAGEA1 Melanoma antigen family A 1 (directs expression of antigen MZ2-E) 7267

(c)

log2-fold changeMuscle Bone

Tumor over bonemuscle

ANO4 Anoctamin 4 (TMEM16D) transmembrane calcium-activated chloride channelfacilitates smooth muscle contraction 9937 8542

SLCO1B3 (OATP1B3) Solute carrier organic anion transporter family member 1B3 9569 9760

MARCH4 Membrane-associated ring finger (C3HC4) 4 E3 ubiquitin ligase located predominantlyto the endoplasmic reticulum 9538 7406

IGF2BP1 Insulin-like growth factor 2 mRNA binding protein 1 binds to and stabilizes mRNA 9440 9631ADAMTS16 ADAMmetallopeptidase with thrombospondin type 1 motif 16 zinc-dependent protease 9260 8450SOX11 SRY (sex determining region Y)-box 11 important in brain development 9178 9954

HAPLN1 Hyaluronan and proteoglycan link protein 1 stabilizes aggregates of aggrecan andhyaluronan giving cartilage its tensile strength and elasticity 8951 8142

MUC15 Mucin 15 cell surface associated 8801 7992HOXB9 Homeobox B9 8773 7378MAGEC2 Melanoma antigen family C 2 8693 8884

ST6GALNAC5 ST6 (alpha-N-acetyl-neuraminyl-23-beta-galactosyl-13)-N-acetylgalactosaminidealpha-26-sialyltransferase 5 7848 8038

C11orf41 Chromosome 11 open reading frame 41 7781 8141MMP1 Matrix metallopeptidase 1 (interstitial collagenase) 7455 7646

(d)

Symbol Name log2-fold change log2-fold changeMuscle Bone

Tumor over bonemuscleFN1 Fibronectin 1 7328 6293 6457 19860COL1A1 Collagen type I alpha 1 6768 3706 5868 11934CCND1 Cyclin D1 5595 3958 5098 9637RGS1 Regulator of G-protein signaling 1 5118 1056 4653 2533ITGBL1 Integrin beta-like 1 (with EGF-like repeat domains) 5071 3177 3895 12415COL1A2 Collagen type I alpha 2 4852 8756 4910 14503MXRA5 Matrix-remodelling associated 5 4834 1352 4631 2685POSTN Periostin osteoblast specific factor 4513 5870 7497 1267PLOD2 Procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 4285 1801 4023 3730COL5A2 Collagen type V alpha 2 4275 1297 4833 1517COL3A1 Collagen type III alpha 1 4003 13118 5801 6500

SEMA3C Sema domain immunoglobulin domain (Ig) shortbasic domain secreted 3954 1164 4215 1675

FBN1 Fibrillin 1 3912 6957 4018 11148AEBP1 AE binding protein 1 3808 3212 4002 4843SIK1 Salt-inducible kinase 1 3805 1219 3566 2484

Sarcoma 9

(d) Continued

Symbol Name log2-fold change log2-fold changeMuscle Bone

SERPINH1 Serpin peptidase inhibitor clade H (heat shock protein47) member 1 3706 1652 4145 2098

ANTXR1 Anthrax toxin receptor 1 3670 3789 3690 6445

(e)

Symbol Name log2-fold change log2-fold changeMuscle Bone

HELLS Helicase lymphoid-specific 5315602 0155898 minus082713 202489

TNFRSF11A Tumor necrosis factor receptor superfamily member 11a andNFKB activator 3017922 003091 1400993 0202453

NFKBID Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor delta 2655352 0 minus147615 0504341

TNFRSF25 Tumor necrosis factor receptor superfamily member 25 2432959 0046388 0163954 0490795

NFKBIE Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor epsilon 2121015 0062395 0311441 043382

TNF Tumor necrosis factor 1847997 0 minus142101 003733

TNFRSF10D Tumor necrosis factor receptor superfamily member 10d decoywith truncated death domain 1754888 0172968 minus01757 1183343

TNFRSF1B Tumor necrosis factor receptor superfamily member 1B 1473438 0709902 minus097011 6729401TNFRSF10A Tumor necrosis factor receptor superfamily member 10a 1411898 0828219 0357701 3004386

NFKBIB Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor beta 1193993 1209816 046055 3484692

TNFRSF10B Tumor necrosis factor receptor superfamily member 10b 1094157 1908597 0425446 5242006TNFRSF21 Tumor necrosis factor receptor superfamily member 21 1055762 3882038 0745815 8305711TNFRSF1A Tumor necrosis factor receptor superfamily member 1A 0875167 3790938 minus011707 130344

NFKBIZ Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor zeta 0575974 3698951 0344657 7493136

RIPK1 Receptor (TNFRSF)-interacting serine-threonine kinase 1 0494467 311749 minus108854 161392NKRF NFKB repressing factor 0475722 2156087 minus086397 9434166NFKB1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 0432959 2054532 minus065865 7568586CHUK Conserved helix-loop-helix ubiquitous kinase 0176126 2961281 minus120204 1330333RELA V-rel reticuloendotheliosis viral oncogene homolog A (avian) 0013848 1263837 0287167 1803044

NFKB2 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 2(p49p100) minus008641 0312993 0485882 0360442

NKAPP1 NFKB activating protein pseudogene 1 minus010692 0825177 minus099449 2655392

TNFRSF10C Tumor necrosis factor receptor superfamily member 10c decoywithout an intracellular domain minus0152 0064676 minus556891 6321515

NKIRAS2 NFKB inhibitor interacting Ras-like 2 minus060768 1095135 minus088758 2299946

NFKBIL1 Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor-like 1 minus08719 0141933 minus008357 0140418

NKIRAS1 NFKB inhibitor interacting Ras-like 1 minus087428 6296399 1467735 2122983TANK TRAF family member-associated NFKB activator minus0983 6777124 minus151908 1695872NKAP NFKB activating protein minus135735 1422458 minus078243 1646287

NFKBIA Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor alpha minus185483 4032743 minus031802 2395275

NKAPL NFKB activating protein-like minus557347 5875743 minus140215 0534643

10 Sarcoma

FAS associating region

DED interacting region

UB12 DZM

UB2domain

UASdomain

UBXdomain

UBAdomain

S289 S291

Aurora ACK II

S181

AKT1ATMGSK3

AuroraPKG

PLK1RSK

STK4CHK1

650566481336260205169110475

(a)

MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRM

6

1

101

100

200

300

400

500

600

650

201

301

401

501

601

667855899998876467765678888887578758999986467 88 8888888888888988888899988888889888877767888777888757

2222454345655744642554435764462443558468643653433344333424435334233333332333332333354335333344334354

LDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITH

9999997797689998787758999999999859996577754788888888875333467888868987688888888888877888855675799987

9585847535375857543447459666955563844544585374435443365845846434457567533353232334334444433337484944

REVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDAT

5888757887577654788887766654677776555468877888765455546887766555688888877777777777776556788888767766

5344456484742545656645594364384534437445444342323455453535435463434323333343434333333334333335433333

EFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESI

5666776654456777888888888888866889999999998848888876677778999999998776686899998589875579999987486899

4635343574354322343434533333343548479747945564424647635575478569766456468999999754233454687457844669

VSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDE

9999888589997557877545444345677876335554467888876899986799876447777677888999999998877555778887777778

6778547999996674433343444333333335466456434433487999895353333464346434365468755846444545454565446565

REARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVF

8888888887788877766545557777777666777777777776678877777766578888888888885799998589975788758987589999

4545445564445344433443354555556653665456544454434444444443344334333333446859596743354745795535796898

DFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKE

99997799988779985788754577888765665678898589998589

67974525444795877564635844444476875837446989897366

(b)

Figure 2 FAF1 structure (a) A schematic of functional domains in FAF1 with annotations (adapted from [6ndash8]) There are two ubiquitin-like domains flanking the S181 site The PDB structure 2DZM identifies the N-terminal one while the C-terminal prediction is by sequencesimilarity Whereas the mutation S181G is not expected to disrupt the protein structure per se this amino acid has a high score as a possiblephosphorylation site for a number of kinases involved inDNAdamage repair (b) Secondary structure prediction of FAF1The residues aroundS181 appear unstructured

reflected in STAT3 constitutive phosphorylation The lack ofphosphorylation in this case suggests that the leiomyosar-coma may not depend on the STAT3 pathway

36 Pharmacogenetic Evaluation Predicting the sensitivityto anticancer drugs is a main goal of molecular analysisFor this the over- or underexpression of genes for drugtransport and metabolism is of key importance Analysis ofthe RNASeq data for these groups of gene products identified

a surprisingly large number of deregulations compared tomuscle or bone (Tables 3(a) and 3(b)) Those alterationsmay affect choices for drug treatment For example the highlevels of glutathione S-transferase may render carmustinethioTEPA cisplatin chlorambucil melphalan nitrogenmus-tard phosphoramide mustard acrolein or steroids ineffec-tive The overexpression ofN-acetyltransferase may compro-mise 5-fluorouracil or taxolThemodest upregulation of onlytwo export transporters (ABC-transporters) and specifically

Sarcoma 11

Bone Tumor

Muscle

(a)

Transcription factor Description Reference

E2F1 Transcriptional activation of cell cycle progression is selectively activated in response to specific cues key downstream target of RB1 can promote proliferation or apoptosis when activated [14]

AP-33 interact in a mutually exclusive manner [15]

CCAC CCAC box is an essential element for muscle-specific transcription from the myoglobin promoter [16]

LIII-BP The L-III transcriptional regulatory element (a carbohydrate-response element) is in the pyruvate kinase L gene necessary for cell type-specific expression and transcriptional stimulation by carbohydrates [17]

ADD-1 (SREBP-1) activates transcription from sterol-regulated elements and from an E-box sequence present in the promoter of fatty acid synthase [18]

PAX6 Member of the paired box gene family transcriptional regulator involved in developmental processes [19]

48kD transcription factor activator protein-3 binds to the core element and recognizes the SV40 enhancer and AP-2 and AP-

(b)

Figure 3 Transcription factor activation Nuclear extracts from tumor muscle and bone were tested for DNA binding activity on a protein-DNA array (a) Transcription factor binding that was high in all 3 tissues and is therefore considered constitutively active is circled in yellow(left to right top to bottom AhRAmt GATA1 GATA2 GATA12 HIF1 and HOXD8910) Transcription factors that show high binding inthe cancer but not in muscle or bone are circled in red (left to right top to bottom E2F1 AP3 LIII-BP PAX6 ADD-1 and CCAC) (b) Thefunctions of transcription factors that display high DNA binding in the cancer but not in muscle or bone are described

the lack of ABCB1 overexpression is favorable for avoidingdrug resistance

37 Population Analysis The above-described results indi-cated that a FAF1 mutation which replaces serine in position181 thus preventing FAF1 phosphorylation and activationmay be a driver for leiomyosarcomagenesis A custom Taq-Man assay confirmed the presence of the somatic mutationin the patient To assess whether this single nucleotidereplacement is common in this type of cancer we analyzedDNA from 29 leiomyosarcomas and 1 blood sample from aleiomyosarcoma patient For comparison to nonsarcomatoustumors 34 breast cancers served as a reference None of themdisplayed amutation in the same locus By contrast there wasa distribution across all leiomyosarcomas in the osteopontin

promoter position minus443 (used as a reference) with 12 CC 10TC and 9 TT

4 Discussion

TheFAF1mutation identified as the likely cause for the cancerunder study gives room for an explanation of the sarcomatoustransformation (Figure 5) DNA damage to mesenchymalcells occurs persistently in an oxidizing environment at 37∘CThese insults are rarely transforming and such an occurrencewould trigger the initiation of programmed cell death inapoptosis-competent cells Intact FAF1 associates with FASand enhances apoptosis mediated through this receptor [12]A loss of function in FAF1 could lead to transformation viaantiapoptosis Whereas the mutation S181G is not expected

12 Sarcoma

Muscle

Bone

Tumor

220

94

6043

29

14

220

94

60

43

29

14

220

94

60

43

29

14

1

7

6

23

24

21

22

25

26

89 1011 12

1516 1719

pH 4 8 pH 4 8

8pH 4

(a)

SwissProt or NCBISpot number Protein identified accession Description

(Structural)6 Transgelin Q01995 Smooth muscle-specific cross-links actin24 Transgelin-2 P37802 Smooth muscle-specific cross-links actin

Vimentin P08670 Intermediate filament in mesenchymal tissue1 Filamin-A P21333 Actin-binding protein regulates the cytoskeleton 21 P06709 Cytoskeleton

(Calcium homeostasis)8 9 Calumenin O43852 Calcium-binding protein in the ER and golgi apparatus26 Protein S100-A11 P31949 Calcium-binding EF hand protein10 Reticulocalbin-3 Q96D15 Calcium-binding protein located in the ER12 P11021 Heat shock 70-kD protein 5 ER lumenal calcium-binding

(Protein processing)25 40S ribosomal protein S12 P25398 Core component of the ribosomal accuracy center7 Glycine-tRNA ligase P41250 Catalyzes the attachment of glycine to tRNA19 P01009 Protease inhibitor23 P01009 Protease inhibitor21 Proteasome activator complex subunit 2 Q9UL46 Proteasome activation10 P07900 Chaperone

(Other)7 Serum albumin P02768 Carrier protein in blood22 Protein disulfide isomerase (C-terminal fragment) P07237 Prolyl hydroxylation in collagen microsomal triglyceride transfer

120573 actin (C-terminal fragment)

78kD glucose-regulated protein

120572-1-antitrypsin120572-1-antitrypsin (C-terminal fragment)

Heat shock protein HSP 90120572 (N-terminal fragment)

11 15ndash17

(b)

Figure 4 Continued

Sarcoma 13

Tumor Muscle

OPN

CD44

STAT3

P-STAT3

Tubulin

75

75

50

15010075

10075

50

(c)

Tumor bone Tumor muscleGene ID Symbol Name Fold change Expression Fold change Expression6876 TAGLN Transgelin 3274 2455 3087 15088407 TAGLN2 Transgelin-2 2164 7338 6975 13037431 VIM Vimentin 1653 295010 4061 696042316 FLNA Filamin A alpha 1304 7791 9005 065160 ACTB Actin beta 0656 973187 5491 67368813 CALU Calumenin 7601 19328 12063 70596282 S100A11 S100 calcium binding protein A11 0931 158914 5071 1686357333 RCN3 Reticulocalbin 3 EF-hand calcium binding domain 2439 0604 6412 01223309 HSPA5 1068 92754 2607 220356696 SPP1 Secreted phosphoprotein 1 7572 46508 547929 0355960 CD44 CD44 molecule (Indian blood group) 2053 26708 15436 20566774 STAT3 Signal transducer and activator of transcription 3 0617 46534 0832 200165925 RB1 Retinoblastoma 1 0736 14772 0442 14268

Heat shock 70kDa protein 5 (glucose-regulated protein 78kDa)

(d)

Figure 4 Protein overexpression (a) 2D protein gel electrophoresis of lysates from muscle (upper left) tumor (upper right) and bone(bottom) in RIPA buffer Red circles indicate the spots that were identified as overexpressed and were further analyzed by mass spectrometry(b) Proteins identified in 2D gel electrophoresis as overexpressed in the tumor in comparison to muscle and bone were analyzed for theiridentity by mass spectrometry The left column indicates the spot number corresponding to the 2D gel The next column contains theprotein name followed by the accession number and a description of the protein functionThe protein functions are grouped into structuralcalcium homeostasis and various others (c) Western blot 10 120583g lysates of tumor muscle and bone in RIPA buffer were loaded per laneand electrophoresed on 10 SDS-polyacrylamide minigels with reducing denaturing sample buffer After transfer to PVDF membranesthey were probed for markers of cancer progression including osteopontin CD44 STAT3 and phospho-STAT3 Antitubulin served as aloading control (d) RNA levels corresponding to the proteins found to be affected in the sarcoma With four exceptions in the tumor-bonecomparison (gray font) upregulated proteins are associated with increased RNA levels STAT3 is not increased on the protein or RNA levelThe reduced level of RB1 expression is consistent with the elevated DNA binding activity of E2F1

to disrupt the structure of the protein this site does scorehigh as a possible phosphorylation site for a number ofkinases involved in DNA damage repair supporting thehypothesis that the cancer cells containing thismutation havelost their ability to respond to transforming DNA damagewith programmed cell death FAF1 antagonizes WNT signal-ing by promoting 120573-catenin degradation in the proteasome[21] a function that may be lost after the point mutationThe elevated DNA binding activity of the protooncogenictranscription factor E2F1 (see Figure 3) could be caused byits interaction with LEF-1 [20] consecutive to persistent

WNT signaling The RNA level of IGF2BP1 (IMP-1) a stress-responsive regulator of mRNA stability is highly upregulatedin the tumor compared to muscle as well as bone (seeTable 2(c)) IGF2BP1 is a transcriptional target of the WNTpathway that regulates NF-120581B activity (see Table 2(e)) andis antiapoptotic [22 23] IGF2BP1 may be upregulated asa consequence of mutated FAF1 not being able to suppressWNT signaling in this specific cancerOf noteWNTpathwayoveractivity may not be required for transformation ratherthe persistence of a WNT pathway signal due to the lack of aFAF1-mediated termination signal may suffice

14 Sarcoma

WNTFrizzled

Axin Dvl

igf2bp1

FAF1

P65 P50

IKK

FAS

120573-Catenin

LEF + E2F1

I120581B

Figure 5 Possible transforming pathway The point mutation inFAF1 is believed to cause a loss of function (crossed out in red)This may lead to an overactivity of the WNT signaling pathwayConsistently E2F1 (activated by LEF1) and igf2bp1 (a transcriptionaltarget of the WNT pathway) have been identified to be upregulatedin the molecular analysis In contrast to the negative regulator FAF1IGF2BP1 is a positive regulator of NF-120581B activityThe overactivity ofthe NF-120581B pathway and the reduced efficacy of FAS signaling can betransforming

The role ofWNT signaling in sarcoma has been subject todebate (eg [24]) In metastatic leiomyosarcoma 120573-Cateninmay accumulate in the nucleus despite a relatively weakexpression of WNT [25] This may be due to WNT signalactivation via noncanonical ligands [26] or to 120573-Cateninbinding the nuclear receptor NR4A2 and releasing it from thecorepressor protein LEF-1 [27] Of note in the cancer understudy here the mRNA level of NR4A2 was overexpressed10-fold compared to bone and 3-fold compared to muscleThe results from this study are consistent with the possibilitythat a lack of termination in the WNT signal rather than itsoveractivation could contribute to sarcomatous transforma-tion Such a mechanism may be reflected in an upregulationof downstream targets even though overexpression of WNTpathway components is not detectable

Other mutations beside FAF1 are less likely to becausative for the cancer EPHA3 was revealed as mutated inthis cancer by DNA exome sequencing and RNASeq EPHA3is a receptor tyrosine kinase that is frequentlymutated in lungcancer Tumor-suppressive effects of wild-type EPHA3 can beoverridden by dominant negative EPHA3 somatic mutations[28]Thismechanism is unlikely to play a role in this sarcomaas the detected mutation is located far N-terminally on theextracellular Ephrin binding domain not on the intracellularkinase domain

FAF1 has been described to act as a tumor suppressor gene[29] Its depletion due to chromosome breakage can affectprognosis in glioblastoma patients [30 31] Single nucleotidepolymorphisms in FAF1 are associated with a risk for gastriccancer [32] While numerous FAF1 mutations are associatedwith various cancers none of these genetic changes in theTCGA database affects the amino acid position 181 (Table 4)

The major upregulations identified in this tumor com-prise muscle-specific gene products (transcription factors

CAAC binding proteomics transgelin transgelin-2 RNAanoctamin-4 and immunohistochemistry smooth mus-cle actin) and calcium-regulating molecules (proteomicscalumenin S100-A11 reticulocalbin-3 and 78 kD glucose-regulated protein) The muscle-specific gene products con-firm this recurrent sarcoma as a leiomyosarcoma (the firsttumor distal to the site of the recurring one had beendiagnosed as an osteosarcoma) Calcium is one of the majorsecond messengers in smooth muscle cells Its uptake isregulated by potential-sensitive ion channels in the cellmembrane and by the activities of various receptors Calciumis stored in the sarcoplasmic reticulum from where it canbe released to facilitate actin-myosin interaction and tensiongeneration Phosphorylation of the myosin light chain by acalmodulin-regulated enzyme is important for contractionThe upregulation of gene products associated with migrationand invasion (osteopontin MMP1 vimentin filamin-A and120573-actin) and gene products for extracellularmatrixmoleculesand their modulators (fibronectin collagen ITGBL1 andMXRA5) reflects the invasive nature of this cancer

The recurrence of a sarcoma after 14 years has twoprobable explanations either it is due to a cancer pre-disposition syndrome based on a germ-line mutation (theage of the patient weakens this hypothesis) or the secondtumor is a metastatic colony of the first that was reactivatedafter dormancy The location of the sarcoma in the sameextremity and proximal to a preceding mesenchymal cancer(and therefore in its natural path of dissemination) impliedthe probability that this was a relapse in a metastatic siteThe different histologic assessment as osteosarcoma in thefirst occurrence and leiomyosarcoma as the second cancerdoes not necessarily negate that Mixed histology [33 34]and transdifferentiation [35ndash37] have been described forsarcomatous tumors Of note however in this scenarioosteosarcoma seems to more commonly follow leiomyosar-coma than precede it Material from the first cancer of thispatient was not accessible to us It is very plausible that thiscould have been a mineralized leiomyosarcoma In thosetumors the differential diagnosis from osteosarcoma can bedifficult [38] The extracompartmental location of the firsttumor supports this interpretation

On the molecular pathology level sarcomas fall intotwo groups comprising tumors with simple karyotypes(with pathogenetic translocations or specific genetic muta-tions) and tumors with very complex karyotypes (overtchromosome and genomic instability with numerous gainsand losses) [39] Some molecular alterations that lead tocarcinogenesis can be defined in absolute termsThey includegain-of-function mutations or chromosome translocationsthat transformprotooncogenes to oncogenes However otherchanges are relative to the normal tissue of origin suchas pathway overactivity or overexpression on the proteinor RNA levels We have combined the analysis of absolutechanges (DNA exome sequence RNA sequence) with theanalysis of relative changes using skeletal muscle and bone asreference organs (protein-DNA array 2D gel electrophoresisand RNA expression levels) This choice was determined inpart by tissue availability after surgery andwas intended to aidin the distinction of osteosarcoma from myosarcoma While

Sarcoma 15

Table 3 Deregulation of genes for drug disposition Analysis of RNASeq for at least twofold overexpression (italic font) or underexpression(bold font) of genes for (a) transport and (b) metabolism in tumor compared to muscle and bone For the export transporters (ABCtransporters) only overexpression is considered relevant for drug resistance Not shown in the table are the underexpressed genes (ABCA7ABCA8 ABCA13 ABCB6 ABCB10 ABCC6 ABCC6P1 ABCC6P2 ABCC8 ABCC11 ABCD2 ABCG2 and ABCG5)

(a) Transport

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

650655 ABCA17P ATP-binding cassette subfamily A (ABC1) member 17 pseudogene 1360 211624 ABCA4 ATP-binding cassette subfamily A (ABC1) member 4 2038 2802

6555 SLC10A2 Solute carrier family 10 (sodiumbile acid cotransporter family)member 2 minus1962 minus2112

345274 SLC10A6 Solute carrier family 10 (sodiumbile acid cotransporter family)member 6 2623 3240

6563 SLC14A1 Solute carrier family 14 (urea transporter) member 1 (Kidd bloodgroup) minus3074 minus3152

6565 SLC15A2 Solute carrier family 15 (H+peptide transporter) member 2 minus2027 minus2152

6566 SLC16A1 Solute carrier family 16 member 1 (monocarboxylic acid transporter1) 1401 2170

117247 SLC16A10 Solute carrier family 16 member 10 (aromatic amino acid transporter) 2113 2848

6567 SLC16A2 Solute carrier family 16 member 2 (monocarboxylic acid transporter8) 3242 3848

10786 SLC17A3 Solute carrier family 17 (sodium phosphate) member 3 minus2868 minus29596571 SLC18A2 Solute carrier family 18 (vesicular monoamine) member 2 minus2087 minus21946573 SLC19A1 Solute carrier family 19 (folate transporter) member 1 minus2099 minus220310560 SLC19A2 Solute carrier family 19 (thiamine transporter) member 2 1820 254880704 SLC19A3 Solute carrier family 19 member 3 minus3331 minus3478387775 SLC22A10 Solute carrier family 22 member 10 5711 60859390 SLC22A13 Solute carrier family 22 (organic anion transporter) member 13 2623 3217

85413 SLC22A16 Solute carrier family 22 (organic cationcarnitine transporter)member 16 minus8101 minus7299

51310 SLC22A17 Solute carrier family 22 member 17 1475 21755002 SLC22A18 Solute carrier family 22 member 18 2038 27226582 SLC22A2 Solute carrier family 22 (organic cation transporter) member 2 4793 5216

6581 SLC22A3 Solute carrier family 22 (extraneuronal monoamine transporter)member 3 2554 3182

6583 SLC22A4 Solute carrier family 22 (organic cationergothioneine transporter)member 4 minus3603 minus3776

151295 SLC23A3 Solute carrier family 23 (nucleobase transporters) member 3 2109 284810478 SLC25A17 Solute carrier family 25 (mitochondrial carrier) member 17 1311 207783733 SLC25A18 Solute carrier family 25 (mitochondrial carrier) member 18 1846 2570

788 SLC25A20 Solute carrier family 25 (carnitineacylcarnitine translocase) member20 minus2105 minus2207

89874 SLC25A21 Solute carrier family 25 (mitochondrial oxodicarboxylate carrier)member 21 minus2962 minus3105

51312 SLC25A37 Solute carrier family 25 member 37 minus4633 minus486651629 SLC25A39 Solute carrier family 25 member 39 minus2585 minus2737203427 SLC25A43 Solute carrier family 25 member 43 1325 208865012 SLC26A10 Solute carrier family 26 member 10 3896 4472115111 SLC26A7 Solute carrier family 26 member 7 3431 4018116369 SLC26A8 Solute carrier family 26 member 8 minus5354 minus5536115019 SLC26A9 Solute carrier family 26 member 9 1623 241911001 SLC27A2 Solute carrier family 27 (fatty acid transporter) member 2 minus4278 minus4487

16 Sarcoma

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

64078 SLC28A3 Solute carrier family 28 (sodium-coupled nucleoside transporter)member 3 minus4543 minus4812

222962 SLC29A4 Solute carrier family 29 (nucleoside transporters) member 4 minus1962 minus204581031 SLC2A10 Solute carrier family 2 (facilitated glucose transporter) member 10 2532 3170

6518 SLC2A5 Solute carrier family 2 (facilitated glucosefructose transporter)member 5 minus3647 minus3821

55532 SLC30A10 Solute carrier family 30 member 10 minus3547 minus37257782 SLC30A4 Solute carrier family 30 (zinc transporter) member 4 2832 33726569 SLC34A1 Solute carrier family 34 (sodium phosphate) member 1 minus4716 minus4941340146 SLC35D3 Solute carrier family 35 member D3 minus5662 minus590654733 SLC35F2 Solute carrier family 35 member F2 1433 2170206358 SLC36A1 Solute carrier family 36 (protonamino acid symporter) member 1 minus2265 minus2345285641 SLC36A3 Solute carrier family 36 (protonamino acid symporter) member 3 minus2284 minus239354020 SLC37A1 Solute carrier family 37 (glycerol-3-phosphate transporter) member 1 minus2112 minus22122542 SLC37A4 Solute carrier family 37 (glucose-6-phosphate transporter) member 4 minus2117 minus2219151258 SLC38A11 Solute carrier family 38 member 11 2410 308555089 SLC38A4 Solute carrier family 38 member 4 1837 254892745 SLC38A5 Solute carrier family 38 member 5 minus1994 minus215291252 SLC39A13 Solute carrier family 39 (zinc transporter) member 13 1301 207023516 SLC39A14 Solute carrier family 39 (zinc transporter) member 14 2569 318829985 SLC39A3 Solute carrier family 39 (zinc transporter) member 3 minus2032 minus2152283375 SLC39A5 Solute carrier family 39 (metal ion transporter) member 5 1623 23707922 SLC39A7 Solute carrier family 39 (zinc transporter) member 7 1273 205830061 SLC40A1 Solute carrier family 40 (iron-regulated transporter) member 1 minus3612 minus379684102 SLC41A2 Solute carrier family 41 member 2 1293 20708501 SLC43A1 Solute carrier family 43 member 1 minus2013 minus215257153 SLC44A2 Solute carrier family 44 member 2 minus2047 minus215250651 SLC45A1 Solute carrier family 45 member 1 2038 2722146802 SLC47A2 Solute carrier family 47 member 2 minus1962 minus20266521 SLC4A1 Solute carrier family 4 anion exchanger member 1 minus6513 minus645357282 SLC4A10 Solute carrier family 4 sodium bicarbonate transporter member 10 minus2640 minus275383959 SLC4A11 Solute carrier family 4 sodium borate transporter member 11 1846 25696508 SLC4A3 Solute carrier family 4 anion exchanger member 3 1261 20458671 SLC4A4 Solute carrier family 4 sodium bicarbonate cotransporter member 4 2445 31139497 SLC4A7 Solute carrier family 4 sodium bicarbonate cotransporter member 7 2717 33076523 SLC5A1 Solute carrier family 5 (sodiumglucose cotransporter) member 1 minus2547 minus2705159963 SLC5A12 Solute carrier family 5 (sodiumglucose cotransporter) member 12 4753 52066527 SLC5A4 Solute carrier family 5 (low affinity glucose cotransporter) member 4 minus3969 minus4249

6540 SLC6A13 Solute carrier family 6 (neurotransmitter transporter GABA)member 13 1328 2092

55117 SLC6A15 Solute carrier family 6 (neutral amino acid transporter) member 15 1623 2333388662 SLC6A17 Solute carrier family 6 member 17 2038 272854716 SLC6A20 Solute carrier family 6 (proline IMINO transporter) member 20 1697 2433

6532 SLC6A4 Solute carrier family 6 (neurotransmitter transporter serotonin)member 4 minus2512 minus2611

6534 SLC6A7 Solute carrier family 6 (neurotransmitter transporter L-proline)member 7 2623 3228

56301 SLC7A10 Solute carrier family 7 (neutral amino acid transporter y+ system)member 10 minus3421 minus3603

Sarcoma 17

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

6547 SLC8A3 Solute carrier family 8 (sodiumcalcium exchanger) member 3 minus2204 minus2309285335 SLC9A10 Solute carrier family 9 member 10 1208 20006549 SLC9A2 Solute carrier family 9 (sodiumhydrogen exchanger) member 2 2038 2706

9368 SLC9A3R1 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 1 minus2760 minus2846

9351 SLC9A3R2 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 2 1889 2619

84679 SLC9A7 Solute carrier family 9 (sodiumhydrogen exchanger) member 7 3347 393510599 SLCO1B1 Solute carrier organic anion transporter family member 1B1 3360 397728234 SLCO1B3 Solute carrier organic anion transporter family member 1B3 9760 95696578 SLCO2A1 Solute carrier organic anion transporter family member 2A1 2432 309628232 SLCO3A1 Solute carrier organic anion transporter family member 3A1 minus2032 minus2152353189 SLCO4C1 Solute carrier organic anion transporter family member 4C1 minus6850 minus6611

(b) Metabolism

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold Cchange

1583 CYP11A1 Cytochrome P450 family 11 subfamily A polypeptide 1 1623 23961589 CYP21A2 Cytochrome P450 family 21 subfamily A polypeptide 2 minus1962 minus20361591 CYP24A1 Cytochrome P450 family 24 subfamily A polypeptide 1 5208 55771592 CYP26A1 Cytochrome P450 family 26 subfamily A polypeptide 1 2623 32571594 CYP27B1 Cytochrome P450 family 27 subfamily B polypeptide 1 1846 2585339761 CYP27C1 Cytochrome P450 family 27 subfamily C polypeptide 1 3585 41781553 CYP2A13 Cytochrome P450 family 2 subfamily A polypeptide 13 minus1962 minus20351580 CYP4B1 Cytochrome P450 family 4 subfamily B polypeptide 1 minus4820 minus506566002 CYP4F12 Cytochrome P450 family 4 subfamily F polypeptide 12 minus6070 minus61958529 CYP4F2 Cytochrome P450 family 4 subfamily F polypeptide 2 minus7769 minus70604051 CYP4F3 Cytochrome P450 family 4 subfamily F polypeptide 3 minus8244 minus749111283 CYP4F8 Cytochrome P450 family 4 subfamily F polypeptide 8 minus5006 minus5270260293 CYP4X1 Cytochrome P450 family 4 subfamily X polypeptide 1 minus2476 minus25809420 CYP7B1 Cytochrome P450 family 7 subfamily B polypeptide 1 2502 31702326 FMO1 Flavin containing monooxygenase 1 4360 48592327 FMO2 Flavin containing monooxygenase 2 (nonfunctional) minus4074 minus43372328 FMO3 Flavin containing monooxygenase 3 minus4036 minus4309388714 FMO6P Flavin containing monooxygenase 6 pseudogene minus2569 minus2737493869 GPX8 Glutathione peroxidase 8 (putative) 2814 33712938 GSTA1 Glutathione S-transferase alpha 1 1623 23632939 GSTA2 Glutathione S-transferase alpha 2 2038 27412941 GSTA4 Glutathione S-transferase alpha 4 1492 21882953 GSTT2 Glutathione S-transferase theta 2 4682 5170653689 GSTT2B Glutathione S-transferase theta 2B (genepseudogene) 3739 430784779 NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439 79969027 NAT8 N-acetyltransferase 8 (GCN5-related putative) minus2769 minus29207358 UGDH UDP-glucose 6-dehydrogenase 2333 301855757 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 1604 227110720 UGT2B11 UDP glucuronosyltransferase 2 family polypeptide B11 minus3586 minus37687367 UGT2B17 UDP glucuronosyltransferase 2 family polypeptide B17 minus2836 minus295954490 UGT2B28 UDP glucuronosyltransferase 2 family polypeptide B28 minus3132 minus3284167127 UGT3A2 UDP glycosyltransferase 3 family polypeptide A2 minus4716 minus4907

18 Sarcoma

Table 4 FAF1 mutations in various cancers FAF1 mutations listed in the TCGA data base were identified without restriction to any type ofcancer For the location of the affected domains on the protein compare Figure 2

AA Mutation Cancer DomainN4S Missense Cutaneous melanomaI10S Missense Stomach adenocarcinoma UBAE21lowast Nonsense Cutaneous melanoma UBAE25K Missense Uterine endometrioid carcinoma UBAV38 splice Splice Stomach adenocarcinoma UBAP86fs FS del Colorectal adenocarcinomaG123 splice Splice Uterine endometrioid carcinoma UB1P136H Missense Colorectal adenocarcinoma UB1T147M Missense Brain lower grade glioma UB1D149Y Missense Uterine endometrioid carcinoma UB1L159V Missense Lung adenocarcinoma UB1K163N Missense Uterine endometrioid carcinoma UB1L170F Missense Cutaneous melanomaG184 splice Splice Colorectal cancerQ187H Missense Stomach adenocarcinomaS214N Missense Colorectal adenocarcinoma UB2R222I Missense Uterine endometrioid carcinoma UB2E238D Missense Lung adenocarcinoma UB2P241S Missense Uterine endometrioid carcinoma UB2T245A Missense Renal clear cell carcinoma UB2M249V Missense Uterine endometrioid carcinoma UB2E280K Missense Brain lower grade gliomaG293lowast Nonsense Colorectal cancerT300I Missense Colorectal adenocarcinomaD305H Missense Lung adenocarcinomaE308Q Missense Lung adenocarcinomaA316V Missense Stomach adenocarcinomaK319fs FS ins Head and neck squamous cell carcinomaR344G Missense Stomach adenocarcinoma UASF353I Missense Cutaneous melanoma UASL379V Missense Breast invasive carcinoma UASC396F Missense Cutaneous melanoma UASS459lowast Nonsense Uterine endometrioid carcinoma UASG469 splice Splice Glioblastoma multiforme UASR509G Missense Lung squamous cell carcinomaE510fs FS del Cutaneous melanomaR516C Missense Uterine endometrioid carcinomaA534V Missense Stomach adenocarcinomaF537L Missense Stomach adenocarcinomaE551lowast Nonsense Lung adenocarcinomaR554W Missense Colorectal cancerS582I Missense Lung adenocarcinoma UBXF585L Missense Uterine endometrioid carcinoma UBXE587lowast Nonsense Lung adenocarcinoma UBXA592V Missense Stomach adenocarcinoma UBXW610lowast Nonsense Breast invasive carcinoma UBXD611Y Missense Lung adenocarcinoma UBXE635fs FS del Brain lower grade glioma UBXP640fs FS del Cutaneous melanoma UBX

Sarcoma 19

a more accurate reference point for a leiomyosarcoma wouldhave been smooth muscle we believe that the comparison tostriated muscle is sufficient to allow the assessment of tumorspecific changes We have measured RNA DNA and proteinwith various assays Similar assessments in the future shouldalso include chromosome analysis for possible translocations

In the first-line defense against cancer the gradualreplacement of conventional chemotherapy with molecularlytargeted agents has opened the possibility to tailor drug treat-ments to particular tumors This transition necessitates thecharacterization of the molecular lesions that are causativefor the transformation of healthy cells into cancerous cellsbecause drugs need to be matched with the underlying carci-nogenic defect to be effective An additional caveat causedby unique genetic changes in the primary tumor can affectdrug transport and metabolism and needs to be taken intoconsideration In this case the RNA levels for genes that regu-late transport andmetabolismwere extensively skewed in thetumor tissue as compared to muscle and bone (see Table 3)implying potential challenges to chemotherapy An advancedmolecular treatment strategy for cancer will rely on themolecular definition of drug target drug transport and drugmetabolism pharmacogenetics in the primary tumor Con-secutive to cancer dissemination it will also require adjust-ments to account for the genetic changes in the metastases

The cost of health care has been under increasing scrutinyThe treatment of cancer patients is expensive in particularwhen hospitalization is required and further in cases ofend-of-life care Avoidable expenditures are generated bysuboptimal treatment decisions that result in low efficacy orhigh toxicity of anticancer regimens Molecular medicine hasthe potential to preempt those problems and reduce wastefulspending Yet it requires the upfront cost of molecular cancerexamination The analysis performed in this study requiredabout $11000- in nonsalary expenses to perform While ithas not identified a drug target it has specified possibleconfines for drug treatment The costs for molecular analysisneed to be weighed against the societal cost derived fromlost productivity in the workforce disrupted lives of familiesand premature deaths While currently limited drug optionsconstitute the major constraint to the approach taken herethe foreseeable future will bring an increasing spectrum ofmolecularly targeted drugs along with faster and cheapertechnologies for the molecular assessment of cancers Theywill facilitate the clinical translation of our approach

Consent

The patient provided informed consent

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by DOD Grant PR094070under University of Cincinnati IRB protocol 04-01-29-01The

author is grateful to Dr Rhett Kovall for helping with thestructural analysis of FAF1

References

[1] G F Weber Molecular Mechanisms of Cancer Springer Dor-drecht The Netherlands 2007

[2] N G Sanerkin ldquoPrimary leiomyosarcoma of the bone and itscomparison with fibrosarcomardquoCancer vol 44 no 4 pp 1375ndash1387 1979

[3] J M Weaver and J A Abraham ldquoLeiomyosarcoma of the Boneand Soft Tissue A Reviewrdquo httpsarcomahelporglearningcenterleiomyosarcomahtml

[4] M A Depristo E Banks R Poplin et al ldquoA framework forvariation discovery and genotyping using next-generationDNAsequencing datardquo Nature Genetics vol 43 no 5 pp 491ndash5012011

[5] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[6] C W Menges D A Altomare and J R Testa ldquoFAS-associatedfactor 1 (FAF1) diverse functions and implications for oncoge-nesisrdquo Cell Cycle vol 8 no 16 pp 2528ndash2534 2009

[7] M Kim J H Lee S Y Lee E Kim and J Chung ldquoCaspar asuppressor of antibacterial immunity in Drosophilardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 44 pp 16358ndash16363 2006

[8] J Song K P Joon J-J Lee et al ldquoStructure and interaction ofubiquitin-associated domain of human Fas-associated factor 1rdquoProtein Science vol 18 no 11 pp 2265ndash2276 2009

[9] P H OrsquoFarrell ldquoHigh resolution two dimensional electrophore-sis of proteinsrdquo Journal of Biological Chemistry vol 250 no 10pp 4007ndash4021 1975

[10] A Burgess-Cassler J J Johansen D A Santek J R Ideand N C Kendrick ldquoComputerized quantitative analysis ofCoomassie-Blue-stained serum proteins separated by two-dimensional electrophoresisrdquo Clinical Chemistry vol 35 no 12pp 2297ndash2304 1989

[11] B R Oakley D R Kirsch and N RMorris ldquoA simplified ultra-sensitive silver stain for detecting proteins in polyacrylamidegelsrdquo Analytical Biochemistry vol 105 no 2 pp 361ndash363 1980

[12] K Chu X Niu and L T Williams ldquoA Fas-associated proteinfactor FAF1 potentiates Fas-mediated apoptosisrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 25 pp 11894ndash11898 1995

[13] B Rikhof S de Jong A J H Suurmeijer C Meijer and W TA van der Graaf ldquoThe insulin-like growth factor system andsarcomasrdquoThe Journal of Pathology vol 217 no 4 pp 469ndash4822009

[14] RGirling J F Partridge L R Bandara et al ldquoAnew componentof the transcription factor DRTF1E2Frdquo Nature vol 362 no6415 pp 83ndash87 1993

[15] F Mercurio and M Karin ldquoTranscription factors AP-3 andAP-2 interact with the SV40 enhancer in a mutually exclusivemannerrdquo EMBO Journal vol 8 no 5 pp 1455ndash1460 1989

[16] R Bassel-Duby M D Hernandez M A Gonzalez J KKrueger and R S Williams ldquoA 40-kilodalton protein bindsspecifically to an upstream sequence element essential for mus-cle-specific transcription of the human myoglobin promoterrdquoMolecular and Cellular Biology vol 12 no 11 pp 5024ndash50321992

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

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Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 7: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

Sarcoma 7

Table 2 RNA induced in the tumor (a) Transcripts overexpressed in the tumor compared to muscle but not bone (b) Transcriptsoverexpressed in the tumor compared to bone but not muscle (c) Transcripts most abundantly overexpressed in the tumor compared tomuscle and bone (d) as (a) but limited to genes expressed at least at the level of 1 unit in the reference tissue (muscle or bone) (e) Alteredexpression of genes associated with NF-120581B activity Analysis of RNASeq for the transcription factor NF-120581B and gene products that regulate itsactivity RNAmessages that are selectively associated with the TNF pathway to NF-120581B activation are marked with bold font log2-fold changeindicates the alteration in the tumor compared to muscle or bone (the respective columns indicate the expression level for each organ)

(a)

log2-fold changeMuscle

Tumor over muscleNRG1 Neuregulin 1 9909KSR2 Kinase suppressor of ras 2 9675MMP13 Matrix metallopeptidase 13 (collagenase 3) 9528SPP1 Secreted phosphoprotein 1 9098INHBA Inhibin beta A 8570COL11A1 Collagen type XI alpha 1 8564MMP9 Matrix metallopeptidase 9 (gelatinase B 92 kda gelatinase 92 kda type IV collagenase) 8552FBN2 Fibrillin 2 8495LRRC15 Leucine rich repeat containing 15 8429MMP11 Matrix metallopeptidase 11 (stromelysin 3) 8336E2F7 E2F transcription factor 7 8314PRAME Preferentially expressed antigen in melanoma 8179PTK7 PTK7 protein tyrosine kinase 7 7996DSCAM Down syndrome cell adhesion molecule 7761RGS4 Regulator of G-protein signaling 4 7633CNIH3 Cornichon homolog 3 (Drosophila) 7632OR10V1 Olfactory receptor family 10 subfamily V member 1 7610CEP55 Centrosomal protein 55 kda 7583WNT5B Wingless-type MMTV integration site family member 5B 7569CA12 Carbonic anhydrase XII 7520

GALNT5 UDP-N-acetyl-alpha-D-galactosaminepolypeptide N-acetylgalactosaminyltransferase 5(GalNAc-T5) 7517

(b)

log2-fold changeBone

Tumor over boneROS1 c-ros oncogene 1 receptor tyrosine kinase 10987GREM1 Gremlin 1 10604NPTX1 Neuronal pentraxin I 8813GJB2 Gap junction protein beta 2 26 kDa 8597CREB3L1 cAMP responsive element binding protein 3-like 1 8506KRT14 Keratin 14 8351SERPINE1 Serpin peptidase inhibitor clade E (nexin plasminogen activator inhibitor type 1) member 1 8288HOXD10 Homeobox D10 8105ALPK2 Alpha-Kinase 2 7783POU3F2 POU class 3 homeobox 2 7530POSTN Periostin osteoblast specific factor 7497NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439STC2 Stanniocalcin 2 7434WNT5A Wingless-type MMTV integration site family member 5A 7412IGFN1 Immunoglobulin-like and fibronectin type III domain containing 1 7387

8 Sarcoma

(b) Continued

log2-fold changeBone

STC1 Stanniocalcin 1 7385FAM180A Family with sequence similarity 180 member A 7315KRT17 Keratin 17 7305BBOX1 Butyrobetaine (gamma) 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 7277MAGEA1 Melanoma antigen family A 1 (directs expression of antigen MZ2-E) 7267

(c)

log2-fold changeMuscle Bone

Tumor over bonemuscle

ANO4 Anoctamin 4 (TMEM16D) transmembrane calcium-activated chloride channelfacilitates smooth muscle contraction 9937 8542

SLCO1B3 (OATP1B3) Solute carrier organic anion transporter family member 1B3 9569 9760

MARCH4 Membrane-associated ring finger (C3HC4) 4 E3 ubiquitin ligase located predominantlyto the endoplasmic reticulum 9538 7406

IGF2BP1 Insulin-like growth factor 2 mRNA binding protein 1 binds to and stabilizes mRNA 9440 9631ADAMTS16 ADAMmetallopeptidase with thrombospondin type 1 motif 16 zinc-dependent protease 9260 8450SOX11 SRY (sex determining region Y)-box 11 important in brain development 9178 9954

HAPLN1 Hyaluronan and proteoglycan link protein 1 stabilizes aggregates of aggrecan andhyaluronan giving cartilage its tensile strength and elasticity 8951 8142

MUC15 Mucin 15 cell surface associated 8801 7992HOXB9 Homeobox B9 8773 7378MAGEC2 Melanoma antigen family C 2 8693 8884

ST6GALNAC5 ST6 (alpha-N-acetyl-neuraminyl-23-beta-galactosyl-13)-N-acetylgalactosaminidealpha-26-sialyltransferase 5 7848 8038

C11orf41 Chromosome 11 open reading frame 41 7781 8141MMP1 Matrix metallopeptidase 1 (interstitial collagenase) 7455 7646

(d)

Symbol Name log2-fold change log2-fold changeMuscle Bone

Tumor over bonemuscleFN1 Fibronectin 1 7328 6293 6457 19860COL1A1 Collagen type I alpha 1 6768 3706 5868 11934CCND1 Cyclin D1 5595 3958 5098 9637RGS1 Regulator of G-protein signaling 1 5118 1056 4653 2533ITGBL1 Integrin beta-like 1 (with EGF-like repeat domains) 5071 3177 3895 12415COL1A2 Collagen type I alpha 2 4852 8756 4910 14503MXRA5 Matrix-remodelling associated 5 4834 1352 4631 2685POSTN Periostin osteoblast specific factor 4513 5870 7497 1267PLOD2 Procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 4285 1801 4023 3730COL5A2 Collagen type V alpha 2 4275 1297 4833 1517COL3A1 Collagen type III alpha 1 4003 13118 5801 6500

SEMA3C Sema domain immunoglobulin domain (Ig) shortbasic domain secreted 3954 1164 4215 1675

FBN1 Fibrillin 1 3912 6957 4018 11148AEBP1 AE binding protein 1 3808 3212 4002 4843SIK1 Salt-inducible kinase 1 3805 1219 3566 2484

Sarcoma 9

(d) Continued

Symbol Name log2-fold change log2-fold changeMuscle Bone

SERPINH1 Serpin peptidase inhibitor clade H (heat shock protein47) member 1 3706 1652 4145 2098

ANTXR1 Anthrax toxin receptor 1 3670 3789 3690 6445

(e)

Symbol Name log2-fold change log2-fold changeMuscle Bone

HELLS Helicase lymphoid-specific 5315602 0155898 minus082713 202489

TNFRSF11A Tumor necrosis factor receptor superfamily member 11a andNFKB activator 3017922 003091 1400993 0202453

NFKBID Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor delta 2655352 0 minus147615 0504341

TNFRSF25 Tumor necrosis factor receptor superfamily member 25 2432959 0046388 0163954 0490795

NFKBIE Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor epsilon 2121015 0062395 0311441 043382

TNF Tumor necrosis factor 1847997 0 minus142101 003733

TNFRSF10D Tumor necrosis factor receptor superfamily member 10d decoywith truncated death domain 1754888 0172968 minus01757 1183343

TNFRSF1B Tumor necrosis factor receptor superfamily member 1B 1473438 0709902 minus097011 6729401TNFRSF10A Tumor necrosis factor receptor superfamily member 10a 1411898 0828219 0357701 3004386

NFKBIB Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor beta 1193993 1209816 046055 3484692

TNFRSF10B Tumor necrosis factor receptor superfamily member 10b 1094157 1908597 0425446 5242006TNFRSF21 Tumor necrosis factor receptor superfamily member 21 1055762 3882038 0745815 8305711TNFRSF1A Tumor necrosis factor receptor superfamily member 1A 0875167 3790938 minus011707 130344

NFKBIZ Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor zeta 0575974 3698951 0344657 7493136

RIPK1 Receptor (TNFRSF)-interacting serine-threonine kinase 1 0494467 311749 minus108854 161392NKRF NFKB repressing factor 0475722 2156087 minus086397 9434166NFKB1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 0432959 2054532 minus065865 7568586CHUK Conserved helix-loop-helix ubiquitous kinase 0176126 2961281 minus120204 1330333RELA V-rel reticuloendotheliosis viral oncogene homolog A (avian) 0013848 1263837 0287167 1803044

NFKB2 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 2(p49p100) minus008641 0312993 0485882 0360442

NKAPP1 NFKB activating protein pseudogene 1 minus010692 0825177 minus099449 2655392

TNFRSF10C Tumor necrosis factor receptor superfamily member 10c decoywithout an intracellular domain minus0152 0064676 minus556891 6321515

NKIRAS2 NFKB inhibitor interacting Ras-like 2 minus060768 1095135 minus088758 2299946

NFKBIL1 Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor-like 1 minus08719 0141933 minus008357 0140418

NKIRAS1 NFKB inhibitor interacting Ras-like 1 minus087428 6296399 1467735 2122983TANK TRAF family member-associated NFKB activator minus0983 6777124 minus151908 1695872NKAP NFKB activating protein minus135735 1422458 minus078243 1646287

NFKBIA Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor alpha minus185483 4032743 minus031802 2395275

NKAPL NFKB activating protein-like minus557347 5875743 minus140215 0534643

10 Sarcoma

FAS associating region

DED interacting region

UB12 DZM

UB2domain

UASdomain

UBXdomain

UBAdomain

S289 S291

Aurora ACK II

S181

AKT1ATMGSK3

AuroraPKG

PLK1RSK

STK4CHK1

650566481336260205169110475

(a)

MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRM

6

1

101

100

200

300

400

500

600

650

201

301

401

501

601

667855899998876467765678888887578758999986467 88 8888888888888988888899988888889888877767888777888757

2222454345655744642554435764462443558468643653433344333424435334233333332333332333354335333344334354

LDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITH

9999997797689998787758999999999859996577754788888888875333467888868987688888888888877888855675799987

9585847535375857543447459666955563844544585374435443365845846434457567533353232334334444433337484944

REVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDAT

5888757887577654788887766654677776555468877888765455546887766555688888877777777777776556788888767766

5344456484742545656645594364384534437445444342323455453535435463434323333343434333333334333335433333

EFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESI

5666776654456777888888888888866889999999998848888876677778999999998776686899998589875579999987486899

4635343574354322343434533333343548479747945564424647635575478569766456468999999754233454687457844669

VSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDE

9999888589997557877545444345677876335554467888876899986799876447777677888999999998877555778887777778

6778547999996674433343444333333335466456434433487999895353333464346434365468755846444545454565446565

REARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVF

8888888887788877766545557777777666777777777776678877777766578888888888885799998589975788758987589999

4545445564445344433443354555556653665456544454434444444443344334333333446859596743354745795535796898

DFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKE

99997799988779985788754577888765665678898589998589

67974525444795877564635844444476875837446989897366

(b)

Figure 2 FAF1 structure (a) A schematic of functional domains in FAF1 with annotations (adapted from [6ndash8]) There are two ubiquitin-like domains flanking the S181 site The PDB structure 2DZM identifies the N-terminal one while the C-terminal prediction is by sequencesimilarity Whereas the mutation S181G is not expected to disrupt the protein structure per se this amino acid has a high score as a possiblephosphorylation site for a number of kinases involved inDNAdamage repair (b) Secondary structure prediction of FAF1The residues aroundS181 appear unstructured

reflected in STAT3 constitutive phosphorylation The lack ofphosphorylation in this case suggests that the leiomyosar-coma may not depend on the STAT3 pathway

36 Pharmacogenetic Evaluation Predicting the sensitivityto anticancer drugs is a main goal of molecular analysisFor this the over- or underexpression of genes for drugtransport and metabolism is of key importance Analysis ofthe RNASeq data for these groups of gene products identified

a surprisingly large number of deregulations compared tomuscle or bone (Tables 3(a) and 3(b)) Those alterationsmay affect choices for drug treatment For example the highlevels of glutathione S-transferase may render carmustinethioTEPA cisplatin chlorambucil melphalan nitrogenmus-tard phosphoramide mustard acrolein or steroids ineffec-tive The overexpression ofN-acetyltransferase may compro-mise 5-fluorouracil or taxolThemodest upregulation of onlytwo export transporters (ABC-transporters) and specifically

Sarcoma 11

Bone Tumor

Muscle

(a)

Transcription factor Description Reference

E2F1 Transcriptional activation of cell cycle progression is selectively activated in response to specific cues key downstream target of RB1 can promote proliferation or apoptosis when activated [14]

AP-33 interact in a mutually exclusive manner [15]

CCAC CCAC box is an essential element for muscle-specific transcription from the myoglobin promoter [16]

LIII-BP The L-III transcriptional regulatory element (a carbohydrate-response element) is in the pyruvate kinase L gene necessary for cell type-specific expression and transcriptional stimulation by carbohydrates [17]

ADD-1 (SREBP-1) activates transcription from sterol-regulated elements and from an E-box sequence present in the promoter of fatty acid synthase [18]

PAX6 Member of the paired box gene family transcriptional regulator involved in developmental processes [19]

48kD transcription factor activator protein-3 binds to the core element and recognizes the SV40 enhancer and AP-2 and AP-

(b)

Figure 3 Transcription factor activation Nuclear extracts from tumor muscle and bone were tested for DNA binding activity on a protein-DNA array (a) Transcription factor binding that was high in all 3 tissues and is therefore considered constitutively active is circled in yellow(left to right top to bottom AhRAmt GATA1 GATA2 GATA12 HIF1 and HOXD8910) Transcription factors that show high binding inthe cancer but not in muscle or bone are circled in red (left to right top to bottom E2F1 AP3 LIII-BP PAX6 ADD-1 and CCAC) (b) Thefunctions of transcription factors that display high DNA binding in the cancer but not in muscle or bone are described

the lack of ABCB1 overexpression is favorable for avoidingdrug resistance

37 Population Analysis The above-described results indi-cated that a FAF1 mutation which replaces serine in position181 thus preventing FAF1 phosphorylation and activationmay be a driver for leiomyosarcomagenesis A custom Taq-Man assay confirmed the presence of the somatic mutationin the patient To assess whether this single nucleotidereplacement is common in this type of cancer we analyzedDNA from 29 leiomyosarcomas and 1 blood sample from aleiomyosarcoma patient For comparison to nonsarcomatoustumors 34 breast cancers served as a reference None of themdisplayed amutation in the same locus By contrast there wasa distribution across all leiomyosarcomas in the osteopontin

promoter position minus443 (used as a reference) with 12 CC 10TC and 9 TT

4 Discussion

TheFAF1mutation identified as the likely cause for the cancerunder study gives room for an explanation of the sarcomatoustransformation (Figure 5) DNA damage to mesenchymalcells occurs persistently in an oxidizing environment at 37∘CThese insults are rarely transforming and such an occurrencewould trigger the initiation of programmed cell death inapoptosis-competent cells Intact FAF1 associates with FASand enhances apoptosis mediated through this receptor [12]A loss of function in FAF1 could lead to transformation viaantiapoptosis Whereas the mutation S181G is not expected

12 Sarcoma

Muscle

Bone

Tumor

220

94

6043

29

14

220

94

60

43

29

14

220

94

60

43

29

14

1

7

6

23

24

21

22

25

26

89 1011 12

1516 1719

pH 4 8 pH 4 8

8pH 4

(a)

SwissProt or NCBISpot number Protein identified accession Description

(Structural)6 Transgelin Q01995 Smooth muscle-specific cross-links actin24 Transgelin-2 P37802 Smooth muscle-specific cross-links actin

Vimentin P08670 Intermediate filament in mesenchymal tissue1 Filamin-A P21333 Actin-binding protein regulates the cytoskeleton 21 P06709 Cytoskeleton

(Calcium homeostasis)8 9 Calumenin O43852 Calcium-binding protein in the ER and golgi apparatus26 Protein S100-A11 P31949 Calcium-binding EF hand protein10 Reticulocalbin-3 Q96D15 Calcium-binding protein located in the ER12 P11021 Heat shock 70-kD protein 5 ER lumenal calcium-binding

(Protein processing)25 40S ribosomal protein S12 P25398 Core component of the ribosomal accuracy center7 Glycine-tRNA ligase P41250 Catalyzes the attachment of glycine to tRNA19 P01009 Protease inhibitor23 P01009 Protease inhibitor21 Proteasome activator complex subunit 2 Q9UL46 Proteasome activation10 P07900 Chaperone

(Other)7 Serum albumin P02768 Carrier protein in blood22 Protein disulfide isomerase (C-terminal fragment) P07237 Prolyl hydroxylation in collagen microsomal triglyceride transfer

120573 actin (C-terminal fragment)

78kD glucose-regulated protein

120572-1-antitrypsin120572-1-antitrypsin (C-terminal fragment)

Heat shock protein HSP 90120572 (N-terminal fragment)

11 15ndash17

(b)

Figure 4 Continued

Sarcoma 13

Tumor Muscle

OPN

CD44

STAT3

P-STAT3

Tubulin

75

75

50

15010075

10075

50

(c)

Tumor bone Tumor muscleGene ID Symbol Name Fold change Expression Fold change Expression6876 TAGLN Transgelin 3274 2455 3087 15088407 TAGLN2 Transgelin-2 2164 7338 6975 13037431 VIM Vimentin 1653 295010 4061 696042316 FLNA Filamin A alpha 1304 7791 9005 065160 ACTB Actin beta 0656 973187 5491 67368813 CALU Calumenin 7601 19328 12063 70596282 S100A11 S100 calcium binding protein A11 0931 158914 5071 1686357333 RCN3 Reticulocalbin 3 EF-hand calcium binding domain 2439 0604 6412 01223309 HSPA5 1068 92754 2607 220356696 SPP1 Secreted phosphoprotein 1 7572 46508 547929 0355960 CD44 CD44 molecule (Indian blood group) 2053 26708 15436 20566774 STAT3 Signal transducer and activator of transcription 3 0617 46534 0832 200165925 RB1 Retinoblastoma 1 0736 14772 0442 14268

Heat shock 70kDa protein 5 (glucose-regulated protein 78kDa)

(d)

Figure 4 Protein overexpression (a) 2D protein gel electrophoresis of lysates from muscle (upper left) tumor (upper right) and bone(bottom) in RIPA buffer Red circles indicate the spots that were identified as overexpressed and were further analyzed by mass spectrometry(b) Proteins identified in 2D gel electrophoresis as overexpressed in the tumor in comparison to muscle and bone were analyzed for theiridentity by mass spectrometry The left column indicates the spot number corresponding to the 2D gel The next column contains theprotein name followed by the accession number and a description of the protein functionThe protein functions are grouped into structuralcalcium homeostasis and various others (c) Western blot 10 120583g lysates of tumor muscle and bone in RIPA buffer were loaded per laneand electrophoresed on 10 SDS-polyacrylamide minigels with reducing denaturing sample buffer After transfer to PVDF membranesthey were probed for markers of cancer progression including osteopontin CD44 STAT3 and phospho-STAT3 Antitubulin served as aloading control (d) RNA levels corresponding to the proteins found to be affected in the sarcoma With four exceptions in the tumor-bonecomparison (gray font) upregulated proteins are associated with increased RNA levels STAT3 is not increased on the protein or RNA levelThe reduced level of RB1 expression is consistent with the elevated DNA binding activity of E2F1

to disrupt the structure of the protein this site does scorehigh as a possible phosphorylation site for a number ofkinases involved in DNA damage repair supporting thehypothesis that the cancer cells containing thismutation havelost their ability to respond to transforming DNA damagewith programmed cell death FAF1 antagonizes WNT signal-ing by promoting 120573-catenin degradation in the proteasome[21] a function that may be lost after the point mutationThe elevated DNA binding activity of the protooncogenictranscription factor E2F1 (see Figure 3) could be caused byits interaction with LEF-1 [20] consecutive to persistent

WNT signaling The RNA level of IGF2BP1 (IMP-1) a stress-responsive regulator of mRNA stability is highly upregulatedin the tumor compared to muscle as well as bone (seeTable 2(c)) IGF2BP1 is a transcriptional target of the WNTpathway that regulates NF-120581B activity (see Table 2(e)) andis antiapoptotic [22 23] IGF2BP1 may be upregulated asa consequence of mutated FAF1 not being able to suppressWNT signaling in this specific cancerOf noteWNTpathwayoveractivity may not be required for transformation ratherthe persistence of a WNT pathway signal due to the lack of aFAF1-mediated termination signal may suffice

14 Sarcoma

WNTFrizzled

Axin Dvl

igf2bp1

FAF1

P65 P50

IKK

FAS

120573-Catenin

LEF + E2F1

I120581B

Figure 5 Possible transforming pathway The point mutation inFAF1 is believed to cause a loss of function (crossed out in red)This may lead to an overactivity of the WNT signaling pathwayConsistently E2F1 (activated by LEF1) and igf2bp1 (a transcriptionaltarget of the WNT pathway) have been identified to be upregulatedin the molecular analysis In contrast to the negative regulator FAF1IGF2BP1 is a positive regulator of NF-120581B activityThe overactivity ofthe NF-120581B pathway and the reduced efficacy of FAS signaling can betransforming

The role ofWNT signaling in sarcoma has been subject todebate (eg [24]) In metastatic leiomyosarcoma 120573-Cateninmay accumulate in the nucleus despite a relatively weakexpression of WNT [25] This may be due to WNT signalactivation via noncanonical ligands [26] or to 120573-Cateninbinding the nuclear receptor NR4A2 and releasing it from thecorepressor protein LEF-1 [27] Of note in the cancer understudy here the mRNA level of NR4A2 was overexpressed10-fold compared to bone and 3-fold compared to muscleThe results from this study are consistent with the possibilitythat a lack of termination in the WNT signal rather than itsoveractivation could contribute to sarcomatous transforma-tion Such a mechanism may be reflected in an upregulationof downstream targets even though overexpression of WNTpathway components is not detectable

Other mutations beside FAF1 are less likely to becausative for the cancer EPHA3 was revealed as mutated inthis cancer by DNA exome sequencing and RNASeq EPHA3is a receptor tyrosine kinase that is frequentlymutated in lungcancer Tumor-suppressive effects of wild-type EPHA3 can beoverridden by dominant negative EPHA3 somatic mutations[28]Thismechanism is unlikely to play a role in this sarcomaas the detected mutation is located far N-terminally on theextracellular Ephrin binding domain not on the intracellularkinase domain

FAF1 has been described to act as a tumor suppressor gene[29] Its depletion due to chromosome breakage can affectprognosis in glioblastoma patients [30 31] Single nucleotidepolymorphisms in FAF1 are associated with a risk for gastriccancer [32] While numerous FAF1 mutations are associatedwith various cancers none of these genetic changes in theTCGA database affects the amino acid position 181 (Table 4)

The major upregulations identified in this tumor com-prise muscle-specific gene products (transcription factors

CAAC binding proteomics transgelin transgelin-2 RNAanoctamin-4 and immunohistochemistry smooth mus-cle actin) and calcium-regulating molecules (proteomicscalumenin S100-A11 reticulocalbin-3 and 78 kD glucose-regulated protein) The muscle-specific gene products con-firm this recurrent sarcoma as a leiomyosarcoma (the firsttumor distal to the site of the recurring one had beendiagnosed as an osteosarcoma) Calcium is one of the majorsecond messengers in smooth muscle cells Its uptake isregulated by potential-sensitive ion channels in the cellmembrane and by the activities of various receptors Calciumis stored in the sarcoplasmic reticulum from where it canbe released to facilitate actin-myosin interaction and tensiongeneration Phosphorylation of the myosin light chain by acalmodulin-regulated enzyme is important for contractionThe upregulation of gene products associated with migrationand invasion (osteopontin MMP1 vimentin filamin-A and120573-actin) and gene products for extracellularmatrixmoleculesand their modulators (fibronectin collagen ITGBL1 andMXRA5) reflects the invasive nature of this cancer

The recurrence of a sarcoma after 14 years has twoprobable explanations either it is due to a cancer pre-disposition syndrome based on a germ-line mutation (theage of the patient weakens this hypothesis) or the secondtumor is a metastatic colony of the first that was reactivatedafter dormancy The location of the sarcoma in the sameextremity and proximal to a preceding mesenchymal cancer(and therefore in its natural path of dissemination) impliedthe probability that this was a relapse in a metastatic siteThe different histologic assessment as osteosarcoma in thefirst occurrence and leiomyosarcoma as the second cancerdoes not necessarily negate that Mixed histology [33 34]and transdifferentiation [35ndash37] have been described forsarcomatous tumors Of note however in this scenarioosteosarcoma seems to more commonly follow leiomyosar-coma than precede it Material from the first cancer of thispatient was not accessible to us It is very plausible that thiscould have been a mineralized leiomyosarcoma In thosetumors the differential diagnosis from osteosarcoma can bedifficult [38] The extracompartmental location of the firsttumor supports this interpretation

On the molecular pathology level sarcomas fall intotwo groups comprising tumors with simple karyotypes(with pathogenetic translocations or specific genetic muta-tions) and tumors with very complex karyotypes (overtchromosome and genomic instability with numerous gainsand losses) [39] Some molecular alterations that lead tocarcinogenesis can be defined in absolute termsThey includegain-of-function mutations or chromosome translocationsthat transformprotooncogenes to oncogenes However otherchanges are relative to the normal tissue of origin suchas pathway overactivity or overexpression on the proteinor RNA levels We have combined the analysis of absolutechanges (DNA exome sequence RNA sequence) with theanalysis of relative changes using skeletal muscle and bone asreference organs (protein-DNA array 2D gel electrophoresisand RNA expression levels) This choice was determined inpart by tissue availability after surgery andwas intended to aidin the distinction of osteosarcoma from myosarcoma While

Sarcoma 15

Table 3 Deregulation of genes for drug disposition Analysis of RNASeq for at least twofold overexpression (italic font) or underexpression(bold font) of genes for (a) transport and (b) metabolism in tumor compared to muscle and bone For the export transporters (ABCtransporters) only overexpression is considered relevant for drug resistance Not shown in the table are the underexpressed genes (ABCA7ABCA8 ABCA13 ABCB6 ABCB10 ABCC6 ABCC6P1 ABCC6P2 ABCC8 ABCC11 ABCD2 ABCG2 and ABCG5)

(a) Transport

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

650655 ABCA17P ATP-binding cassette subfamily A (ABC1) member 17 pseudogene 1360 211624 ABCA4 ATP-binding cassette subfamily A (ABC1) member 4 2038 2802

6555 SLC10A2 Solute carrier family 10 (sodiumbile acid cotransporter family)member 2 minus1962 minus2112

345274 SLC10A6 Solute carrier family 10 (sodiumbile acid cotransporter family)member 6 2623 3240

6563 SLC14A1 Solute carrier family 14 (urea transporter) member 1 (Kidd bloodgroup) minus3074 minus3152

6565 SLC15A2 Solute carrier family 15 (H+peptide transporter) member 2 minus2027 minus2152

6566 SLC16A1 Solute carrier family 16 member 1 (monocarboxylic acid transporter1) 1401 2170

117247 SLC16A10 Solute carrier family 16 member 10 (aromatic amino acid transporter) 2113 2848

6567 SLC16A2 Solute carrier family 16 member 2 (monocarboxylic acid transporter8) 3242 3848

10786 SLC17A3 Solute carrier family 17 (sodium phosphate) member 3 minus2868 minus29596571 SLC18A2 Solute carrier family 18 (vesicular monoamine) member 2 minus2087 minus21946573 SLC19A1 Solute carrier family 19 (folate transporter) member 1 minus2099 minus220310560 SLC19A2 Solute carrier family 19 (thiamine transporter) member 2 1820 254880704 SLC19A3 Solute carrier family 19 member 3 minus3331 minus3478387775 SLC22A10 Solute carrier family 22 member 10 5711 60859390 SLC22A13 Solute carrier family 22 (organic anion transporter) member 13 2623 3217

85413 SLC22A16 Solute carrier family 22 (organic cationcarnitine transporter)member 16 minus8101 minus7299

51310 SLC22A17 Solute carrier family 22 member 17 1475 21755002 SLC22A18 Solute carrier family 22 member 18 2038 27226582 SLC22A2 Solute carrier family 22 (organic cation transporter) member 2 4793 5216

6581 SLC22A3 Solute carrier family 22 (extraneuronal monoamine transporter)member 3 2554 3182

6583 SLC22A4 Solute carrier family 22 (organic cationergothioneine transporter)member 4 minus3603 minus3776

151295 SLC23A3 Solute carrier family 23 (nucleobase transporters) member 3 2109 284810478 SLC25A17 Solute carrier family 25 (mitochondrial carrier) member 17 1311 207783733 SLC25A18 Solute carrier family 25 (mitochondrial carrier) member 18 1846 2570

788 SLC25A20 Solute carrier family 25 (carnitineacylcarnitine translocase) member20 minus2105 minus2207

89874 SLC25A21 Solute carrier family 25 (mitochondrial oxodicarboxylate carrier)member 21 minus2962 minus3105

51312 SLC25A37 Solute carrier family 25 member 37 minus4633 minus486651629 SLC25A39 Solute carrier family 25 member 39 minus2585 minus2737203427 SLC25A43 Solute carrier family 25 member 43 1325 208865012 SLC26A10 Solute carrier family 26 member 10 3896 4472115111 SLC26A7 Solute carrier family 26 member 7 3431 4018116369 SLC26A8 Solute carrier family 26 member 8 minus5354 minus5536115019 SLC26A9 Solute carrier family 26 member 9 1623 241911001 SLC27A2 Solute carrier family 27 (fatty acid transporter) member 2 minus4278 minus4487

16 Sarcoma

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

64078 SLC28A3 Solute carrier family 28 (sodium-coupled nucleoside transporter)member 3 minus4543 minus4812

222962 SLC29A4 Solute carrier family 29 (nucleoside transporters) member 4 minus1962 minus204581031 SLC2A10 Solute carrier family 2 (facilitated glucose transporter) member 10 2532 3170

6518 SLC2A5 Solute carrier family 2 (facilitated glucosefructose transporter)member 5 minus3647 minus3821

55532 SLC30A10 Solute carrier family 30 member 10 minus3547 minus37257782 SLC30A4 Solute carrier family 30 (zinc transporter) member 4 2832 33726569 SLC34A1 Solute carrier family 34 (sodium phosphate) member 1 minus4716 minus4941340146 SLC35D3 Solute carrier family 35 member D3 minus5662 minus590654733 SLC35F2 Solute carrier family 35 member F2 1433 2170206358 SLC36A1 Solute carrier family 36 (protonamino acid symporter) member 1 minus2265 minus2345285641 SLC36A3 Solute carrier family 36 (protonamino acid symporter) member 3 minus2284 minus239354020 SLC37A1 Solute carrier family 37 (glycerol-3-phosphate transporter) member 1 minus2112 minus22122542 SLC37A4 Solute carrier family 37 (glucose-6-phosphate transporter) member 4 minus2117 minus2219151258 SLC38A11 Solute carrier family 38 member 11 2410 308555089 SLC38A4 Solute carrier family 38 member 4 1837 254892745 SLC38A5 Solute carrier family 38 member 5 minus1994 minus215291252 SLC39A13 Solute carrier family 39 (zinc transporter) member 13 1301 207023516 SLC39A14 Solute carrier family 39 (zinc transporter) member 14 2569 318829985 SLC39A3 Solute carrier family 39 (zinc transporter) member 3 minus2032 minus2152283375 SLC39A5 Solute carrier family 39 (metal ion transporter) member 5 1623 23707922 SLC39A7 Solute carrier family 39 (zinc transporter) member 7 1273 205830061 SLC40A1 Solute carrier family 40 (iron-regulated transporter) member 1 minus3612 minus379684102 SLC41A2 Solute carrier family 41 member 2 1293 20708501 SLC43A1 Solute carrier family 43 member 1 minus2013 minus215257153 SLC44A2 Solute carrier family 44 member 2 minus2047 minus215250651 SLC45A1 Solute carrier family 45 member 1 2038 2722146802 SLC47A2 Solute carrier family 47 member 2 minus1962 minus20266521 SLC4A1 Solute carrier family 4 anion exchanger member 1 minus6513 minus645357282 SLC4A10 Solute carrier family 4 sodium bicarbonate transporter member 10 minus2640 minus275383959 SLC4A11 Solute carrier family 4 sodium borate transporter member 11 1846 25696508 SLC4A3 Solute carrier family 4 anion exchanger member 3 1261 20458671 SLC4A4 Solute carrier family 4 sodium bicarbonate cotransporter member 4 2445 31139497 SLC4A7 Solute carrier family 4 sodium bicarbonate cotransporter member 7 2717 33076523 SLC5A1 Solute carrier family 5 (sodiumglucose cotransporter) member 1 minus2547 minus2705159963 SLC5A12 Solute carrier family 5 (sodiumglucose cotransporter) member 12 4753 52066527 SLC5A4 Solute carrier family 5 (low affinity glucose cotransporter) member 4 minus3969 minus4249

6540 SLC6A13 Solute carrier family 6 (neurotransmitter transporter GABA)member 13 1328 2092

55117 SLC6A15 Solute carrier family 6 (neutral amino acid transporter) member 15 1623 2333388662 SLC6A17 Solute carrier family 6 member 17 2038 272854716 SLC6A20 Solute carrier family 6 (proline IMINO transporter) member 20 1697 2433

6532 SLC6A4 Solute carrier family 6 (neurotransmitter transporter serotonin)member 4 minus2512 minus2611

6534 SLC6A7 Solute carrier family 6 (neurotransmitter transporter L-proline)member 7 2623 3228

56301 SLC7A10 Solute carrier family 7 (neutral amino acid transporter y+ system)member 10 minus3421 minus3603

Sarcoma 17

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

6547 SLC8A3 Solute carrier family 8 (sodiumcalcium exchanger) member 3 minus2204 minus2309285335 SLC9A10 Solute carrier family 9 member 10 1208 20006549 SLC9A2 Solute carrier family 9 (sodiumhydrogen exchanger) member 2 2038 2706

9368 SLC9A3R1 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 1 minus2760 minus2846

9351 SLC9A3R2 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 2 1889 2619

84679 SLC9A7 Solute carrier family 9 (sodiumhydrogen exchanger) member 7 3347 393510599 SLCO1B1 Solute carrier organic anion transporter family member 1B1 3360 397728234 SLCO1B3 Solute carrier organic anion transporter family member 1B3 9760 95696578 SLCO2A1 Solute carrier organic anion transporter family member 2A1 2432 309628232 SLCO3A1 Solute carrier organic anion transporter family member 3A1 minus2032 minus2152353189 SLCO4C1 Solute carrier organic anion transporter family member 4C1 minus6850 minus6611

(b) Metabolism

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold Cchange

1583 CYP11A1 Cytochrome P450 family 11 subfamily A polypeptide 1 1623 23961589 CYP21A2 Cytochrome P450 family 21 subfamily A polypeptide 2 minus1962 minus20361591 CYP24A1 Cytochrome P450 family 24 subfamily A polypeptide 1 5208 55771592 CYP26A1 Cytochrome P450 family 26 subfamily A polypeptide 1 2623 32571594 CYP27B1 Cytochrome P450 family 27 subfamily B polypeptide 1 1846 2585339761 CYP27C1 Cytochrome P450 family 27 subfamily C polypeptide 1 3585 41781553 CYP2A13 Cytochrome P450 family 2 subfamily A polypeptide 13 minus1962 minus20351580 CYP4B1 Cytochrome P450 family 4 subfamily B polypeptide 1 minus4820 minus506566002 CYP4F12 Cytochrome P450 family 4 subfamily F polypeptide 12 minus6070 minus61958529 CYP4F2 Cytochrome P450 family 4 subfamily F polypeptide 2 minus7769 minus70604051 CYP4F3 Cytochrome P450 family 4 subfamily F polypeptide 3 minus8244 minus749111283 CYP4F8 Cytochrome P450 family 4 subfamily F polypeptide 8 minus5006 minus5270260293 CYP4X1 Cytochrome P450 family 4 subfamily X polypeptide 1 minus2476 minus25809420 CYP7B1 Cytochrome P450 family 7 subfamily B polypeptide 1 2502 31702326 FMO1 Flavin containing monooxygenase 1 4360 48592327 FMO2 Flavin containing monooxygenase 2 (nonfunctional) minus4074 minus43372328 FMO3 Flavin containing monooxygenase 3 minus4036 minus4309388714 FMO6P Flavin containing monooxygenase 6 pseudogene minus2569 minus2737493869 GPX8 Glutathione peroxidase 8 (putative) 2814 33712938 GSTA1 Glutathione S-transferase alpha 1 1623 23632939 GSTA2 Glutathione S-transferase alpha 2 2038 27412941 GSTA4 Glutathione S-transferase alpha 4 1492 21882953 GSTT2 Glutathione S-transferase theta 2 4682 5170653689 GSTT2B Glutathione S-transferase theta 2B (genepseudogene) 3739 430784779 NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439 79969027 NAT8 N-acetyltransferase 8 (GCN5-related putative) minus2769 minus29207358 UGDH UDP-glucose 6-dehydrogenase 2333 301855757 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 1604 227110720 UGT2B11 UDP glucuronosyltransferase 2 family polypeptide B11 minus3586 minus37687367 UGT2B17 UDP glucuronosyltransferase 2 family polypeptide B17 minus2836 minus295954490 UGT2B28 UDP glucuronosyltransferase 2 family polypeptide B28 minus3132 minus3284167127 UGT3A2 UDP glycosyltransferase 3 family polypeptide A2 minus4716 minus4907

18 Sarcoma

Table 4 FAF1 mutations in various cancers FAF1 mutations listed in the TCGA data base were identified without restriction to any type ofcancer For the location of the affected domains on the protein compare Figure 2

AA Mutation Cancer DomainN4S Missense Cutaneous melanomaI10S Missense Stomach adenocarcinoma UBAE21lowast Nonsense Cutaneous melanoma UBAE25K Missense Uterine endometrioid carcinoma UBAV38 splice Splice Stomach adenocarcinoma UBAP86fs FS del Colorectal adenocarcinomaG123 splice Splice Uterine endometrioid carcinoma UB1P136H Missense Colorectal adenocarcinoma UB1T147M Missense Brain lower grade glioma UB1D149Y Missense Uterine endometrioid carcinoma UB1L159V Missense Lung adenocarcinoma UB1K163N Missense Uterine endometrioid carcinoma UB1L170F Missense Cutaneous melanomaG184 splice Splice Colorectal cancerQ187H Missense Stomach adenocarcinomaS214N Missense Colorectal adenocarcinoma UB2R222I Missense Uterine endometrioid carcinoma UB2E238D Missense Lung adenocarcinoma UB2P241S Missense Uterine endometrioid carcinoma UB2T245A Missense Renal clear cell carcinoma UB2M249V Missense Uterine endometrioid carcinoma UB2E280K Missense Brain lower grade gliomaG293lowast Nonsense Colorectal cancerT300I Missense Colorectal adenocarcinomaD305H Missense Lung adenocarcinomaE308Q Missense Lung adenocarcinomaA316V Missense Stomach adenocarcinomaK319fs FS ins Head and neck squamous cell carcinomaR344G Missense Stomach adenocarcinoma UASF353I Missense Cutaneous melanoma UASL379V Missense Breast invasive carcinoma UASC396F Missense Cutaneous melanoma UASS459lowast Nonsense Uterine endometrioid carcinoma UASG469 splice Splice Glioblastoma multiforme UASR509G Missense Lung squamous cell carcinomaE510fs FS del Cutaneous melanomaR516C Missense Uterine endometrioid carcinomaA534V Missense Stomach adenocarcinomaF537L Missense Stomach adenocarcinomaE551lowast Nonsense Lung adenocarcinomaR554W Missense Colorectal cancerS582I Missense Lung adenocarcinoma UBXF585L Missense Uterine endometrioid carcinoma UBXE587lowast Nonsense Lung adenocarcinoma UBXA592V Missense Stomach adenocarcinoma UBXW610lowast Nonsense Breast invasive carcinoma UBXD611Y Missense Lung adenocarcinoma UBXE635fs FS del Brain lower grade glioma UBXP640fs FS del Cutaneous melanoma UBX

Sarcoma 19

a more accurate reference point for a leiomyosarcoma wouldhave been smooth muscle we believe that the comparison tostriated muscle is sufficient to allow the assessment of tumorspecific changes We have measured RNA DNA and proteinwith various assays Similar assessments in the future shouldalso include chromosome analysis for possible translocations

In the first-line defense against cancer the gradualreplacement of conventional chemotherapy with molecularlytargeted agents has opened the possibility to tailor drug treat-ments to particular tumors This transition necessitates thecharacterization of the molecular lesions that are causativefor the transformation of healthy cells into cancerous cellsbecause drugs need to be matched with the underlying carci-nogenic defect to be effective An additional caveat causedby unique genetic changes in the primary tumor can affectdrug transport and metabolism and needs to be taken intoconsideration In this case the RNA levels for genes that regu-late transport andmetabolismwere extensively skewed in thetumor tissue as compared to muscle and bone (see Table 3)implying potential challenges to chemotherapy An advancedmolecular treatment strategy for cancer will rely on themolecular definition of drug target drug transport and drugmetabolism pharmacogenetics in the primary tumor Con-secutive to cancer dissemination it will also require adjust-ments to account for the genetic changes in the metastases

The cost of health care has been under increasing scrutinyThe treatment of cancer patients is expensive in particularwhen hospitalization is required and further in cases ofend-of-life care Avoidable expenditures are generated bysuboptimal treatment decisions that result in low efficacy orhigh toxicity of anticancer regimens Molecular medicine hasthe potential to preempt those problems and reduce wastefulspending Yet it requires the upfront cost of molecular cancerexamination The analysis performed in this study requiredabout $11000- in nonsalary expenses to perform While ithas not identified a drug target it has specified possibleconfines for drug treatment The costs for molecular analysisneed to be weighed against the societal cost derived fromlost productivity in the workforce disrupted lives of familiesand premature deaths While currently limited drug optionsconstitute the major constraint to the approach taken herethe foreseeable future will bring an increasing spectrum ofmolecularly targeted drugs along with faster and cheapertechnologies for the molecular assessment of cancers Theywill facilitate the clinical translation of our approach

Consent

The patient provided informed consent

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by DOD Grant PR094070under University of Cincinnati IRB protocol 04-01-29-01The

author is grateful to Dr Rhett Kovall for helping with thestructural analysis of FAF1

References

[1] G F Weber Molecular Mechanisms of Cancer Springer Dor-drecht The Netherlands 2007

[2] N G Sanerkin ldquoPrimary leiomyosarcoma of the bone and itscomparison with fibrosarcomardquoCancer vol 44 no 4 pp 1375ndash1387 1979

[3] J M Weaver and J A Abraham ldquoLeiomyosarcoma of the Boneand Soft Tissue A Reviewrdquo httpsarcomahelporglearningcenterleiomyosarcomahtml

[4] M A Depristo E Banks R Poplin et al ldquoA framework forvariation discovery and genotyping using next-generationDNAsequencing datardquo Nature Genetics vol 43 no 5 pp 491ndash5012011

[5] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[6] C W Menges D A Altomare and J R Testa ldquoFAS-associatedfactor 1 (FAF1) diverse functions and implications for oncoge-nesisrdquo Cell Cycle vol 8 no 16 pp 2528ndash2534 2009

[7] M Kim J H Lee S Y Lee E Kim and J Chung ldquoCaspar asuppressor of antibacterial immunity in Drosophilardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 44 pp 16358ndash16363 2006

[8] J Song K P Joon J-J Lee et al ldquoStructure and interaction ofubiquitin-associated domain of human Fas-associated factor 1rdquoProtein Science vol 18 no 11 pp 2265ndash2276 2009

[9] P H OrsquoFarrell ldquoHigh resolution two dimensional electrophore-sis of proteinsrdquo Journal of Biological Chemistry vol 250 no 10pp 4007ndash4021 1975

[10] A Burgess-Cassler J J Johansen D A Santek J R Ideand N C Kendrick ldquoComputerized quantitative analysis ofCoomassie-Blue-stained serum proteins separated by two-dimensional electrophoresisrdquo Clinical Chemistry vol 35 no 12pp 2297ndash2304 1989

[11] B R Oakley D R Kirsch and N RMorris ldquoA simplified ultra-sensitive silver stain for detecting proteins in polyacrylamidegelsrdquo Analytical Biochemistry vol 105 no 2 pp 361ndash363 1980

[12] K Chu X Niu and L T Williams ldquoA Fas-associated proteinfactor FAF1 potentiates Fas-mediated apoptosisrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 25 pp 11894ndash11898 1995

[13] B Rikhof S de Jong A J H Suurmeijer C Meijer and W TA van der Graaf ldquoThe insulin-like growth factor system andsarcomasrdquoThe Journal of Pathology vol 217 no 4 pp 469ndash4822009

[14] RGirling J F Partridge L R Bandara et al ldquoAnew componentof the transcription factor DRTF1E2Frdquo Nature vol 362 no6415 pp 83ndash87 1993

[15] F Mercurio and M Karin ldquoTranscription factors AP-3 andAP-2 interact with the SV40 enhancer in a mutually exclusivemannerrdquo EMBO Journal vol 8 no 5 pp 1455ndash1460 1989

[16] R Bassel-Duby M D Hernandez M A Gonzalez J KKrueger and R S Williams ldquoA 40-kilodalton protein bindsspecifically to an upstream sequence element essential for mus-cle-specific transcription of the human myoglobin promoterrdquoMolecular and Cellular Biology vol 12 no 11 pp 5024ndash50321992

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 8: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

8 Sarcoma

(b) Continued

log2-fold changeBone

STC1 Stanniocalcin 1 7385FAM180A Family with sequence similarity 180 member A 7315KRT17 Keratin 17 7305BBOX1 Butyrobetaine (gamma) 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 7277MAGEA1 Melanoma antigen family A 1 (directs expression of antigen MZ2-E) 7267

(c)

log2-fold changeMuscle Bone

Tumor over bonemuscle

ANO4 Anoctamin 4 (TMEM16D) transmembrane calcium-activated chloride channelfacilitates smooth muscle contraction 9937 8542

SLCO1B3 (OATP1B3) Solute carrier organic anion transporter family member 1B3 9569 9760

MARCH4 Membrane-associated ring finger (C3HC4) 4 E3 ubiquitin ligase located predominantlyto the endoplasmic reticulum 9538 7406

IGF2BP1 Insulin-like growth factor 2 mRNA binding protein 1 binds to and stabilizes mRNA 9440 9631ADAMTS16 ADAMmetallopeptidase with thrombospondin type 1 motif 16 zinc-dependent protease 9260 8450SOX11 SRY (sex determining region Y)-box 11 important in brain development 9178 9954

HAPLN1 Hyaluronan and proteoglycan link protein 1 stabilizes aggregates of aggrecan andhyaluronan giving cartilage its tensile strength and elasticity 8951 8142

MUC15 Mucin 15 cell surface associated 8801 7992HOXB9 Homeobox B9 8773 7378MAGEC2 Melanoma antigen family C 2 8693 8884

ST6GALNAC5 ST6 (alpha-N-acetyl-neuraminyl-23-beta-galactosyl-13)-N-acetylgalactosaminidealpha-26-sialyltransferase 5 7848 8038

C11orf41 Chromosome 11 open reading frame 41 7781 8141MMP1 Matrix metallopeptidase 1 (interstitial collagenase) 7455 7646

(d)

Symbol Name log2-fold change log2-fold changeMuscle Bone

Tumor over bonemuscleFN1 Fibronectin 1 7328 6293 6457 19860COL1A1 Collagen type I alpha 1 6768 3706 5868 11934CCND1 Cyclin D1 5595 3958 5098 9637RGS1 Regulator of G-protein signaling 1 5118 1056 4653 2533ITGBL1 Integrin beta-like 1 (with EGF-like repeat domains) 5071 3177 3895 12415COL1A2 Collagen type I alpha 2 4852 8756 4910 14503MXRA5 Matrix-remodelling associated 5 4834 1352 4631 2685POSTN Periostin osteoblast specific factor 4513 5870 7497 1267PLOD2 Procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 4285 1801 4023 3730COL5A2 Collagen type V alpha 2 4275 1297 4833 1517COL3A1 Collagen type III alpha 1 4003 13118 5801 6500

SEMA3C Sema domain immunoglobulin domain (Ig) shortbasic domain secreted 3954 1164 4215 1675

FBN1 Fibrillin 1 3912 6957 4018 11148AEBP1 AE binding protein 1 3808 3212 4002 4843SIK1 Salt-inducible kinase 1 3805 1219 3566 2484

Sarcoma 9

(d) Continued

Symbol Name log2-fold change log2-fold changeMuscle Bone

SERPINH1 Serpin peptidase inhibitor clade H (heat shock protein47) member 1 3706 1652 4145 2098

ANTXR1 Anthrax toxin receptor 1 3670 3789 3690 6445

(e)

Symbol Name log2-fold change log2-fold changeMuscle Bone

HELLS Helicase lymphoid-specific 5315602 0155898 minus082713 202489

TNFRSF11A Tumor necrosis factor receptor superfamily member 11a andNFKB activator 3017922 003091 1400993 0202453

NFKBID Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor delta 2655352 0 minus147615 0504341

TNFRSF25 Tumor necrosis factor receptor superfamily member 25 2432959 0046388 0163954 0490795

NFKBIE Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor epsilon 2121015 0062395 0311441 043382

TNF Tumor necrosis factor 1847997 0 minus142101 003733

TNFRSF10D Tumor necrosis factor receptor superfamily member 10d decoywith truncated death domain 1754888 0172968 minus01757 1183343

TNFRSF1B Tumor necrosis factor receptor superfamily member 1B 1473438 0709902 minus097011 6729401TNFRSF10A Tumor necrosis factor receptor superfamily member 10a 1411898 0828219 0357701 3004386

NFKBIB Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor beta 1193993 1209816 046055 3484692

TNFRSF10B Tumor necrosis factor receptor superfamily member 10b 1094157 1908597 0425446 5242006TNFRSF21 Tumor necrosis factor receptor superfamily member 21 1055762 3882038 0745815 8305711TNFRSF1A Tumor necrosis factor receptor superfamily member 1A 0875167 3790938 minus011707 130344

NFKBIZ Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor zeta 0575974 3698951 0344657 7493136

RIPK1 Receptor (TNFRSF)-interacting serine-threonine kinase 1 0494467 311749 minus108854 161392NKRF NFKB repressing factor 0475722 2156087 minus086397 9434166NFKB1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 0432959 2054532 minus065865 7568586CHUK Conserved helix-loop-helix ubiquitous kinase 0176126 2961281 minus120204 1330333RELA V-rel reticuloendotheliosis viral oncogene homolog A (avian) 0013848 1263837 0287167 1803044

NFKB2 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 2(p49p100) minus008641 0312993 0485882 0360442

NKAPP1 NFKB activating protein pseudogene 1 minus010692 0825177 minus099449 2655392

TNFRSF10C Tumor necrosis factor receptor superfamily member 10c decoywithout an intracellular domain minus0152 0064676 minus556891 6321515

NKIRAS2 NFKB inhibitor interacting Ras-like 2 minus060768 1095135 minus088758 2299946

NFKBIL1 Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor-like 1 minus08719 0141933 minus008357 0140418

NKIRAS1 NFKB inhibitor interacting Ras-like 1 minus087428 6296399 1467735 2122983TANK TRAF family member-associated NFKB activator minus0983 6777124 minus151908 1695872NKAP NFKB activating protein minus135735 1422458 minus078243 1646287

NFKBIA Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor alpha minus185483 4032743 minus031802 2395275

NKAPL NFKB activating protein-like minus557347 5875743 minus140215 0534643

10 Sarcoma

FAS associating region

DED interacting region

UB12 DZM

UB2domain

UASdomain

UBXdomain

UBAdomain

S289 S291

Aurora ACK II

S181

AKT1ATMGSK3

AuroraPKG

PLK1RSK

STK4CHK1

650566481336260205169110475

(a)

MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRM

6

1

101

100

200

300

400

500

600

650

201

301

401

501

601

667855899998876467765678888887578758999986467 88 8888888888888988888899988888889888877767888777888757

2222454345655744642554435764462443558468643653433344333424435334233333332333332333354335333344334354

LDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITH

9999997797689998787758999999999859996577754788888888875333467888868987688888888888877888855675799987

9585847535375857543447459666955563844544585374435443365845846434457567533353232334334444433337484944

REVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDAT

5888757887577654788887766654677776555468877888765455546887766555688888877777777777776556788888767766

5344456484742545656645594364384534437445444342323455453535435463434323333343434333333334333335433333

EFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESI

5666776654456777888888888888866889999999998848888876677778999999998776686899998589875579999987486899

4635343574354322343434533333343548479747945564424647635575478569766456468999999754233454687457844669

VSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDE

9999888589997557877545444345677876335554467888876899986799876447777677888999999998877555778887777778

6778547999996674433343444333333335466456434433487999895353333464346434365468755846444545454565446565

REARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVF

8888888887788877766545557777777666777777777776678877777766578888888888885799998589975788758987589999

4545445564445344433443354555556653665456544454434444444443344334333333446859596743354745795535796898

DFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKE

99997799988779985788754577888765665678898589998589

67974525444795877564635844444476875837446989897366

(b)

Figure 2 FAF1 structure (a) A schematic of functional domains in FAF1 with annotations (adapted from [6ndash8]) There are two ubiquitin-like domains flanking the S181 site The PDB structure 2DZM identifies the N-terminal one while the C-terminal prediction is by sequencesimilarity Whereas the mutation S181G is not expected to disrupt the protein structure per se this amino acid has a high score as a possiblephosphorylation site for a number of kinases involved inDNAdamage repair (b) Secondary structure prediction of FAF1The residues aroundS181 appear unstructured

reflected in STAT3 constitutive phosphorylation The lack ofphosphorylation in this case suggests that the leiomyosar-coma may not depend on the STAT3 pathway

36 Pharmacogenetic Evaluation Predicting the sensitivityto anticancer drugs is a main goal of molecular analysisFor this the over- or underexpression of genes for drugtransport and metabolism is of key importance Analysis ofthe RNASeq data for these groups of gene products identified

a surprisingly large number of deregulations compared tomuscle or bone (Tables 3(a) and 3(b)) Those alterationsmay affect choices for drug treatment For example the highlevels of glutathione S-transferase may render carmustinethioTEPA cisplatin chlorambucil melphalan nitrogenmus-tard phosphoramide mustard acrolein or steroids ineffec-tive The overexpression ofN-acetyltransferase may compro-mise 5-fluorouracil or taxolThemodest upregulation of onlytwo export transporters (ABC-transporters) and specifically

Sarcoma 11

Bone Tumor

Muscle

(a)

Transcription factor Description Reference

E2F1 Transcriptional activation of cell cycle progression is selectively activated in response to specific cues key downstream target of RB1 can promote proliferation or apoptosis when activated [14]

AP-33 interact in a mutually exclusive manner [15]

CCAC CCAC box is an essential element for muscle-specific transcription from the myoglobin promoter [16]

LIII-BP The L-III transcriptional regulatory element (a carbohydrate-response element) is in the pyruvate kinase L gene necessary for cell type-specific expression and transcriptional stimulation by carbohydrates [17]

ADD-1 (SREBP-1) activates transcription from sterol-regulated elements and from an E-box sequence present in the promoter of fatty acid synthase [18]

PAX6 Member of the paired box gene family transcriptional regulator involved in developmental processes [19]

48kD transcription factor activator protein-3 binds to the core element and recognizes the SV40 enhancer and AP-2 and AP-

(b)

Figure 3 Transcription factor activation Nuclear extracts from tumor muscle and bone were tested for DNA binding activity on a protein-DNA array (a) Transcription factor binding that was high in all 3 tissues and is therefore considered constitutively active is circled in yellow(left to right top to bottom AhRAmt GATA1 GATA2 GATA12 HIF1 and HOXD8910) Transcription factors that show high binding inthe cancer but not in muscle or bone are circled in red (left to right top to bottom E2F1 AP3 LIII-BP PAX6 ADD-1 and CCAC) (b) Thefunctions of transcription factors that display high DNA binding in the cancer but not in muscle or bone are described

the lack of ABCB1 overexpression is favorable for avoidingdrug resistance

37 Population Analysis The above-described results indi-cated that a FAF1 mutation which replaces serine in position181 thus preventing FAF1 phosphorylation and activationmay be a driver for leiomyosarcomagenesis A custom Taq-Man assay confirmed the presence of the somatic mutationin the patient To assess whether this single nucleotidereplacement is common in this type of cancer we analyzedDNA from 29 leiomyosarcomas and 1 blood sample from aleiomyosarcoma patient For comparison to nonsarcomatoustumors 34 breast cancers served as a reference None of themdisplayed amutation in the same locus By contrast there wasa distribution across all leiomyosarcomas in the osteopontin

promoter position minus443 (used as a reference) with 12 CC 10TC and 9 TT

4 Discussion

TheFAF1mutation identified as the likely cause for the cancerunder study gives room for an explanation of the sarcomatoustransformation (Figure 5) DNA damage to mesenchymalcells occurs persistently in an oxidizing environment at 37∘CThese insults are rarely transforming and such an occurrencewould trigger the initiation of programmed cell death inapoptosis-competent cells Intact FAF1 associates with FASand enhances apoptosis mediated through this receptor [12]A loss of function in FAF1 could lead to transformation viaantiapoptosis Whereas the mutation S181G is not expected

12 Sarcoma

Muscle

Bone

Tumor

220

94

6043

29

14

220

94

60

43

29

14

220

94

60

43

29

14

1

7

6

23

24

21

22

25

26

89 1011 12

1516 1719

pH 4 8 pH 4 8

8pH 4

(a)

SwissProt or NCBISpot number Protein identified accession Description

(Structural)6 Transgelin Q01995 Smooth muscle-specific cross-links actin24 Transgelin-2 P37802 Smooth muscle-specific cross-links actin

Vimentin P08670 Intermediate filament in mesenchymal tissue1 Filamin-A P21333 Actin-binding protein regulates the cytoskeleton 21 P06709 Cytoskeleton

(Calcium homeostasis)8 9 Calumenin O43852 Calcium-binding protein in the ER and golgi apparatus26 Protein S100-A11 P31949 Calcium-binding EF hand protein10 Reticulocalbin-3 Q96D15 Calcium-binding protein located in the ER12 P11021 Heat shock 70-kD protein 5 ER lumenal calcium-binding

(Protein processing)25 40S ribosomal protein S12 P25398 Core component of the ribosomal accuracy center7 Glycine-tRNA ligase P41250 Catalyzes the attachment of glycine to tRNA19 P01009 Protease inhibitor23 P01009 Protease inhibitor21 Proteasome activator complex subunit 2 Q9UL46 Proteasome activation10 P07900 Chaperone

(Other)7 Serum albumin P02768 Carrier protein in blood22 Protein disulfide isomerase (C-terminal fragment) P07237 Prolyl hydroxylation in collagen microsomal triglyceride transfer

120573 actin (C-terminal fragment)

78kD glucose-regulated protein

120572-1-antitrypsin120572-1-antitrypsin (C-terminal fragment)

Heat shock protein HSP 90120572 (N-terminal fragment)

11 15ndash17

(b)

Figure 4 Continued

Sarcoma 13

Tumor Muscle

OPN

CD44

STAT3

P-STAT3

Tubulin

75

75

50

15010075

10075

50

(c)

Tumor bone Tumor muscleGene ID Symbol Name Fold change Expression Fold change Expression6876 TAGLN Transgelin 3274 2455 3087 15088407 TAGLN2 Transgelin-2 2164 7338 6975 13037431 VIM Vimentin 1653 295010 4061 696042316 FLNA Filamin A alpha 1304 7791 9005 065160 ACTB Actin beta 0656 973187 5491 67368813 CALU Calumenin 7601 19328 12063 70596282 S100A11 S100 calcium binding protein A11 0931 158914 5071 1686357333 RCN3 Reticulocalbin 3 EF-hand calcium binding domain 2439 0604 6412 01223309 HSPA5 1068 92754 2607 220356696 SPP1 Secreted phosphoprotein 1 7572 46508 547929 0355960 CD44 CD44 molecule (Indian blood group) 2053 26708 15436 20566774 STAT3 Signal transducer and activator of transcription 3 0617 46534 0832 200165925 RB1 Retinoblastoma 1 0736 14772 0442 14268

Heat shock 70kDa protein 5 (glucose-regulated protein 78kDa)

(d)

Figure 4 Protein overexpression (a) 2D protein gel electrophoresis of lysates from muscle (upper left) tumor (upper right) and bone(bottom) in RIPA buffer Red circles indicate the spots that were identified as overexpressed and were further analyzed by mass spectrometry(b) Proteins identified in 2D gel electrophoresis as overexpressed in the tumor in comparison to muscle and bone were analyzed for theiridentity by mass spectrometry The left column indicates the spot number corresponding to the 2D gel The next column contains theprotein name followed by the accession number and a description of the protein functionThe protein functions are grouped into structuralcalcium homeostasis and various others (c) Western blot 10 120583g lysates of tumor muscle and bone in RIPA buffer were loaded per laneand electrophoresed on 10 SDS-polyacrylamide minigels with reducing denaturing sample buffer After transfer to PVDF membranesthey were probed for markers of cancer progression including osteopontin CD44 STAT3 and phospho-STAT3 Antitubulin served as aloading control (d) RNA levels corresponding to the proteins found to be affected in the sarcoma With four exceptions in the tumor-bonecomparison (gray font) upregulated proteins are associated with increased RNA levels STAT3 is not increased on the protein or RNA levelThe reduced level of RB1 expression is consistent with the elevated DNA binding activity of E2F1

to disrupt the structure of the protein this site does scorehigh as a possible phosphorylation site for a number ofkinases involved in DNA damage repair supporting thehypothesis that the cancer cells containing thismutation havelost their ability to respond to transforming DNA damagewith programmed cell death FAF1 antagonizes WNT signal-ing by promoting 120573-catenin degradation in the proteasome[21] a function that may be lost after the point mutationThe elevated DNA binding activity of the protooncogenictranscription factor E2F1 (see Figure 3) could be caused byits interaction with LEF-1 [20] consecutive to persistent

WNT signaling The RNA level of IGF2BP1 (IMP-1) a stress-responsive regulator of mRNA stability is highly upregulatedin the tumor compared to muscle as well as bone (seeTable 2(c)) IGF2BP1 is a transcriptional target of the WNTpathway that regulates NF-120581B activity (see Table 2(e)) andis antiapoptotic [22 23] IGF2BP1 may be upregulated asa consequence of mutated FAF1 not being able to suppressWNT signaling in this specific cancerOf noteWNTpathwayoveractivity may not be required for transformation ratherthe persistence of a WNT pathway signal due to the lack of aFAF1-mediated termination signal may suffice

14 Sarcoma

WNTFrizzled

Axin Dvl

igf2bp1

FAF1

P65 P50

IKK

FAS

120573-Catenin

LEF + E2F1

I120581B

Figure 5 Possible transforming pathway The point mutation inFAF1 is believed to cause a loss of function (crossed out in red)This may lead to an overactivity of the WNT signaling pathwayConsistently E2F1 (activated by LEF1) and igf2bp1 (a transcriptionaltarget of the WNT pathway) have been identified to be upregulatedin the molecular analysis In contrast to the negative regulator FAF1IGF2BP1 is a positive regulator of NF-120581B activityThe overactivity ofthe NF-120581B pathway and the reduced efficacy of FAS signaling can betransforming

The role ofWNT signaling in sarcoma has been subject todebate (eg [24]) In metastatic leiomyosarcoma 120573-Cateninmay accumulate in the nucleus despite a relatively weakexpression of WNT [25] This may be due to WNT signalactivation via noncanonical ligands [26] or to 120573-Cateninbinding the nuclear receptor NR4A2 and releasing it from thecorepressor protein LEF-1 [27] Of note in the cancer understudy here the mRNA level of NR4A2 was overexpressed10-fold compared to bone and 3-fold compared to muscleThe results from this study are consistent with the possibilitythat a lack of termination in the WNT signal rather than itsoveractivation could contribute to sarcomatous transforma-tion Such a mechanism may be reflected in an upregulationof downstream targets even though overexpression of WNTpathway components is not detectable

Other mutations beside FAF1 are less likely to becausative for the cancer EPHA3 was revealed as mutated inthis cancer by DNA exome sequencing and RNASeq EPHA3is a receptor tyrosine kinase that is frequentlymutated in lungcancer Tumor-suppressive effects of wild-type EPHA3 can beoverridden by dominant negative EPHA3 somatic mutations[28]Thismechanism is unlikely to play a role in this sarcomaas the detected mutation is located far N-terminally on theextracellular Ephrin binding domain not on the intracellularkinase domain

FAF1 has been described to act as a tumor suppressor gene[29] Its depletion due to chromosome breakage can affectprognosis in glioblastoma patients [30 31] Single nucleotidepolymorphisms in FAF1 are associated with a risk for gastriccancer [32] While numerous FAF1 mutations are associatedwith various cancers none of these genetic changes in theTCGA database affects the amino acid position 181 (Table 4)

The major upregulations identified in this tumor com-prise muscle-specific gene products (transcription factors

CAAC binding proteomics transgelin transgelin-2 RNAanoctamin-4 and immunohistochemistry smooth mus-cle actin) and calcium-regulating molecules (proteomicscalumenin S100-A11 reticulocalbin-3 and 78 kD glucose-regulated protein) The muscle-specific gene products con-firm this recurrent sarcoma as a leiomyosarcoma (the firsttumor distal to the site of the recurring one had beendiagnosed as an osteosarcoma) Calcium is one of the majorsecond messengers in smooth muscle cells Its uptake isregulated by potential-sensitive ion channels in the cellmembrane and by the activities of various receptors Calciumis stored in the sarcoplasmic reticulum from where it canbe released to facilitate actin-myosin interaction and tensiongeneration Phosphorylation of the myosin light chain by acalmodulin-regulated enzyme is important for contractionThe upregulation of gene products associated with migrationand invasion (osteopontin MMP1 vimentin filamin-A and120573-actin) and gene products for extracellularmatrixmoleculesand their modulators (fibronectin collagen ITGBL1 andMXRA5) reflects the invasive nature of this cancer

The recurrence of a sarcoma after 14 years has twoprobable explanations either it is due to a cancer pre-disposition syndrome based on a germ-line mutation (theage of the patient weakens this hypothesis) or the secondtumor is a metastatic colony of the first that was reactivatedafter dormancy The location of the sarcoma in the sameextremity and proximal to a preceding mesenchymal cancer(and therefore in its natural path of dissemination) impliedthe probability that this was a relapse in a metastatic siteThe different histologic assessment as osteosarcoma in thefirst occurrence and leiomyosarcoma as the second cancerdoes not necessarily negate that Mixed histology [33 34]and transdifferentiation [35ndash37] have been described forsarcomatous tumors Of note however in this scenarioosteosarcoma seems to more commonly follow leiomyosar-coma than precede it Material from the first cancer of thispatient was not accessible to us It is very plausible that thiscould have been a mineralized leiomyosarcoma In thosetumors the differential diagnosis from osteosarcoma can bedifficult [38] The extracompartmental location of the firsttumor supports this interpretation

On the molecular pathology level sarcomas fall intotwo groups comprising tumors with simple karyotypes(with pathogenetic translocations or specific genetic muta-tions) and tumors with very complex karyotypes (overtchromosome and genomic instability with numerous gainsand losses) [39] Some molecular alterations that lead tocarcinogenesis can be defined in absolute termsThey includegain-of-function mutations or chromosome translocationsthat transformprotooncogenes to oncogenes However otherchanges are relative to the normal tissue of origin suchas pathway overactivity or overexpression on the proteinor RNA levels We have combined the analysis of absolutechanges (DNA exome sequence RNA sequence) with theanalysis of relative changes using skeletal muscle and bone asreference organs (protein-DNA array 2D gel electrophoresisand RNA expression levels) This choice was determined inpart by tissue availability after surgery andwas intended to aidin the distinction of osteosarcoma from myosarcoma While

Sarcoma 15

Table 3 Deregulation of genes for drug disposition Analysis of RNASeq for at least twofold overexpression (italic font) or underexpression(bold font) of genes for (a) transport and (b) metabolism in tumor compared to muscle and bone For the export transporters (ABCtransporters) only overexpression is considered relevant for drug resistance Not shown in the table are the underexpressed genes (ABCA7ABCA8 ABCA13 ABCB6 ABCB10 ABCC6 ABCC6P1 ABCC6P2 ABCC8 ABCC11 ABCD2 ABCG2 and ABCG5)

(a) Transport

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

650655 ABCA17P ATP-binding cassette subfamily A (ABC1) member 17 pseudogene 1360 211624 ABCA4 ATP-binding cassette subfamily A (ABC1) member 4 2038 2802

6555 SLC10A2 Solute carrier family 10 (sodiumbile acid cotransporter family)member 2 minus1962 minus2112

345274 SLC10A6 Solute carrier family 10 (sodiumbile acid cotransporter family)member 6 2623 3240

6563 SLC14A1 Solute carrier family 14 (urea transporter) member 1 (Kidd bloodgroup) minus3074 minus3152

6565 SLC15A2 Solute carrier family 15 (H+peptide transporter) member 2 minus2027 minus2152

6566 SLC16A1 Solute carrier family 16 member 1 (monocarboxylic acid transporter1) 1401 2170

117247 SLC16A10 Solute carrier family 16 member 10 (aromatic amino acid transporter) 2113 2848

6567 SLC16A2 Solute carrier family 16 member 2 (monocarboxylic acid transporter8) 3242 3848

10786 SLC17A3 Solute carrier family 17 (sodium phosphate) member 3 minus2868 minus29596571 SLC18A2 Solute carrier family 18 (vesicular monoamine) member 2 minus2087 minus21946573 SLC19A1 Solute carrier family 19 (folate transporter) member 1 minus2099 minus220310560 SLC19A2 Solute carrier family 19 (thiamine transporter) member 2 1820 254880704 SLC19A3 Solute carrier family 19 member 3 minus3331 minus3478387775 SLC22A10 Solute carrier family 22 member 10 5711 60859390 SLC22A13 Solute carrier family 22 (organic anion transporter) member 13 2623 3217

85413 SLC22A16 Solute carrier family 22 (organic cationcarnitine transporter)member 16 minus8101 minus7299

51310 SLC22A17 Solute carrier family 22 member 17 1475 21755002 SLC22A18 Solute carrier family 22 member 18 2038 27226582 SLC22A2 Solute carrier family 22 (organic cation transporter) member 2 4793 5216

6581 SLC22A3 Solute carrier family 22 (extraneuronal monoamine transporter)member 3 2554 3182

6583 SLC22A4 Solute carrier family 22 (organic cationergothioneine transporter)member 4 minus3603 minus3776

151295 SLC23A3 Solute carrier family 23 (nucleobase transporters) member 3 2109 284810478 SLC25A17 Solute carrier family 25 (mitochondrial carrier) member 17 1311 207783733 SLC25A18 Solute carrier family 25 (mitochondrial carrier) member 18 1846 2570

788 SLC25A20 Solute carrier family 25 (carnitineacylcarnitine translocase) member20 minus2105 minus2207

89874 SLC25A21 Solute carrier family 25 (mitochondrial oxodicarboxylate carrier)member 21 minus2962 minus3105

51312 SLC25A37 Solute carrier family 25 member 37 minus4633 minus486651629 SLC25A39 Solute carrier family 25 member 39 minus2585 minus2737203427 SLC25A43 Solute carrier family 25 member 43 1325 208865012 SLC26A10 Solute carrier family 26 member 10 3896 4472115111 SLC26A7 Solute carrier family 26 member 7 3431 4018116369 SLC26A8 Solute carrier family 26 member 8 minus5354 minus5536115019 SLC26A9 Solute carrier family 26 member 9 1623 241911001 SLC27A2 Solute carrier family 27 (fatty acid transporter) member 2 minus4278 minus4487

16 Sarcoma

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

64078 SLC28A3 Solute carrier family 28 (sodium-coupled nucleoside transporter)member 3 minus4543 minus4812

222962 SLC29A4 Solute carrier family 29 (nucleoside transporters) member 4 minus1962 minus204581031 SLC2A10 Solute carrier family 2 (facilitated glucose transporter) member 10 2532 3170

6518 SLC2A5 Solute carrier family 2 (facilitated glucosefructose transporter)member 5 minus3647 minus3821

55532 SLC30A10 Solute carrier family 30 member 10 minus3547 minus37257782 SLC30A4 Solute carrier family 30 (zinc transporter) member 4 2832 33726569 SLC34A1 Solute carrier family 34 (sodium phosphate) member 1 minus4716 minus4941340146 SLC35D3 Solute carrier family 35 member D3 minus5662 minus590654733 SLC35F2 Solute carrier family 35 member F2 1433 2170206358 SLC36A1 Solute carrier family 36 (protonamino acid symporter) member 1 minus2265 minus2345285641 SLC36A3 Solute carrier family 36 (protonamino acid symporter) member 3 minus2284 minus239354020 SLC37A1 Solute carrier family 37 (glycerol-3-phosphate transporter) member 1 minus2112 minus22122542 SLC37A4 Solute carrier family 37 (glucose-6-phosphate transporter) member 4 minus2117 minus2219151258 SLC38A11 Solute carrier family 38 member 11 2410 308555089 SLC38A4 Solute carrier family 38 member 4 1837 254892745 SLC38A5 Solute carrier family 38 member 5 minus1994 minus215291252 SLC39A13 Solute carrier family 39 (zinc transporter) member 13 1301 207023516 SLC39A14 Solute carrier family 39 (zinc transporter) member 14 2569 318829985 SLC39A3 Solute carrier family 39 (zinc transporter) member 3 minus2032 minus2152283375 SLC39A5 Solute carrier family 39 (metal ion transporter) member 5 1623 23707922 SLC39A7 Solute carrier family 39 (zinc transporter) member 7 1273 205830061 SLC40A1 Solute carrier family 40 (iron-regulated transporter) member 1 minus3612 minus379684102 SLC41A2 Solute carrier family 41 member 2 1293 20708501 SLC43A1 Solute carrier family 43 member 1 minus2013 minus215257153 SLC44A2 Solute carrier family 44 member 2 minus2047 minus215250651 SLC45A1 Solute carrier family 45 member 1 2038 2722146802 SLC47A2 Solute carrier family 47 member 2 minus1962 minus20266521 SLC4A1 Solute carrier family 4 anion exchanger member 1 minus6513 minus645357282 SLC4A10 Solute carrier family 4 sodium bicarbonate transporter member 10 minus2640 minus275383959 SLC4A11 Solute carrier family 4 sodium borate transporter member 11 1846 25696508 SLC4A3 Solute carrier family 4 anion exchanger member 3 1261 20458671 SLC4A4 Solute carrier family 4 sodium bicarbonate cotransporter member 4 2445 31139497 SLC4A7 Solute carrier family 4 sodium bicarbonate cotransporter member 7 2717 33076523 SLC5A1 Solute carrier family 5 (sodiumglucose cotransporter) member 1 minus2547 minus2705159963 SLC5A12 Solute carrier family 5 (sodiumglucose cotransporter) member 12 4753 52066527 SLC5A4 Solute carrier family 5 (low affinity glucose cotransporter) member 4 minus3969 minus4249

6540 SLC6A13 Solute carrier family 6 (neurotransmitter transporter GABA)member 13 1328 2092

55117 SLC6A15 Solute carrier family 6 (neutral amino acid transporter) member 15 1623 2333388662 SLC6A17 Solute carrier family 6 member 17 2038 272854716 SLC6A20 Solute carrier family 6 (proline IMINO transporter) member 20 1697 2433

6532 SLC6A4 Solute carrier family 6 (neurotransmitter transporter serotonin)member 4 minus2512 minus2611

6534 SLC6A7 Solute carrier family 6 (neurotransmitter transporter L-proline)member 7 2623 3228

56301 SLC7A10 Solute carrier family 7 (neutral amino acid transporter y+ system)member 10 minus3421 minus3603

Sarcoma 17

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

6547 SLC8A3 Solute carrier family 8 (sodiumcalcium exchanger) member 3 minus2204 minus2309285335 SLC9A10 Solute carrier family 9 member 10 1208 20006549 SLC9A2 Solute carrier family 9 (sodiumhydrogen exchanger) member 2 2038 2706

9368 SLC9A3R1 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 1 minus2760 minus2846

9351 SLC9A3R2 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 2 1889 2619

84679 SLC9A7 Solute carrier family 9 (sodiumhydrogen exchanger) member 7 3347 393510599 SLCO1B1 Solute carrier organic anion transporter family member 1B1 3360 397728234 SLCO1B3 Solute carrier organic anion transporter family member 1B3 9760 95696578 SLCO2A1 Solute carrier organic anion transporter family member 2A1 2432 309628232 SLCO3A1 Solute carrier organic anion transporter family member 3A1 minus2032 minus2152353189 SLCO4C1 Solute carrier organic anion transporter family member 4C1 minus6850 minus6611

(b) Metabolism

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold Cchange

1583 CYP11A1 Cytochrome P450 family 11 subfamily A polypeptide 1 1623 23961589 CYP21A2 Cytochrome P450 family 21 subfamily A polypeptide 2 minus1962 minus20361591 CYP24A1 Cytochrome P450 family 24 subfamily A polypeptide 1 5208 55771592 CYP26A1 Cytochrome P450 family 26 subfamily A polypeptide 1 2623 32571594 CYP27B1 Cytochrome P450 family 27 subfamily B polypeptide 1 1846 2585339761 CYP27C1 Cytochrome P450 family 27 subfamily C polypeptide 1 3585 41781553 CYP2A13 Cytochrome P450 family 2 subfamily A polypeptide 13 minus1962 minus20351580 CYP4B1 Cytochrome P450 family 4 subfamily B polypeptide 1 minus4820 minus506566002 CYP4F12 Cytochrome P450 family 4 subfamily F polypeptide 12 minus6070 minus61958529 CYP4F2 Cytochrome P450 family 4 subfamily F polypeptide 2 minus7769 minus70604051 CYP4F3 Cytochrome P450 family 4 subfamily F polypeptide 3 minus8244 minus749111283 CYP4F8 Cytochrome P450 family 4 subfamily F polypeptide 8 minus5006 minus5270260293 CYP4X1 Cytochrome P450 family 4 subfamily X polypeptide 1 minus2476 minus25809420 CYP7B1 Cytochrome P450 family 7 subfamily B polypeptide 1 2502 31702326 FMO1 Flavin containing monooxygenase 1 4360 48592327 FMO2 Flavin containing monooxygenase 2 (nonfunctional) minus4074 minus43372328 FMO3 Flavin containing monooxygenase 3 minus4036 minus4309388714 FMO6P Flavin containing monooxygenase 6 pseudogene minus2569 minus2737493869 GPX8 Glutathione peroxidase 8 (putative) 2814 33712938 GSTA1 Glutathione S-transferase alpha 1 1623 23632939 GSTA2 Glutathione S-transferase alpha 2 2038 27412941 GSTA4 Glutathione S-transferase alpha 4 1492 21882953 GSTT2 Glutathione S-transferase theta 2 4682 5170653689 GSTT2B Glutathione S-transferase theta 2B (genepseudogene) 3739 430784779 NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439 79969027 NAT8 N-acetyltransferase 8 (GCN5-related putative) minus2769 minus29207358 UGDH UDP-glucose 6-dehydrogenase 2333 301855757 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 1604 227110720 UGT2B11 UDP glucuronosyltransferase 2 family polypeptide B11 minus3586 minus37687367 UGT2B17 UDP glucuronosyltransferase 2 family polypeptide B17 minus2836 minus295954490 UGT2B28 UDP glucuronosyltransferase 2 family polypeptide B28 minus3132 minus3284167127 UGT3A2 UDP glycosyltransferase 3 family polypeptide A2 minus4716 minus4907

18 Sarcoma

Table 4 FAF1 mutations in various cancers FAF1 mutations listed in the TCGA data base were identified without restriction to any type ofcancer For the location of the affected domains on the protein compare Figure 2

AA Mutation Cancer DomainN4S Missense Cutaneous melanomaI10S Missense Stomach adenocarcinoma UBAE21lowast Nonsense Cutaneous melanoma UBAE25K Missense Uterine endometrioid carcinoma UBAV38 splice Splice Stomach adenocarcinoma UBAP86fs FS del Colorectal adenocarcinomaG123 splice Splice Uterine endometrioid carcinoma UB1P136H Missense Colorectal adenocarcinoma UB1T147M Missense Brain lower grade glioma UB1D149Y Missense Uterine endometrioid carcinoma UB1L159V Missense Lung adenocarcinoma UB1K163N Missense Uterine endometrioid carcinoma UB1L170F Missense Cutaneous melanomaG184 splice Splice Colorectal cancerQ187H Missense Stomach adenocarcinomaS214N Missense Colorectal adenocarcinoma UB2R222I Missense Uterine endometrioid carcinoma UB2E238D Missense Lung adenocarcinoma UB2P241S Missense Uterine endometrioid carcinoma UB2T245A Missense Renal clear cell carcinoma UB2M249V Missense Uterine endometrioid carcinoma UB2E280K Missense Brain lower grade gliomaG293lowast Nonsense Colorectal cancerT300I Missense Colorectal adenocarcinomaD305H Missense Lung adenocarcinomaE308Q Missense Lung adenocarcinomaA316V Missense Stomach adenocarcinomaK319fs FS ins Head and neck squamous cell carcinomaR344G Missense Stomach adenocarcinoma UASF353I Missense Cutaneous melanoma UASL379V Missense Breast invasive carcinoma UASC396F Missense Cutaneous melanoma UASS459lowast Nonsense Uterine endometrioid carcinoma UASG469 splice Splice Glioblastoma multiforme UASR509G Missense Lung squamous cell carcinomaE510fs FS del Cutaneous melanomaR516C Missense Uterine endometrioid carcinomaA534V Missense Stomach adenocarcinomaF537L Missense Stomach adenocarcinomaE551lowast Nonsense Lung adenocarcinomaR554W Missense Colorectal cancerS582I Missense Lung adenocarcinoma UBXF585L Missense Uterine endometrioid carcinoma UBXE587lowast Nonsense Lung adenocarcinoma UBXA592V Missense Stomach adenocarcinoma UBXW610lowast Nonsense Breast invasive carcinoma UBXD611Y Missense Lung adenocarcinoma UBXE635fs FS del Brain lower grade glioma UBXP640fs FS del Cutaneous melanoma UBX

Sarcoma 19

a more accurate reference point for a leiomyosarcoma wouldhave been smooth muscle we believe that the comparison tostriated muscle is sufficient to allow the assessment of tumorspecific changes We have measured RNA DNA and proteinwith various assays Similar assessments in the future shouldalso include chromosome analysis for possible translocations

In the first-line defense against cancer the gradualreplacement of conventional chemotherapy with molecularlytargeted agents has opened the possibility to tailor drug treat-ments to particular tumors This transition necessitates thecharacterization of the molecular lesions that are causativefor the transformation of healthy cells into cancerous cellsbecause drugs need to be matched with the underlying carci-nogenic defect to be effective An additional caveat causedby unique genetic changes in the primary tumor can affectdrug transport and metabolism and needs to be taken intoconsideration In this case the RNA levels for genes that regu-late transport andmetabolismwere extensively skewed in thetumor tissue as compared to muscle and bone (see Table 3)implying potential challenges to chemotherapy An advancedmolecular treatment strategy for cancer will rely on themolecular definition of drug target drug transport and drugmetabolism pharmacogenetics in the primary tumor Con-secutive to cancer dissemination it will also require adjust-ments to account for the genetic changes in the metastases

The cost of health care has been under increasing scrutinyThe treatment of cancer patients is expensive in particularwhen hospitalization is required and further in cases ofend-of-life care Avoidable expenditures are generated bysuboptimal treatment decisions that result in low efficacy orhigh toxicity of anticancer regimens Molecular medicine hasthe potential to preempt those problems and reduce wastefulspending Yet it requires the upfront cost of molecular cancerexamination The analysis performed in this study requiredabout $11000- in nonsalary expenses to perform While ithas not identified a drug target it has specified possibleconfines for drug treatment The costs for molecular analysisneed to be weighed against the societal cost derived fromlost productivity in the workforce disrupted lives of familiesand premature deaths While currently limited drug optionsconstitute the major constraint to the approach taken herethe foreseeable future will bring an increasing spectrum ofmolecularly targeted drugs along with faster and cheapertechnologies for the molecular assessment of cancers Theywill facilitate the clinical translation of our approach

Consent

The patient provided informed consent

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by DOD Grant PR094070under University of Cincinnati IRB protocol 04-01-29-01The

author is grateful to Dr Rhett Kovall for helping with thestructural analysis of FAF1

References

[1] G F Weber Molecular Mechanisms of Cancer Springer Dor-drecht The Netherlands 2007

[2] N G Sanerkin ldquoPrimary leiomyosarcoma of the bone and itscomparison with fibrosarcomardquoCancer vol 44 no 4 pp 1375ndash1387 1979

[3] J M Weaver and J A Abraham ldquoLeiomyosarcoma of the Boneand Soft Tissue A Reviewrdquo httpsarcomahelporglearningcenterleiomyosarcomahtml

[4] M A Depristo E Banks R Poplin et al ldquoA framework forvariation discovery and genotyping using next-generationDNAsequencing datardquo Nature Genetics vol 43 no 5 pp 491ndash5012011

[5] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[6] C W Menges D A Altomare and J R Testa ldquoFAS-associatedfactor 1 (FAF1) diverse functions and implications for oncoge-nesisrdquo Cell Cycle vol 8 no 16 pp 2528ndash2534 2009

[7] M Kim J H Lee S Y Lee E Kim and J Chung ldquoCaspar asuppressor of antibacterial immunity in Drosophilardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 44 pp 16358ndash16363 2006

[8] J Song K P Joon J-J Lee et al ldquoStructure and interaction ofubiquitin-associated domain of human Fas-associated factor 1rdquoProtein Science vol 18 no 11 pp 2265ndash2276 2009

[9] P H OrsquoFarrell ldquoHigh resolution two dimensional electrophore-sis of proteinsrdquo Journal of Biological Chemistry vol 250 no 10pp 4007ndash4021 1975

[10] A Burgess-Cassler J J Johansen D A Santek J R Ideand N C Kendrick ldquoComputerized quantitative analysis ofCoomassie-Blue-stained serum proteins separated by two-dimensional electrophoresisrdquo Clinical Chemistry vol 35 no 12pp 2297ndash2304 1989

[11] B R Oakley D R Kirsch and N RMorris ldquoA simplified ultra-sensitive silver stain for detecting proteins in polyacrylamidegelsrdquo Analytical Biochemistry vol 105 no 2 pp 361ndash363 1980

[12] K Chu X Niu and L T Williams ldquoA Fas-associated proteinfactor FAF1 potentiates Fas-mediated apoptosisrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 25 pp 11894ndash11898 1995

[13] B Rikhof S de Jong A J H Suurmeijer C Meijer and W TA van der Graaf ldquoThe insulin-like growth factor system andsarcomasrdquoThe Journal of Pathology vol 217 no 4 pp 469ndash4822009

[14] RGirling J F Partridge L R Bandara et al ldquoAnew componentof the transcription factor DRTF1E2Frdquo Nature vol 362 no6415 pp 83ndash87 1993

[15] F Mercurio and M Karin ldquoTranscription factors AP-3 andAP-2 interact with the SV40 enhancer in a mutually exclusivemannerrdquo EMBO Journal vol 8 no 5 pp 1455ndash1460 1989

[16] R Bassel-Duby M D Hernandez M A Gonzalez J KKrueger and R S Williams ldquoA 40-kilodalton protein bindsspecifically to an upstream sequence element essential for mus-cle-specific transcription of the human myoglobin promoterrdquoMolecular and Cellular Biology vol 12 no 11 pp 5024ndash50321992

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 9: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

Sarcoma 9

(d) Continued

Symbol Name log2-fold change log2-fold changeMuscle Bone

SERPINH1 Serpin peptidase inhibitor clade H (heat shock protein47) member 1 3706 1652 4145 2098

ANTXR1 Anthrax toxin receptor 1 3670 3789 3690 6445

(e)

Symbol Name log2-fold change log2-fold changeMuscle Bone

HELLS Helicase lymphoid-specific 5315602 0155898 minus082713 202489

TNFRSF11A Tumor necrosis factor receptor superfamily member 11a andNFKB activator 3017922 003091 1400993 0202453

NFKBID Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor delta 2655352 0 minus147615 0504341

TNFRSF25 Tumor necrosis factor receptor superfamily member 25 2432959 0046388 0163954 0490795

NFKBIE Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor epsilon 2121015 0062395 0311441 043382

TNF Tumor necrosis factor 1847997 0 minus142101 003733

TNFRSF10D Tumor necrosis factor receptor superfamily member 10d decoywith truncated death domain 1754888 0172968 minus01757 1183343

TNFRSF1B Tumor necrosis factor receptor superfamily member 1B 1473438 0709902 minus097011 6729401TNFRSF10A Tumor necrosis factor receptor superfamily member 10a 1411898 0828219 0357701 3004386

NFKBIB Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor beta 1193993 1209816 046055 3484692

TNFRSF10B Tumor necrosis factor receptor superfamily member 10b 1094157 1908597 0425446 5242006TNFRSF21 Tumor necrosis factor receptor superfamily member 21 1055762 3882038 0745815 8305711TNFRSF1A Tumor necrosis factor receptor superfamily member 1A 0875167 3790938 minus011707 130344

NFKBIZ Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor zeta 0575974 3698951 0344657 7493136

RIPK1 Receptor (TNFRSF)-interacting serine-threonine kinase 1 0494467 311749 minus108854 161392NKRF NFKB repressing factor 0475722 2156087 minus086397 9434166NFKB1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 0432959 2054532 minus065865 7568586CHUK Conserved helix-loop-helix ubiquitous kinase 0176126 2961281 minus120204 1330333RELA V-rel reticuloendotheliosis viral oncogene homolog A (avian) 0013848 1263837 0287167 1803044

NFKB2 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 2(p49p100) minus008641 0312993 0485882 0360442

NKAPP1 NFKB activating protein pseudogene 1 minus010692 0825177 minus099449 2655392

TNFRSF10C Tumor necrosis factor receptor superfamily member 10c decoywithout an intracellular domain minus0152 0064676 minus556891 6321515

NKIRAS2 NFKB inhibitor interacting Ras-like 2 minus060768 1095135 minus088758 2299946

NFKBIL1 Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor-like 1 minus08719 0141933 minus008357 0140418

NKIRAS1 NFKB inhibitor interacting Ras-like 1 minus087428 6296399 1467735 2122983TANK TRAF family member-associated NFKB activator minus0983 6777124 minus151908 1695872NKAP NFKB activating protein minus135735 1422458 minus078243 1646287

NFKBIA Nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor alpha minus185483 4032743 minus031802 2395275

NKAPL NFKB activating protein-like minus557347 5875743 minus140215 0534643

10 Sarcoma

FAS associating region

DED interacting region

UB12 DZM

UB2domain

UASdomain

UBXdomain

UBAdomain

S289 S291

Aurora ACK II

S181

AKT1ATMGSK3

AuroraPKG

PLK1RSK

STK4CHK1

650566481336260205169110475

(a)

MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRM

6

1

101

100

200

300

400

500

600

650

201

301

401

501

601

667855899998876467765678888887578758999986467 88 8888888888888988888899988888889888877767888777888757

2222454345655744642554435764462443558468643653433344333424435334233333332333332333354335333344334354

LDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITH

9999997797689998787758999999999859996577754788888888875333467888868987688888888888877888855675799987

9585847535375857543447459666955563844544585374435443365845846434457567533353232334334444433337484944

REVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDAT

5888757887577654788887766654677776555468877888765455546887766555688888877777777777776556788888767766

5344456484742545656645594364384534437445444342323455453535435463434323333343434333333334333335433333

EFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESI

5666776654456777888888888888866889999999998848888876677778999999998776686899998589875579999987486899

4635343574354322343434533333343548479747945564424647635575478569766456468999999754233454687457844669

VSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDE

9999888589997557877545444345677876335554467888876899986799876447777677888999999998877555778887777778

6778547999996674433343444333333335466456434433487999895353333464346434365468755846444545454565446565

REARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVF

8888888887788877766545557777777666777777777776678877777766578888888888885799998589975788758987589999

4545445564445344433443354555556653665456544454434444444443344334333333446859596743354745795535796898

DFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKE

99997799988779985788754577888765665678898589998589

67974525444795877564635844444476875837446989897366

(b)

Figure 2 FAF1 structure (a) A schematic of functional domains in FAF1 with annotations (adapted from [6ndash8]) There are two ubiquitin-like domains flanking the S181 site The PDB structure 2DZM identifies the N-terminal one while the C-terminal prediction is by sequencesimilarity Whereas the mutation S181G is not expected to disrupt the protein structure per se this amino acid has a high score as a possiblephosphorylation site for a number of kinases involved inDNAdamage repair (b) Secondary structure prediction of FAF1The residues aroundS181 appear unstructured

reflected in STAT3 constitutive phosphorylation The lack ofphosphorylation in this case suggests that the leiomyosar-coma may not depend on the STAT3 pathway

36 Pharmacogenetic Evaluation Predicting the sensitivityto anticancer drugs is a main goal of molecular analysisFor this the over- or underexpression of genes for drugtransport and metabolism is of key importance Analysis ofthe RNASeq data for these groups of gene products identified

a surprisingly large number of deregulations compared tomuscle or bone (Tables 3(a) and 3(b)) Those alterationsmay affect choices for drug treatment For example the highlevels of glutathione S-transferase may render carmustinethioTEPA cisplatin chlorambucil melphalan nitrogenmus-tard phosphoramide mustard acrolein or steroids ineffec-tive The overexpression ofN-acetyltransferase may compro-mise 5-fluorouracil or taxolThemodest upregulation of onlytwo export transporters (ABC-transporters) and specifically

Sarcoma 11

Bone Tumor

Muscle

(a)

Transcription factor Description Reference

E2F1 Transcriptional activation of cell cycle progression is selectively activated in response to specific cues key downstream target of RB1 can promote proliferation or apoptosis when activated [14]

AP-33 interact in a mutually exclusive manner [15]

CCAC CCAC box is an essential element for muscle-specific transcription from the myoglobin promoter [16]

LIII-BP The L-III transcriptional regulatory element (a carbohydrate-response element) is in the pyruvate kinase L gene necessary for cell type-specific expression and transcriptional stimulation by carbohydrates [17]

ADD-1 (SREBP-1) activates transcription from sterol-regulated elements and from an E-box sequence present in the promoter of fatty acid synthase [18]

PAX6 Member of the paired box gene family transcriptional regulator involved in developmental processes [19]

48kD transcription factor activator protein-3 binds to the core element and recognizes the SV40 enhancer and AP-2 and AP-

(b)

Figure 3 Transcription factor activation Nuclear extracts from tumor muscle and bone were tested for DNA binding activity on a protein-DNA array (a) Transcription factor binding that was high in all 3 tissues and is therefore considered constitutively active is circled in yellow(left to right top to bottom AhRAmt GATA1 GATA2 GATA12 HIF1 and HOXD8910) Transcription factors that show high binding inthe cancer but not in muscle or bone are circled in red (left to right top to bottom E2F1 AP3 LIII-BP PAX6 ADD-1 and CCAC) (b) Thefunctions of transcription factors that display high DNA binding in the cancer but not in muscle or bone are described

the lack of ABCB1 overexpression is favorable for avoidingdrug resistance

37 Population Analysis The above-described results indi-cated that a FAF1 mutation which replaces serine in position181 thus preventing FAF1 phosphorylation and activationmay be a driver for leiomyosarcomagenesis A custom Taq-Man assay confirmed the presence of the somatic mutationin the patient To assess whether this single nucleotidereplacement is common in this type of cancer we analyzedDNA from 29 leiomyosarcomas and 1 blood sample from aleiomyosarcoma patient For comparison to nonsarcomatoustumors 34 breast cancers served as a reference None of themdisplayed amutation in the same locus By contrast there wasa distribution across all leiomyosarcomas in the osteopontin

promoter position minus443 (used as a reference) with 12 CC 10TC and 9 TT

4 Discussion

TheFAF1mutation identified as the likely cause for the cancerunder study gives room for an explanation of the sarcomatoustransformation (Figure 5) DNA damage to mesenchymalcells occurs persistently in an oxidizing environment at 37∘CThese insults are rarely transforming and such an occurrencewould trigger the initiation of programmed cell death inapoptosis-competent cells Intact FAF1 associates with FASand enhances apoptosis mediated through this receptor [12]A loss of function in FAF1 could lead to transformation viaantiapoptosis Whereas the mutation S181G is not expected

12 Sarcoma

Muscle

Bone

Tumor

220

94

6043

29

14

220

94

60

43

29

14

220

94

60

43

29

14

1

7

6

23

24

21

22

25

26

89 1011 12

1516 1719

pH 4 8 pH 4 8

8pH 4

(a)

SwissProt or NCBISpot number Protein identified accession Description

(Structural)6 Transgelin Q01995 Smooth muscle-specific cross-links actin24 Transgelin-2 P37802 Smooth muscle-specific cross-links actin

Vimentin P08670 Intermediate filament in mesenchymal tissue1 Filamin-A P21333 Actin-binding protein regulates the cytoskeleton 21 P06709 Cytoskeleton

(Calcium homeostasis)8 9 Calumenin O43852 Calcium-binding protein in the ER and golgi apparatus26 Protein S100-A11 P31949 Calcium-binding EF hand protein10 Reticulocalbin-3 Q96D15 Calcium-binding protein located in the ER12 P11021 Heat shock 70-kD protein 5 ER lumenal calcium-binding

(Protein processing)25 40S ribosomal protein S12 P25398 Core component of the ribosomal accuracy center7 Glycine-tRNA ligase P41250 Catalyzes the attachment of glycine to tRNA19 P01009 Protease inhibitor23 P01009 Protease inhibitor21 Proteasome activator complex subunit 2 Q9UL46 Proteasome activation10 P07900 Chaperone

(Other)7 Serum albumin P02768 Carrier protein in blood22 Protein disulfide isomerase (C-terminal fragment) P07237 Prolyl hydroxylation in collagen microsomal triglyceride transfer

120573 actin (C-terminal fragment)

78kD glucose-regulated protein

120572-1-antitrypsin120572-1-antitrypsin (C-terminal fragment)

Heat shock protein HSP 90120572 (N-terminal fragment)

11 15ndash17

(b)

Figure 4 Continued

Sarcoma 13

Tumor Muscle

OPN

CD44

STAT3

P-STAT3

Tubulin

75

75

50

15010075

10075

50

(c)

Tumor bone Tumor muscleGene ID Symbol Name Fold change Expression Fold change Expression6876 TAGLN Transgelin 3274 2455 3087 15088407 TAGLN2 Transgelin-2 2164 7338 6975 13037431 VIM Vimentin 1653 295010 4061 696042316 FLNA Filamin A alpha 1304 7791 9005 065160 ACTB Actin beta 0656 973187 5491 67368813 CALU Calumenin 7601 19328 12063 70596282 S100A11 S100 calcium binding protein A11 0931 158914 5071 1686357333 RCN3 Reticulocalbin 3 EF-hand calcium binding domain 2439 0604 6412 01223309 HSPA5 1068 92754 2607 220356696 SPP1 Secreted phosphoprotein 1 7572 46508 547929 0355960 CD44 CD44 molecule (Indian blood group) 2053 26708 15436 20566774 STAT3 Signal transducer and activator of transcription 3 0617 46534 0832 200165925 RB1 Retinoblastoma 1 0736 14772 0442 14268

Heat shock 70kDa protein 5 (glucose-regulated protein 78kDa)

(d)

Figure 4 Protein overexpression (a) 2D protein gel electrophoresis of lysates from muscle (upper left) tumor (upper right) and bone(bottom) in RIPA buffer Red circles indicate the spots that were identified as overexpressed and were further analyzed by mass spectrometry(b) Proteins identified in 2D gel electrophoresis as overexpressed in the tumor in comparison to muscle and bone were analyzed for theiridentity by mass spectrometry The left column indicates the spot number corresponding to the 2D gel The next column contains theprotein name followed by the accession number and a description of the protein functionThe protein functions are grouped into structuralcalcium homeostasis and various others (c) Western blot 10 120583g lysates of tumor muscle and bone in RIPA buffer were loaded per laneand electrophoresed on 10 SDS-polyacrylamide minigels with reducing denaturing sample buffer After transfer to PVDF membranesthey were probed for markers of cancer progression including osteopontin CD44 STAT3 and phospho-STAT3 Antitubulin served as aloading control (d) RNA levels corresponding to the proteins found to be affected in the sarcoma With four exceptions in the tumor-bonecomparison (gray font) upregulated proteins are associated with increased RNA levels STAT3 is not increased on the protein or RNA levelThe reduced level of RB1 expression is consistent with the elevated DNA binding activity of E2F1

to disrupt the structure of the protein this site does scorehigh as a possible phosphorylation site for a number ofkinases involved in DNA damage repair supporting thehypothesis that the cancer cells containing thismutation havelost their ability to respond to transforming DNA damagewith programmed cell death FAF1 antagonizes WNT signal-ing by promoting 120573-catenin degradation in the proteasome[21] a function that may be lost after the point mutationThe elevated DNA binding activity of the protooncogenictranscription factor E2F1 (see Figure 3) could be caused byits interaction with LEF-1 [20] consecutive to persistent

WNT signaling The RNA level of IGF2BP1 (IMP-1) a stress-responsive regulator of mRNA stability is highly upregulatedin the tumor compared to muscle as well as bone (seeTable 2(c)) IGF2BP1 is a transcriptional target of the WNTpathway that regulates NF-120581B activity (see Table 2(e)) andis antiapoptotic [22 23] IGF2BP1 may be upregulated asa consequence of mutated FAF1 not being able to suppressWNT signaling in this specific cancerOf noteWNTpathwayoveractivity may not be required for transformation ratherthe persistence of a WNT pathway signal due to the lack of aFAF1-mediated termination signal may suffice

14 Sarcoma

WNTFrizzled

Axin Dvl

igf2bp1

FAF1

P65 P50

IKK

FAS

120573-Catenin

LEF + E2F1

I120581B

Figure 5 Possible transforming pathway The point mutation inFAF1 is believed to cause a loss of function (crossed out in red)This may lead to an overactivity of the WNT signaling pathwayConsistently E2F1 (activated by LEF1) and igf2bp1 (a transcriptionaltarget of the WNT pathway) have been identified to be upregulatedin the molecular analysis In contrast to the negative regulator FAF1IGF2BP1 is a positive regulator of NF-120581B activityThe overactivity ofthe NF-120581B pathway and the reduced efficacy of FAS signaling can betransforming

The role ofWNT signaling in sarcoma has been subject todebate (eg [24]) In metastatic leiomyosarcoma 120573-Cateninmay accumulate in the nucleus despite a relatively weakexpression of WNT [25] This may be due to WNT signalactivation via noncanonical ligands [26] or to 120573-Cateninbinding the nuclear receptor NR4A2 and releasing it from thecorepressor protein LEF-1 [27] Of note in the cancer understudy here the mRNA level of NR4A2 was overexpressed10-fold compared to bone and 3-fold compared to muscleThe results from this study are consistent with the possibilitythat a lack of termination in the WNT signal rather than itsoveractivation could contribute to sarcomatous transforma-tion Such a mechanism may be reflected in an upregulationof downstream targets even though overexpression of WNTpathway components is not detectable

Other mutations beside FAF1 are less likely to becausative for the cancer EPHA3 was revealed as mutated inthis cancer by DNA exome sequencing and RNASeq EPHA3is a receptor tyrosine kinase that is frequentlymutated in lungcancer Tumor-suppressive effects of wild-type EPHA3 can beoverridden by dominant negative EPHA3 somatic mutations[28]Thismechanism is unlikely to play a role in this sarcomaas the detected mutation is located far N-terminally on theextracellular Ephrin binding domain not on the intracellularkinase domain

FAF1 has been described to act as a tumor suppressor gene[29] Its depletion due to chromosome breakage can affectprognosis in glioblastoma patients [30 31] Single nucleotidepolymorphisms in FAF1 are associated with a risk for gastriccancer [32] While numerous FAF1 mutations are associatedwith various cancers none of these genetic changes in theTCGA database affects the amino acid position 181 (Table 4)

The major upregulations identified in this tumor com-prise muscle-specific gene products (transcription factors

CAAC binding proteomics transgelin transgelin-2 RNAanoctamin-4 and immunohistochemistry smooth mus-cle actin) and calcium-regulating molecules (proteomicscalumenin S100-A11 reticulocalbin-3 and 78 kD glucose-regulated protein) The muscle-specific gene products con-firm this recurrent sarcoma as a leiomyosarcoma (the firsttumor distal to the site of the recurring one had beendiagnosed as an osteosarcoma) Calcium is one of the majorsecond messengers in smooth muscle cells Its uptake isregulated by potential-sensitive ion channels in the cellmembrane and by the activities of various receptors Calciumis stored in the sarcoplasmic reticulum from where it canbe released to facilitate actin-myosin interaction and tensiongeneration Phosphorylation of the myosin light chain by acalmodulin-regulated enzyme is important for contractionThe upregulation of gene products associated with migrationand invasion (osteopontin MMP1 vimentin filamin-A and120573-actin) and gene products for extracellularmatrixmoleculesand their modulators (fibronectin collagen ITGBL1 andMXRA5) reflects the invasive nature of this cancer

The recurrence of a sarcoma after 14 years has twoprobable explanations either it is due to a cancer pre-disposition syndrome based on a germ-line mutation (theage of the patient weakens this hypothesis) or the secondtumor is a metastatic colony of the first that was reactivatedafter dormancy The location of the sarcoma in the sameextremity and proximal to a preceding mesenchymal cancer(and therefore in its natural path of dissemination) impliedthe probability that this was a relapse in a metastatic siteThe different histologic assessment as osteosarcoma in thefirst occurrence and leiomyosarcoma as the second cancerdoes not necessarily negate that Mixed histology [33 34]and transdifferentiation [35ndash37] have been described forsarcomatous tumors Of note however in this scenarioosteosarcoma seems to more commonly follow leiomyosar-coma than precede it Material from the first cancer of thispatient was not accessible to us It is very plausible that thiscould have been a mineralized leiomyosarcoma In thosetumors the differential diagnosis from osteosarcoma can bedifficult [38] The extracompartmental location of the firsttumor supports this interpretation

On the molecular pathology level sarcomas fall intotwo groups comprising tumors with simple karyotypes(with pathogenetic translocations or specific genetic muta-tions) and tumors with very complex karyotypes (overtchromosome and genomic instability with numerous gainsand losses) [39] Some molecular alterations that lead tocarcinogenesis can be defined in absolute termsThey includegain-of-function mutations or chromosome translocationsthat transformprotooncogenes to oncogenes However otherchanges are relative to the normal tissue of origin suchas pathway overactivity or overexpression on the proteinor RNA levels We have combined the analysis of absolutechanges (DNA exome sequence RNA sequence) with theanalysis of relative changes using skeletal muscle and bone asreference organs (protein-DNA array 2D gel electrophoresisand RNA expression levels) This choice was determined inpart by tissue availability after surgery andwas intended to aidin the distinction of osteosarcoma from myosarcoma While

Sarcoma 15

Table 3 Deregulation of genes for drug disposition Analysis of RNASeq for at least twofold overexpression (italic font) or underexpression(bold font) of genes for (a) transport and (b) metabolism in tumor compared to muscle and bone For the export transporters (ABCtransporters) only overexpression is considered relevant for drug resistance Not shown in the table are the underexpressed genes (ABCA7ABCA8 ABCA13 ABCB6 ABCB10 ABCC6 ABCC6P1 ABCC6P2 ABCC8 ABCC11 ABCD2 ABCG2 and ABCG5)

(a) Transport

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

650655 ABCA17P ATP-binding cassette subfamily A (ABC1) member 17 pseudogene 1360 211624 ABCA4 ATP-binding cassette subfamily A (ABC1) member 4 2038 2802

6555 SLC10A2 Solute carrier family 10 (sodiumbile acid cotransporter family)member 2 minus1962 minus2112

345274 SLC10A6 Solute carrier family 10 (sodiumbile acid cotransporter family)member 6 2623 3240

6563 SLC14A1 Solute carrier family 14 (urea transporter) member 1 (Kidd bloodgroup) minus3074 minus3152

6565 SLC15A2 Solute carrier family 15 (H+peptide transporter) member 2 minus2027 minus2152

6566 SLC16A1 Solute carrier family 16 member 1 (monocarboxylic acid transporter1) 1401 2170

117247 SLC16A10 Solute carrier family 16 member 10 (aromatic amino acid transporter) 2113 2848

6567 SLC16A2 Solute carrier family 16 member 2 (monocarboxylic acid transporter8) 3242 3848

10786 SLC17A3 Solute carrier family 17 (sodium phosphate) member 3 minus2868 minus29596571 SLC18A2 Solute carrier family 18 (vesicular monoamine) member 2 minus2087 minus21946573 SLC19A1 Solute carrier family 19 (folate transporter) member 1 minus2099 minus220310560 SLC19A2 Solute carrier family 19 (thiamine transporter) member 2 1820 254880704 SLC19A3 Solute carrier family 19 member 3 minus3331 minus3478387775 SLC22A10 Solute carrier family 22 member 10 5711 60859390 SLC22A13 Solute carrier family 22 (organic anion transporter) member 13 2623 3217

85413 SLC22A16 Solute carrier family 22 (organic cationcarnitine transporter)member 16 minus8101 minus7299

51310 SLC22A17 Solute carrier family 22 member 17 1475 21755002 SLC22A18 Solute carrier family 22 member 18 2038 27226582 SLC22A2 Solute carrier family 22 (organic cation transporter) member 2 4793 5216

6581 SLC22A3 Solute carrier family 22 (extraneuronal monoamine transporter)member 3 2554 3182

6583 SLC22A4 Solute carrier family 22 (organic cationergothioneine transporter)member 4 minus3603 minus3776

151295 SLC23A3 Solute carrier family 23 (nucleobase transporters) member 3 2109 284810478 SLC25A17 Solute carrier family 25 (mitochondrial carrier) member 17 1311 207783733 SLC25A18 Solute carrier family 25 (mitochondrial carrier) member 18 1846 2570

788 SLC25A20 Solute carrier family 25 (carnitineacylcarnitine translocase) member20 minus2105 minus2207

89874 SLC25A21 Solute carrier family 25 (mitochondrial oxodicarboxylate carrier)member 21 minus2962 minus3105

51312 SLC25A37 Solute carrier family 25 member 37 minus4633 minus486651629 SLC25A39 Solute carrier family 25 member 39 minus2585 minus2737203427 SLC25A43 Solute carrier family 25 member 43 1325 208865012 SLC26A10 Solute carrier family 26 member 10 3896 4472115111 SLC26A7 Solute carrier family 26 member 7 3431 4018116369 SLC26A8 Solute carrier family 26 member 8 minus5354 minus5536115019 SLC26A9 Solute carrier family 26 member 9 1623 241911001 SLC27A2 Solute carrier family 27 (fatty acid transporter) member 2 minus4278 minus4487

16 Sarcoma

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

64078 SLC28A3 Solute carrier family 28 (sodium-coupled nucleoside transporter)member 3 minus4543 minus4812

222962 SLC29A4 Solute carrier family 29 (nucleoside transporters) member 4 minus1962 minus204581031 SLC2A10 Solute carrier family 2 (facilitated glucose transporter) member 10 2532 3170

6518 SLC2A5 Solute carrier family 2 (facilitated glucosefructose transporter)member 5 minus3647 minus3821

55532 SLC30A10 Solute carrier family 30 member 10 minus3547 minus37257782 SLC30A4 Solute carrier family 30 (zinc transporter) member 4 2832 33726569 SLC34A1 Solute carrier family 34 (sodium phosphate) member 1 minus4716 minus4941340146 SLC35D3 Solute carrier family 35 member D3 minus5662 minus590654733 SLC35F2 Solute carrier family 35 member F2 1433 2170206358 SLC36A1 Solute carrier family 36 (protonamino acid symporter) member 1 minus2265 minus2345285641 SLC36A3 Solute carrier family 36 (protonamino acid symporter) member 3 minus2284 minus239354020 SLC37A1 Solute carrier family 37 (glycerol-3-phosphate transporter) member 1 minus2112 minus22122542 SLC37A4 Solute carrier family 37 (glucose-6-phosphate transporter) member 4 minus2117 minus2219151258 SLC38A11 Solute carrier family 38 member 11 2410 308555089 SLC38A4 Solute carrier family 38 member 4 1837 254892745 SLC38A5 Solute carrier family 38 member 5 minus1994 minus215291252 SLC39A13 Solute carrier family 39 (zinc transporter) member 13 1301 207023516 SLC39A14 Solute carrier family 39 (zinc transporter) member 14 2569 318829985 SLC39A3 Solute carrier family 39 (zinc transporter) member 3 minus2032 minus2152283375 SLC39A5 Solute carrier family 39 (metal ion transporter) member 5 1623 23707922 SLC39A7 Solute carrier family 39 (zinc transporter) member 7 1273 205830061 SLC40A1 Solute carrier family 40 (iron-regulated transporter) member 1 minus3612 minus379684102 SLC41A2 Solute carrier family 41 member 2 1293 20708501 SLC43A1 Solute carrier family 43 member 1 minus2013 minus215257153 SLC44A2 Solute carrier family 44 member 2 minus2047 minus215250651 SLC45A1 Solute carrier family 45 member 1 2038 2722146802 SLC47A2 Solute carrier family 47 member 2 minus1962 minus20266521 SLC4A1 Solute carrier family 4 anion exchanger member 1 minus6513 minus645357282 SLC4A10 Solute carrier family 4 sodium bicarbonate transporter member 10 minus2640 minus275383959 SLC4A11 Solute carrier family 4 sodium borate transporter member 11 1846 25696508 SLC4A3 Solute carrier family 4 anion exchanger member 3 1261 20458671 SLC4A4 Solute carrier family 4 sodium bicarbonate cotransporter member 4 2445 31139497 SLC4A7 Solute carrier family 4 sodium bicarbonate cotransporter member 7 2717 33076523 SLC5A1 Solute carrier family 5 (sodiumglucose cotransporter) member 1 minus2547 minus2705159963 SLC5A12 Solute carrier family 5 (sodiumglucose cotransporter) member 12 4753 52066527 SLC5A4 Solute carrier family 5 (low affinity glucose cotransporter) member 4 minus3969 minus4249

6540 SLC6A13 Solute carrier family 6 (neurotransmitter transporter GABA)member 13 1328 2092

55117 SLC6A15 Solute carrier family 6 (neutral amino acid transporter) member 15 1623 2333388662 SLC6A17 Solute carrier family 6 member 17 2038 272854716 SLC6A20 Solute carrier family 6 (proline IMINO transporter) member 20 1697 2433

6532 SLC6A4 Solute carrier family 6 (neurotransmitter transporter serotonin)member 4 minus2512 minus2611

6534 SLC6A7 Solute carrier family 6 (neurotransmitter transporter L-proline)member 7 2623 3228

56301 SLC7A10 Solute carrier family 7 (neutral amino acid transporter y+ system)member 10 minus3421 minus3603

Sarcoma 17

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

6547 SLC8A3 Solute carrier family 8 (sodiumcalcium exchanger) member 3 minus2204 minus2309285335 SLC9A10 Solute carrier family 9 member 10 1208 20006549 SLC9A2 Solute carrier family 9 (sodiumhydrogen exchanger) member 2 2038 2706

9368 SLC9A3R1 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 1 minus2760 minus2846

9351 SLC9A3R2 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 2 1889 2619

84679 SLC9A7 Solute carrier family 9 (sodiumhydrogen exchanger) member 7 3347 393510599 SLCO1B1 Solute carrier organic anion transporter family member 1B1 3360 397728234 SLCO1B3 Solute carrier organic anion transporter family member 1B3 9760 95696578 SLCO2A1 Solute carrier organic anion transporter family member 2A1 2432 309628232 SLCO3A1 Solute carrier organic anion transporter family member 3A1 minus2032 minus2152353189 SLCO4C1 Solute carrier organic anion transporter family member 4C1 minus6850 minus6611

(b) Metabolism

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold Cchange

1583 CYP11A1 Cytochrome P450 family 11 subfamily A polypeptide 1 1623 23961589 CYP21A2 Cytochrome P450 family 21 subfamily A polypeptide 2 minus1962 minus20361591 CYP24A1 Cytochrome P450 family 24 subfamily A polypeptide 1 5208 55771592 CYP26A1 Cytochrome P450 family 26 subfamily A polypeptide 1 2623 32571594 CYP27B1 Cytochrome P450 family 27 subfamily B polypeptide 1 1846 2585339761 CYP27C1 Cytochrome P450 family 27 subfamily C polypeptide 1 3585 41781553 CYP2A13 Cytochrome P450 family 2 subfamily A polypeptide 13 minus1962 minus20351580 CYP4B1 Cytochrome P450 family 4 subfamily B polypeptide 1 minus4820 minus506566002 CYP4F12 Cytochrome P450 family 4 subfamily F polypeptide 12 minus6070 minus61958529 CYP4F2 Cytochrome P450 family 4 subfamily F polypeptide 2 minus7769 minus70604051 CYP4F3 Cytochrome P450 family 4 subfamily F polypeptide 3 minus8244 minus749111283 CYP4F8 Cytochrome P450 family 4 subfamily F polypeptide 8 minus5006 minus5270260293 CYP4X1 Cytochrome P450 family 4 subfamily X polypeptide 1 minus2476 minus25809420 CYP7B1 Cytochrome P450 family 7 subfamily B polypeptide 1 2502 31702326 FMO1 Flavin containing monooxygenase 1 4360 48592327 FMO2 Flavin containing monooxygenase 2 (nonfunctional) minus4074 minus43372328 FMO3 Flavin containing monooxygenase 3 minus4036 minus4309388714 FMO6P Flavin containing monooxygenase 6 pseudogene minus2569 minus2737493869 GPX8 Glutathione peroxidase 8 (putative) 2814 33712938 GSTA1 Glutathione S-transferase alpha 1 1623 23632939 GSTA2 Glutathione S-transferase alpha 2 2038 27412941 GSTA4 Glutathione S-transferase alpha 4 1492 21882953 GSTT2 Glutathione S-transferase theta 2 4682 5170653689 GSTT2B Glutathione S-transferase theta 2B (genepseudogene) 3739 430784779 NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439 79969027 NAT8 N-acetyltransferase 8 (GCN5-related putative) minus2769 minus29207358 UGDH UDP-glucose 6-dehydrogenase 2333 301855757 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 1604 227110720 UGT2B11 UDP glucuronosyltransferase 2 family polypeptide B11 minus3586 minus37687367 UGT2B17 UDP glucuronosyltransferase 2 family polypeptide B17 minus2836 minus295954490 UGT2B28 UDP glucuronosyltransferase 2 family polypeptide B28 minus3132 minus3284167127 UGT3A2 UDP glycosyltransferase 3 family polypeptide A2 minus4716 minus4907

18 Sarcoma

Table 4 FAF1 mutations in various cancers FAF1 mutations listed in the TCGA data base were identified without restriction to any type ofcancer For the location of the affected domains on the protein compare Figure 2

AA Mutation Cancer DomainN4S Missense Cutaneous melanomaI10S Missense Stomach adenocarcinoma UBAE21lowast Nonsense Cutaneous melanoma UBAE25K Missense Uterine endometrioid carcinoma UBAV38 splice Splice Stomach adenocarcinoma UBAP86fs FS del Colorectal adenocarcinomaG123 splice Splice Uterine endometrioid carcinoma UB1P136H Missense Colorectal adenocarcinoma UB1T147M Missense Brain lower grade glioma UB1D149Y Missense Uterine endometrioid carcinoma UB1L159V Missense Lung adenocarcinoma UB1K163N Missense Uterine endometrioid carcinoma UB1L170F Missense Cutaneous melanomaG184 splice Splice Colorectal cancerQ187H Missense Stomach adenocarcinomaS214N Missense Colorectal adenocarcinoma UB2R222I Missense Uterine endometrioid carcinoma UB2E238D Missense Lung adenocarcinoma UB2P241S Missense Uterine endometrioid carcinoma UB2T245A Missense Renal clear cell carcinoma UB2M249V Missense Uterine endometrioid carcinoma UB2E280K Missense Brain lower grade gliomaG293lowast Nonsense Colorectal cancerT300I Missense Colorectal adenocarcinomaD305H Missense Lung adenocarcinomaE308Q Missense Lung adenocarcinomaA316V Missense Stomach adenocarcinomaK319fs FS ins Head and neck squamous cell carcinomaR344G Missense Stomach adenocarcinoma UASF353I Missense Cutaneous melanoma UASL379V Missense Breast invasive carcinoma UASC396F Missense Cutaneous melanoma UASS459lowast Nonsense Uterine endometrioid carcinoma UASG469 splice Splice Glioblastoma multiforme UASR509G Missense Lung squamous cell carcinomaE510fs FS del Cutaneous melanomaR516C Missense Uterine endometrioid carcinomaA534V Missense Stomach adenocarcinomaF537L Missense Stomach adenocarcinomaE551lowast Nonsense Lung adenocarcinomaR554W Missense Colorectal cancerS582I Missense Lung adenocarcinoma UBXF585L Missense Uterine endometrioid carcinoma UBXE587lowast Nonsense Lung adenocarcinoma UBXA592V Missense Stomach adenocarcinoma UBXW610lowast Nonsense Breast invasive carcinoma UBXD611Y Missense Lung adenocarcinoma UBXE635fs FS del Brain lower grade glioma UBXP640fs FS del Cutaneous melanoma UBX

Sarcoma 19

a more accurate reference point for a leiomyosarcoma wouldhave been smooth muscle we believe that the comparison tostriated muscle is sufficient to allow the assessment of tumorspecific changes We have measured RNA DNA and proteinwith various assays Similar assessments in the future shouldalso include chromosome analysis for possible translocations

In the first-line defense against cancer the gradualreplacement of conventional chemotherapy with molecularlytargeted agents has opened the possibility to tailor drug treat-ments to particular tumors This transition necessitates thecharacterization of the molecular lesions that are causativefor the transformation of healthy cells into cancerous cellsbecause drugs need to be matched with the underlying carci-nogenic defect to be effective An additional caveat causedby unique genetic changes in the primary tumor can affectdrug transport and metabolism and needs to be taken intoconsideration In this case the RNA levels for genes that regu-late transport andmetabolismwere extensively skewed in thetumor tissue as compared to muscle and bone (see Table 3)implying potential challenges to chemotherapy An advancedmolecular treatment strategy for cancer will rely on themolecular definition of drug target drug transport and drugmetabolism pharmacogenetics in the primary tumor Con-secutive to cancer dissemination it will also require adjust-ments to account for the genetic changes in the metastases

The cost of health care has been under increasing scrutinyThe treatment of cancer patients is expensive in particularwhen hospitalization is required and further in cases ofend-of-life care Avoidable expenditures are generated bysuboptimal treatment decisions that result in low efficacy orhigh toxicity of anticancer regimens Molecular medicine hasthe potential to preempt those problems and reduce wastefulspending Yet it requires the upfront cost of molecular cancerexamination The analysis performed in this study requiredabout $11000- in nonsalary expenses to perform While ithas not identified a drug target it has specified possibleconfines for drug treatment The costs for molecular analysisneed to be weighed against the societal cost derived fromlost productivity in the workforce disrupted lives of familiesand premature deaths While currently limited drug optionsconstitute the major constraint to the approach taken herethe foreseeable future will bring an increasing spectrum ofmolecularly targeted drugs along with faster and cheapertechnologies for the molecular assessment of cancers Theywill facilitate the clinical translation of our approach

Consent

The patient provided informed consent

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by DOD Grant PR094070under University of Cincinnati IRB protocol 04-01-29-01The

author is grateful to Dr Rhett Kovall for helping with thestructural analysis of FAF1

References

[1] G F Weber Molecular Mechanisms of Cancer Springer Dor-drecht The Netherlands 2007

[2] N G Sanerkin ldquoPrimary leiomyosarcoma of the bone and itscomparison with fibrosarcomardquoCancer vol 44 no 4 pp 1375ndash1387 1979

[3] J M Weaver and J A Abraham ldquoLeiomyosarcoma of the Boneand Soft Tissue A Reviewrdquo httpsarcomahelporglearningcenterleiomyosarcomahtml

[4] M A Depristo E Banks R Poplin et al ldquoA framework forvariation discovery and genotyping using next-generationDNAsequencing datardquo Nature Genetics vol 43 no 5 pp 491ndash5012011

[5] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[6] C W Menges D A Altomare and J R Testa ldquoFAS-associatedfactor 1 (FAF1) diverse functions and implications for oncoge-nesisrdquo Cell Cycle vol 8 no 16 pp 2528ndash2534 2009

[7] M Kim J H Lee S Y Lee E Kim and J Chung ldquoCaspar asuppressor of antibacterial immunity in Drosophilardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 44 pp 16358ndash16363 2006

[8] J Song K P Joon J-J Lee et al ldquoStructure and interaction ofubiquitin-associated domain of human Fas-associated factor 1rdquoProtein Science vol 18 no 11 pp 2265ndash2276 2009

[9] P H OrsquoFarrell ldquoHigh resolution two dimensional electrophore-sis of proteinsrdquo Journal of Biological Chemistry vol 250 no 10pp 4007ndash4021 1975

[10] A Burgess-Cassler J J Johansen D A Santek J R Ideand N C Kendrick ldquoComputerized quantitative analysis ofCoomassie-Blue-stained serum proteins separated by two-dimensional electrophoresisrdquo Clinical Chemistry vol 35 no 12pp 2297ndash2304 1989

[11] B R Oakley D R Kirsch and N RMorris ldquoA simplified ultra-sensitive silver stain for detecting proteins in polyacrylamidegelsrdquo Analytical Biochemistry vol 105 no 2 pp 361ndash363 1980

[12] K Chu X Niu and L T Williams ldquoA Fas-associated proteinfactor FAF1 potentiates Fas-mediated apoptosisrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 25 pp 11894ndash11898 1995

[13] B Rikhof S de Jong A J H Suurmeijer C Meijer and W TA van der Graaf ldquoThe insulin-like growth factor system andsarcomasrdquoThe Journal of Pathology vol 217 no 4 pp 469ndash4822009

[14] RGirling J F Partridge L R Bandara et al ldquoAnew componentof the transcription factor DRTF1E2Frdquo Nature vol 362 no6415 pp 83ndash87 1993

[15] F Mercurio and M Karin ldquoTranscription factors AP-3 andAP-2 interact with the SV40 enhancer in a mutually exclusivemannerrdquo EMBO Journal vol 8 no 5 pp 1455ndash1460 1989

[16] R Bassel-Duby M D Hernandez M A Gonzalez J KKrueger and R S Williams ldquoA 40-kilodalton protein bindsspecifically to an upstream sequence element essential for mus-cle-specific transcription of the human myoglobin promoterrdquoMolecular and Cellular Biology vol 12 no 11 pp 5024ndash50321992

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 10: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

10 Sarcoma

FAS associating region

DED interacting region

UB12 DZM

UB2domain

UASdomain

UBXdomain

UBAdomain

S289 S291

Aurora ACK II

S181

AKT1ATMGSK3

AuroraPKG

PLK1RSK

STK4CHK1

650566481336260205169110475

(a)

MASNMDREMILADFQACTGIENIDEAITLLEQNNWDLVAAINGVIPQENGILQSEYGGETIPGPAFNPASHPASAPTSSSSSAFRPVMPSRQIVERQPRM

6

1

101

100

200

300

400

500

600

650

201

301

401

501

601

667855899998876467765678888887578758999986467 88 8888888888888988888899988888889888877767888777888757

2222454345655744642554435764462443558468643653433344333424435334233333332333332333354335333344334354

LDFRVEYRDRNVDVVLEDTCTVGEIKQILENELQIPVSKMLLKGWKTGDVEDSTVLKSLHLPKNNSLYVLTPDLPPPSSSSHAGALQESLNQNFMLIITH

9999997797689998787758999999999859996577754788888888875333467888868987688888888888877888855675799987

9585847535375857543447459666955563844544585374435443365845846434457567533353232334334444433337484944

REVQREYNLNFSGSSTIQEVKRNVYDLTSIPVRHQLWEGWPTSATDDSMCLAESGLSYPCHRLTVGRRSSPAQTREQSEEQITDVHMVSDSDGDDFEDAT

5888757887577654788887766654677776555468877888765455546887766555688888877777777777776556788888767766

5344456484742545656645594364384534437445444342323455453535435463434323333343434333333334333335433333

EFGVDDGEVFGMASSALRKSPMMPENAENEGDALLQFTAEFSSRYGDCHPVFFIGSLEAAFQEAFYVKARDRKLLAIYLHHDESVLTNVFCSQMLCAESI

5666776654456777888888888888866889999999998848888876677778999999998776686899998589875579999987486899

4635343574354322343434533333343548479747945564424647635575478569766456468999999754233454687457844669

VSYLSQNFITWAWDLTKDSNRARFLTMCNRHFGSVVAQTIRTQKTDQFPLFLIIMGKRSSNEVLNVIQGNTTVDELMMRLMAAMEIFTAQQQEDIKDEDE

9999888589997557877545444345677876335554467888876899986799876447777677888999999998877555778887777778

6778547999996674433343444333333335466456434433487999895353333464346434365468755846444545454565446565

REARENVKREQDEAYRLSLEADRAKREAHEREMAEQFRLEQIRKEQEEEREAIRLSLEQALPPEPKEENAEPVSKLRIRTPSGEFLERRFLASNKLQIVF

8888888887788877766545557777777666777777777776678877777766578888888888885799998589975788758987589999

4545445564445344433443354555556653665456544454434444444443344334333333446859596743354745795535796898

DFVASKGFPWDEYKLLSTFPRRDVTQLDPNKSLLEVKLFPQETLFLEAKE

99997799988779985788754577888765665678898589998589

67974525444795877564635844444476875837446989897366

(b)

Figure 2 FAF1 structure (a) A schematic of functional domains in FAF1 with annotations (adapted from [6ndash8]) There are two ubiquitin-like domains flanking the S181 site The PDB structure 2DZM identifies the N-terminal one while the C-terminal prediction is by sequencesimilarity Whereas the mutation S181G is not expected to disrupt the protein structure per se this amino acid has a high score as a possiblephosphorylation site for a number of kinases involved inDNAdamage repair (b) Secondary structure prediction of FAF1The residues aroundS181 appear unstructured

reflected in STAT3 constitutive phosphorylation The lack ofphosphorylation in this case suggests that the leiomyosar-coma may not depend on the STAT3 pathway

36 Pharmacogenetic Evaluation Predicting the sensitivityto anticancer drugs is a main goal of molecular analysisFor this the over- or underexpression of genes for drugtransport and metabolism is of key importance Analysis ofthe RNASeq data for these groups of gene products identified

a surprisingly large number of deregulations compared tomuscle or bone (Tables 3(a) and 3(b)) Those alterationsmay affect choices for drug treatment For example the highlevels of glutathione S-transferase may render carmustinethioTEPA cisplatin chlorambucil melphalan nitrogenmus-tard phosphoramide mustard acrolein or steroids ineffec-tive The overexpression ofN-acetyltransferase may compro-mise 5-fluorouracil or taxolThemodest upregulation of onlytwo export transporters (ABC-transporters) and specifically

Sarcoma 11

Bone Tumor

Muscle

(a)

Transcription factor Description Reference

E2F1 Transcriptional activation of cell cycle progression is selectively activated in response to specific cues key downstream target of RB1 can promote proliferation or apoptosis when activated [14]

AP-33 interact in a mutually exclusive manner [15]

CCAC CCAC box is an essential element for muscle-specific transcription from the myoglobin promoter [16]

LIII-BP The L-III transcriptional regulatory element (a carbohydrate-response element) is in the pyruvate kinase L gene necessary for cell type-specific expression and transcriptional stimulation by carbohydrates [17]

ADD-1 (SREBP-1) activates transcription from sterol-regulated elements and from an E-box sequence present in the promoter of fatty acid synthase [18]

PAX6 Member of the paired box gene family transcriptional regulator involved in developmental processes [19]

48kD transcription factor activator protein-3 binds to the core element and recognizes the SV40 enhancer and AP-2 and AP-

(b)

Figure 3 Transcription factor activation Nuclear extracts from tumor muscle and bone were tested for DNA binding activity on a protein-DNA array (a) Transcription factor binding that was high in all 3 tissues and is therefore considered constitutively active is circled in yellow(left to right top to bottom AhRAmt GATA1 GATA2 GATA12 HIF1 and HOXD8910) Transcription factors that show high binding inthe cancer but not in muscle or bone are circled in red (left to right top to bottom E2F1 AP3 LIII-BP PAX6 ADD-1 and CCAC) (b) Thefunctions of transcription factors that display high DNA binding in the cancer but not in muscle or bone are described

the lack of ABCB1 overexpression is favorable for avoidingdrug resistance

37 Population Analysis The above-described results indi-cated that a FAF1 mutation which replaces serine in position181 thus preventing FAF1 phosphorylation and activationmay be a driver for leiomyosarcomagenesis A custom Taq-Man assay confirmed the presence of the somatic mutationin the patient To assess whether this single nucleotidereplacement is common in this type of cancer we analyzedDNA from 29 leiomyosarcomas and 1 blood sample from aleiomyosarcoma patient For comparison to nonsarcomatoustumors 34 breast cancers served as a reference None of themdisplayed amutation in the same locus By contrast there wasa distribution across all leiomyosarcomas in the osteopontin

promoter position minus443 (used as a reference) with 12 CC 10TC and 9 TT

4 Discussion

TheFAF1mutation identified as the likely cause for the cancerunder study gives room for an explanation of the sarcomatoustransformation (Figure 5) DNA damage to mesenchymalcells occurs persistently in an oxidizing environment at 37∘CThese insults are rarely transforming and such an occurrencewould trigger the initiation of programmed cell death inapoptosis-competent cells Intact FAF1 associates with FASand enhances apoptosis mediated through this receptor [12]A loss of function in FAF1 could lead to transformation viaantiapoptosis Whereas the mutation S181G is not expected

12 Sarcoma

Muscle

Bone

Tumor

220

94

6043

29

14

220

94

60

43

29

14

220

94

60

43

29

14

1

7

6

23

24

21

22

25

26

89 1011 12

1516 1719

pH 4 8 pH 4 8

8pH 4

(a)

SwissProt or NCBISpot number Protein identified accession Description

(Structural)6 Transgelin Q01995 Smooth muscle-specific cross-links actin24 Transgelin-2 P37802 Smooth muscle-specific cross-links actin

Vimentin P08670 Intermediate filament in mesenchymal tissue1 Filamin-A P21333 Actin-binding protein regulates the cytoskeleton 21 P06709 Cytoskeleton

(Calcium homeostasis)8 9 Calumenin O43852 Calcium-binding protein in the ER and golgi apparatus26 Protein S100-A11 P31949 Calcium-binding EF hand protein10 Reticulocalbin-3 Q96D15 Calcium-binding protein located in the ER12 P11021 Heat shock 70-kD protein 5 ER lumenal calcium-binding

(Protein processing)25 40S ribosomal protein S12 P25398 Core component of the ribosomal accuracy center7 Glycine-tRNA ligase P41250 Catalyzes the attachment of glycine to tRNA19 P01009 Protease inhibitor23 P01009 Protease inhibitor21 Proteasome activator complex subunit 2 Q9UL46 Proteasome activation10 P07900 Chaperone

(Other)7 Serum albumin P02768 Carrier protein in blood22 Protein disulfide isomerase (C-terminal fragment) P07237 Prolyl hydroxylation in collagen microsomal triglyceride transfer

120573 actin (C-terminal fragment)

78kD glucose-regulated protein

120572-1-antitrypsin120572-1-antitrypsin (C-terminal fragment)

Heat shock protein HSP 90120572 (N-terminal fragment)

11 15ndash17

(b)

Figure 4 Continued

Sarcoma 13

Tumor Muscle

OPN

CD44

STAT3

P-STAT3

Tubulin

75

75

50

15010075

10075

50

(c)

Tumor bone Tumor muscleGene ID Symbol Name Fold change Expression Fold change Expression6876 TAGLN Transgelin 3274 2455 3087 15088407 TAGLN2 Transgelin-2 2164 7338 6975 13037431 VIM Vimentin 1653 295010 4061 696042316 FLNA Filamin A alpha 1304 7791 9005 065160 ACTB Actin beta 0656 973187 5491 67368813 CALU Calumenin 7601 19328 12063 70596282 S100A11 S100 calcium binding protein A11 0931 158914 5071 1686357333 RCN3 Reticulocalbin 3 EF-hand calcium binding domain 2439 0604 6412 01223309 HSPA5 1068 92754 2607 220356696 SPP1 Secreted phosphoprotein 1 7572 46508 547929 0355960 CD44 CD44 molecule (Indian blood group) 2053 26708 15436 20566774 STAT3 Signal transducer and activator of transcription 3 0617 46534 0832 200165925 RB1 Retinoblastoma 1 0736 14772 0442 14268

Heat shock 70kDa protein 5 (glucose-regulated protein 78kDa)

(d)

Figure 4 Protein overexpression (a) 2D protein gel electrophoresis of lysates from muscle (upper left) tumor (upper right) and bone(bottom) in RIPA buffer Red circles indicate the spots that were identified as overexpressed and were further analyzed by mass spectrometry(b) Proteins identified in 2D gel electrophoresis as overexpressed in the tumor in comparison to muscle and bone were analyzed for theiridentity by mass spectrometry The left column indicates the spot number corresponding to the 2D gel The next column contains theprotein name followed by the accession number and a description of the protein functionThe protein functions are grouped into structuralcalcium homeostasis and various others (c) Western blot 10 120583g lysates of tumor muscle and bone in RIPA buffer were loaded per laneand electrophoresed on 10 SDS-polyacrylamide minigels with reducing denaturing sample buffer After transfer to PVDF membranesthey were probed for markers of cancer progression including osteopontin CD44 STAT3 and phospho-STAT3 Antitubulin served as aloading control (d) RNA levels corresponding to the proteins found to be affected in the sarcoma With four exceptions in the tumor-bonecomparison (gray font) upregulated proteins are associated with increased RNA levels STAT3 is not increased on the protein or RNA levelThe reduced level of RB1 expression is consistent with the elevated DNA binding activity of E2F1

to disrupt the structure of the protein this site does scorehigh as a possible phosphorylation site for a number ofkinases involved in DNA damage repair supporting thehypothesis that the cancer cells containing thismutation havelost their ability to respond to transforming DNA damagewith programmed cell death FAF1 antagonizes WNT signal-ing by promoting 120573-catenin degradation in the proteasome[21] a function that may be lost after the point mutationThe elevated DNA binding activity of the protooncogenictranscription factor E2F1 (see Figure 3) could be caused byits interaction with LEF-1 [20] consecutive to persistent

WNT signaling The RNA level of IGF2BP1 (IMP-1) a stress-responsive regulator of mRNA stability is highly upregulatedin the tumor compared to muscle as well as bone (seeTable 2(c)) IGF2BP1 is a transcriptional target of the WNTpathway that regulates NF-120581B activity (see Table 2(e)) andis antiapoptotic [22 23] IGF2BP1 may be upregulated asa consequence of mutated FAF1 not being able to suppressWNT signaling in this specific cancerOf noteWNTpathwayoveractivity may not be required for transformation ratherthe persistence of a WNT pathway signal due to the lack of aFAF1-mediated termination signal may suffice

14 Sarcoma

WNTFrizzled

Axin Dvl

igf2bp1

FAF1

P65 P50

IKK

FAS

120573-Catenin

LEF + E2F1

I120581B

Figure 5 Possible transforming pathway The point mutation inFAF1 is believed to cause a loss of function (crossed out in red)This may lead to an overactivity of the WNT signaling pathwayConsistently E2F1 (activated by LEF1) and igf2bp1 (a transcriptionaltarget of the WNT pathway) have been identified to be upregulatedin the molecular analysis In contrast to the negative regulator FAF1IGF2BP1 is a positive regulator of NF-120581B activityThe overactivity ofthe NF-120581B pathway and the reduced efficacy of FAS signaling can betransforming

The role ofWNT signaling in sarcoma has been subject todebate (eg [24]) In metastatic leiomyosarcoma 120573-Cateninmay accumulate in the nucleus despite a relatively weakexpression of WNT [25] This may be due to WNT signalactivation via noncanonical ligands [26] or to 120573-Cateninbinding the nuclear receptor NR4A2 and releasing it from thecorepressor protein LEF-1 [27] Of note in the cancer understudy here the mRNA level of NR4A2 was overexpressed10-fold compared to bone and 3-fold compared to muscleThe results from this study are consistent with the possibilitythat a lack of termination in the WNT signal rather than itsoveractivation could contribute to sarcomatous transforma-tion Such a mechanism may be reflected in an upregulationof downstream targets even though overexpression of WNTpathway components is not detectable

Other mutations beside FAF1 are less likely to becausative for the cancer EPHA3 was revealed as mutated inthis cancer by DNA exome sequencing and RNASeq EPHA3is a receptor tyrosine kinase that is frequentlymutated in lungcancer Tumor-suppressive effects of wild-type EPHA3 can beoverridden by dominant negative EPHA3 somatic mutations[28]Thismechanism is unlikely to play a role in this sarcomaas the detected mutation is located far N-terminally on theextracellular Ephrin binding domain not on the intracellularkinase domain

FAF1 has been described to act as a tumor suppressor gene[29] Its depletion due to chromosome breakage can affectprognosis in glioblastoma patients [30 31] Single nucleotidepolymorphisms in FAF1 are associated with a risk for gastriccancer [32] While numerous FAF1 mutations are associatedwith various cancers none of these genetic changes in theTCGA database affects the amino acid position 181 (Table 4)

The major upregulations identified in this tumor com-prise muscle-specific gene products (transcription factors

CAAC binding proteomics transgelin transgelin-2 RNAanoctamin-4 and immunohistochemistry smooth mus-cle actin) and calcium-regulating molecules (proteomicscalumenin S100-A11 reticulocalbin-3 and 78 kD glucose-regulated protein) The muscle-specific gene products con-firm this recurrent sarcoma as a leiomyosarcoma (the firsttumor distal to the site of the recurring one had beendiagnosed as an osteosarcoma) Calcium is one of the majorsecond messengers in smooth muscle cells Its uptake isregulated by potential-sensitive ion channels in the cellmembrane and by the activities of various receptors Calciumis stored in the sarcoplasmic reticulum from where it canbe released to facilitate actin-myosin interaction and tensiongeneration Phosphorylation of the myosin light chain by acalmodulin-regulated enzyme is important for contractionThe upregulation of gene products associated with migrationand invasion (osteopontin MMP1 vimentin filamin-A and120573-actin) and gene products for extracellularmatrixmoleculesand their modulators (fibronectin collagen ITGBL1 andMXRA5) reflects the invasive nature of this cancer

The recurrence of a sarcoma after 14 years has twoprobable explanations either it is due to a cancer pre-disposition syndrome based on a germ-line mutation (theage of the patient weakens this hypothesis) or the secondtumor is a metastatic colony of the first that was reactivatedafter dormancy The location of the sarcoma in the sameextremity and proximal to a preceding mesenchymal cancer(and therefore in its natural path of dissemination) impliedthe probability that this was a relapse in a metastatic siteThe different histologic assessment as osteosarcoma in thefirst occurrence and leiomyosarcoma as the second cancerdoes not necessarily negate that Mixed histology [33 34]and transdifferentiation [35ndash37] have been described forsarcomatous tumors Of note however in this scenarioosteosarcoma seems to more commonly follow leiomyosar-coma than precede it Material from the first cancer of thispatient was not accessible to us It is very plausible that thiscould have been a mineralized leiomyosarcoma In thosetumors the differential diagnosis from osteosarcoma can bedifficult [38] The extracompartmental location of the firsttumor supports this interpretation

On the molecular pathology level sarcomas fall intotwo groups comprising tumors with simple karyotypes(with pathogenetic translocations or specific genetic muta-tions) and tumors with very complex karyotypes (overtchromosome and genomic instability with numerous gainsand losses) [39] Some molecular alterations that lead tocarcinogenesis can be defined in absolute termsThey includegain-of-function mutations or chromosome translocationsthat transformprotooncogenes to oncogenes However otherchanges are relative to the normal tissue of origin suchas pathway overactivity or overexpression on the proteinor RNA levels We have combined the analysis of absolutechanges (DNA exome sequence RNA sequence) with theanalysis of relative changes using skeletal muscle and bone asreference organs (protein-DNA array 2D gel electrophoresisand RNA expression levels) This choice was determined inpart by tissue availability after surgery andwas intended to aidin the distinction of osteosarcoma from myosarcoma While

Sarcoma 15

Table 3 Deregulation of genes for drug disposition Analysis of RNASeq for at least twofold overexpression (italic font) or underexpression(bold font) of genes for (a) transport and (b) metabolism in tumor compared to muscle and bone For the export transporters (ABCtransporters) only overexpression is considered relevant for drug resistance Not shown in the table are the underexpressed genes (ABCA7ABCA8 ABCA13 ABCB6 ABCB10 ABCC6 ABCC6P1 ABCC6P2 ABCC8 ABCC11 ABCD2 ABCG2 and ABCG5)

(a) Transport

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

650655 ABCA17P ATP-binding cassette subfamily A (ABC1) member 17 pseudogene 1360 211624 ABCA4 ATP-binding cassette subfamily A (ABC1) member 4 2038 2802

6555 SLC10A2 Solute carrier family 10 (sodiumbile acid cotransporter family)member 2 minus1962 minus2112

345274 SLC10A6 Solute carrier family 10 (sodiumbile acid cotransporter family)member 6 2623 3240

6563 SLC14A1 Solute carrier family 14 (urea transporter) member 1 (Kidd bloodgroup) minus3074 minus3152

6565 SLC15A2 Solute carrier family 15 (H+peptide transporter) member 2 minus2027 minus2152

6566 SLC16A1 Solute carrier family 16 member 1 (monocarboxylic acid transporter1) 1401 2170

117247 SLC16A10 Solute carrier family 16 member 10 (aromatic amino acid transporter) 2113 2848

6567 SLC16A2 Solute carrier family 16 member 2 (monocarboxylic acid transporter8) 3242 3848

10786 SLC17A3 Solute carrier family 17 (sodium phosphate) member 3 minus2868 minus29596571 SLC18A2 Solute carrier family 18 (vesicular monoamine) member 2 minus2087 minus21946573 SLC19A1 Solute carrier family 19 (folate transporter) member 1 minus2099 minus220310560 SLC19A2 Solute carrier family 19 (thiamine transporter) member 2 1820 254880704 SLC19A3 Solute carrier family 19 member 3 minus3331 minus3478387775 SLC22A10 Solute carrier family 22 member 10 5711 60859390 SLC22A13 Solute carrier family 22 (organic anion transporter) member 13 2623 3217

85413 SLC22A16 Solute carrier family 22 (organic cationcarnitine transporter)member 16 minus8101 minus7299

51310 SLC22A17 Solute carrier family 22 member 17 1475 21755002 SLC22A18 Solute carrier family 22 member 18 2038 27226582 SLC22A2 Solute carrier family 22 (organic cation transporter) member 2 4793 5216

6581 SLC22A3 Solute carrier family 22 (extraneuronal monoamine transporter)member 3 2554 3182

6583 SLC22A4 Solute carrier family 22 (organic cationergothioneine transporter)member 4 minus3603 minus3776

151295 SLC23A3 Solute carrier family 23 (nucleobase transporters) member 3 2109 284810478 SLC25A17 Solute carrier family 25 (mitochondrial carrier) member 17 1311 207783733 SLC25A18 Solute carrier family 25 (mitochondrial carrier) member 18 1846 2570

788 SLC25A20 Solute carrier family 25 (carnitineacylcarnitine translocase) member20 minus2105 minus2207

89874 SLC25A21 Solute carrier family 25 (mitochondrial oxodicarboxylate carrier)member 21 minus2962 minus3105

51312 SLC25A37 Solute carrier family 25 member 37 minus4633 minus486651629 SLC25A39 Solute carrier family 25 member 39 minus2585 minus2737203427 SLC25A43 Solute carrier family 25 member 43 1325 208865012 SLC26A10 Solute carrier family 26 member 10 3896 4472115111 SLC26A7 Solute carrier family 26 member 7 3431 4018116369 SLC26A8 Solute carrier family 26 member 8 minus5354 minus5536115019 SLC26A9 Solute carrier family 26 member 9 1623 241911001 SLC27A2 Solute carrier family 27 (fatty acid transporter) member 2 minus4278 minus4487

16 Sarcoma

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

64078 SLC28A3 Solute carrier family 28 (sodium-coupled nucleoside transporter)member 3 minus4543 minus4812

222962 SLC29A4 Solute carrier family 29 (nucleoside transporters) member 4 minus1962 minus204581031 SLC2A10 Solute carrier family 2 (facilitated glucose transporter) member 10 2532 3170

6518 SLC2A5 Solute carrier family 2 (facilitated glucosefructose transporter)member 5 minus3647 minus3821

55532 SLC30A10 Solute carrier family 30 member 10 minus3547 minus37257782 SLC30A4 Solute carrier family 30 (zinc transporter) member 4 2832 33726569 SLC34A1 Solute carrier family 34 (sodium phosphate) member 1 minus4716 minus4941340146 SLC35D3 Solute carrier family 35 member D3 minus5662 minus590654733 SLC35F2 Solute carrier family 35 member F2 1433 2170206358 SLC36A1 Solute carrier family 36 (protonamino acid symporter) member 1 minus2265 minus2345285641 SLC36A3 Solute carrier family 36 (protonamino acid symporter) member 3 minus2284 minus239354020 SLC37A1 Solute carrier family 37 (glycerol-3-phosphate transporter) member 1 minus2112 minus22122542 SLC37A4 Solute carrier family 37 (glucose-6-phosphate transporter) member 4 minus2117 minus2219151258 SLC38A11 Solute carrier family 38 member 11 2410 308555089 SLC38A4 Solute carrier family 38 member 4 1837 254892745 SLC38A5 Solute carrier family 38 member 5 minus1994 minus215291252 SLC39A13 Solute carrier family 39 (zinc transporter) member 13 1301 207023516 SLC39A14 Solute carrier family 39 (zinc transporter) member 14 2569 318829985 SLC39A3 Solute carrier family 39 (zinc transporter) member 3 minus2032 minus2152283375 SLC39A5 Solute carrier family 39 (metal ion transporter) member 5 1623 23707922 SLC39A7 Solute carrier family 39 (zinc transporter) member 7 1273 205830061 SLC40A1 Solute carrier family 40 (iron-regulated transporter) member 1 minus3612 minus379684102 SLC41A2 Solute carrier family 41 member 2 1293 20708501 SLC43A1 Solute carrier family 43 member 1 minus2013 minus215257153 SLC44A2 Solute carrier family 44 member 2 minus2047 minus215250651 SLC45A1 Solute carrier family 45 member 1 2038 2722146802 SLC47A2 Solute carrier family 47 member 2 minus1962 minus20266521 SLC4A1 Solute carrier family 4 anion exchanger member 1 minus6513 minus645357282 SLC4A10 Solute carrier family 4 sodium bicarbonate transporter member 10 minus2640 minus275383959 SLC4A11 Solute carrier family 4 sodium borate transporter member 11 1846 25696508 SLC4A3 Solute carrier family 4 anion exchanger member 3 1261 20458671 SLC4A4 Solute carrier family 4 sodium bicarbonate cotransporter member 4 2445 31139497 SLC4A7 Solute carrier family 4 sodium bicarbonate cotransporter member 7 2717 33076523 SLC5A1 Solute carrier family 5 (sodiumglucose cotransporter) member 1 minus2547 minus2705159963 SLC5A12 Solute carrier family 5 (sodiumglucose cotransporter) member 12 4753 52066527 SLC5A4 Solute carrier family 5 (low affinity glucose cotransporter) member 4 minus3969 minus4249

6540 SLC6A13 Solute carrier family 6 (neurotransmitter transporter GABA)member 13 1328 2092

55117 SLC6A15 Solute carrier family 6 (neutral amino acid transporter) member 15 1623 2333388662 SLC6A17 Solute carrier family 6 member 17 2038 272854716 SLC6A20 Solute carrier family 6 (proline IMINO transporter) member 20 1697 2433

6532 SLC6A4 Solute carrier family 6 (neurotransmitter transporter serotonin)member 4 minus2512 minus2611

6534 SLC6A7 Solute carrier family 6 (neurotransmitter transporter L-proline)member 7 2623 3228

56301 SLC7A10 Solute carrier family 7 (neutral amino acid transporter y+ system)member 10 minus3421 minus3603

Sarcoma 17

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

6547 SLC8A3 Solute carrier family 8 (sodiumcalcium exchanger) member 3 minus2204 minus2309285335 SLC9A10 Solute carrier family 9 member 10 1208 20006549 SLC9A2 Solute carrier family 9 (sodiumhydrogen exchanger) member 2 2038 2706

9368 SLC9A3R1 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 1 minus2760 minus2846

9351 SLC9A3R2 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 2 1889 2619

84679 SLC9A7 Solute carrier family 9 (sodiumhydrogen exchanger) member 7 3347 393510599 SLCO1B1 Solute carrier organic anion transporter family member 1B1 3360 397728234 SLCO1B3 Solute carrier organic anion transporter family member 1B3 9760 95696578 SLCO2A1 Solute carrier organic anion transporter family member 2A1 2432 309628232 SLCO3A1 Solute carrier organic anion transporter family member 3A1 minus2032 minus2152353189 SLCO4C1 Solute carrier organic anion transporter family member 4C1 minus6850 minus6611

(b) Metabolism

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold Cchange

1583 CYP11A1 Cytochrome P450 family 11 subfamily A polypeptide 1 1623 23961589 CYP21A2 Cytochrome P450 family 21 subfamily A polypeptide 2 minus1962 minus20361591 CYP24A1 Cytochrome P450 family 24 subfamily A polypeptide 1 5208 55771592 CYP26A1 Cytochrome P450 family 26 subfamily A polypeptide 1 2623 32571594 CYP27B1 Cytochrome P450 family 27 subfamily B polypeptide 1 1846 2585339761 CYP27C1 Cytochrome P450 family 27 subfamily C polypeptide 1 3585 41781553 CYP2A13 Cytochrome P450 family 2 subfamily A polypeptide 13 minus1962 minus20351580 CYP4B1 Cytochrome P450 family 4 subfamily B polypeptide 1 minus4820 minus506566002 CYP4F12 Cytochrome P450 family 4 subfamily F polypeptide 12 minus6070 minus61958529 CYP4F2 Cytochrome P450 family 4 subfamily F polypeptide 2 minus7769 minus70604051 CYP4F3 Cytochrome P450 family 4 subfamily F polypeptide 3 minus8244 minus749111283 CYP4F8 Cytochrome P450 family 4 subfamily F polypeptide 8 minus5006 minus5270260293 CYP4X1 Cytochrome P450 family 4 subfamily X polypeptide 1 minus2476 minus25809420 CYP7B1 Cytochrome P450 family 7 subfamily B polypeptide 1 2502 31702326 FMO1 Flavin containing monooxygenase 1 4360 48592327 FMO2 Flavin containing monooxygenase 2 (nonfunctional) minus4074 minus43372328 FMO3 Flavin containing monooxygenase 3 minus4036 minus4309388714 FMO6P Flavin containing monooxygenase 6 pseudogene minus2569 minus2737493869 GPX8 Glutathione peroxidase 8 (putative) 2814 33712938 GSTA1 Glutathione S-transferase alpha 1 1623 23632939 GSTA2 Glutathione S-transferase alpha 2 2038 27412941 GSTA4 Glutathione S-transferase alpha 4 1492 21882953 GSTT2 Glutathione S-transferase theta 2 4682 5170653689 GSTT2B Glutathione S-transferase theta 2B (genepseudogene) 3739 430784779 NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439 79969027 NAT8 N-acetyltransferase 8 (GCN5-related putative) minus2769 minus29207358 UGDH UDP-glucose 6-dehydrogenase 2333 301855757 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 1604 227110720 UGT2B11 UDP glucuronosyltransferase 2 family polypeptide B11 minus3586 minus37687367 UGT2B17 UDP glucuronosyltransferase 2 family polypeptide B17 minus2836 minus295954490 UGT2B28 UDP glucuronosyltransferase 2 family polypeptide B28 minus3132 minus3284167127 UGT3A2 UDP glycosyltransferase 3 family polypeptide A2 minus4716 minus4907

18 Sarcoma

Table 4 FAF1 mutations in various cancers FAF1 mutations listed in the TCGA data base were identified without restriction to any type ofcancer For the location of the affected domains on the protein compare Figure 2

AA Mutation Cancer DomainN4S Missense Cutaneous melanomaI10S Missense Stomach adenocarcinoma UBAE21lowast Nonsense Cutaneous melanoma UBAE25K Missense Uterine endometrioid carcinoma UBAV38 splice Splice Stomach adenocarcinoma UBAP86fs FS del Colorectal adenocarcinomaG123 splice Splice Uterine endometrioid carcinoma UB1P136H Missense Colorectal adenocarcinoma UB1T147M Missense Brain lower grade glioma UB1D149Y Missense Uterine endometrioid carcinoma UB1L159V Missense Lung adenocarcinoma UB1K163N Missense Uterine endometrioid carcinoma UB1L170F Missense Cutaneous melanomaG184 splice Splice Colorectal cancerQ187H Missense Stomach adenocarcinomaS214N Missense Colorectal adenocarcinoma UB2R222I Missense Uterine endometrioid carcinoma UB2E238D Missense Lung adenocarcinoma UB2P241S Missense Uterine endometrioid carcinoma UB2T245A Missense Renal clear cell carcinoma UB2M249V Missense Uterine endometrioid carcinoma UB2E280K Missense Brain lower grade gliomaG293lowast Nonsense Colorectal cancerT300I Missense Colorectal adenocarcinomaD305H Missense Lung adenocarcinomaE308Q Missense Lung adenocarcinomaA316V Missense Stomach adenocarcinomaK319fs FS ins Head and neck squamous cell carcinomaR344G Missense Stomach adenocarcinoma UASF353I Missense Cutaneous melanoma UASL379V Missense Breast invasive carcinoma UASC396F Missense Cutaneous melanoma UASS459lowast Nonsense Uterine endometrioid carcinoma UASG469 splice Splice Glioblastoma multiforme UASR509G Missense Lung squamous cell carcinomaE510fs FS del Cutaneous melanomaR516C Missense Uterine endometrioid carcinomaA534V Missense Stomach adenocarcinomaF537L Missense Stomach adenocarcinomaE551lowast Nonsense Lung adenocarcinomaR554W Missense Colorectal cancerS582I Missense Lung adenocarcinoma UBXF585L Missense Uterine endometrioid carcinoma UBXE587lowast Nonsense Lung adenocarcinoma UBXA592V Missense Stomach adenocarcinoma UBXW610lowast Nonsense Breast invasive carcinoma UBXD611Y Missense Lung adenocarcinoma UBXE635fs FS del Brain lower grade glioma UBXP640fs FS del Cutaneous melanoma UBX

Sarcoma 19

a more accurate reference point for a leiomyosarcoma wouldhave been smooth muscle we believe that the comparison tostriated muscle is sufficient to allow the assessment of tumorspecific changes We have measured RNA DNA and proteinwith various assays Similar assessments in the future shouldalso include chromosome analysis for possible translocations

In the first-line defense against cancer the gradualreplacement of conventional chemotherapy with molecularlytargeted agents has opened the possibility to tailor drug treat-ments to particular tumors This transition necessitates thecharacterization of the molecular lesions that are causativefor the transformation of healthy cells into cancerous cellsbecause drugs need to be matched with the underlying carci-nogenic defect to be effective An additional caveat causedby unique genetic changes in the primary tumor can affectdrug transport and metabolism and needs to be taken intoconsideration In this case the RNA levels for genes that regu-late transport andmetabolismwere extensively skewed in thetumor tissue as compared to muscle and bone (see Table 3)implying potential challenges to chemotherapy An advancedmolecular treatment strategy for cancer will rely on themolecular definition of drug target drug transport and drugmetabolism pharmacogenetics in the primary tumor Con-secutive to cancer dissemination it will also require adjust-ments to account for the genetic changes in the metastases

The cost of health care has been under increasing scrutinyThe treatment of cancer patients is expensive in particularwhen hospitalization is required and further in cases ofend-of-life care Avoidable expenditures are generated bysuboptimal treatment decisions that result in low efficacy orhigh toxicity of anticancer regimens Molecular medicine hasthe potential to preempt those problems and reduce wastefulspending Yet it requires the upfront cost of molecular cancerexamination The analysis performed in this study requiredabout $11000- in nonsalary expenses to perform While ithas not identified a drug target it has specified possibleconfines for drug treatment The costs for molecular analysisneed to be weighed against the societal cost derived fromlost productivity in the workforce disrupted lives of familiesand premature deaths While currently limited drug optionsconstitute the major constraint to the approach taken herethe foreseeable future will bring an increasing spectrum ofmolecularly targeted drugs along with faster and cheapertechnologies for the molecular assessment of cancers Theywill facilitate the clinical translation of our approach

Consent

The patient provided informed consent

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by DOD Grant PR094070under University of Cincinnati IRB protocol 04-01-29-01The

author is grateful to Dr Rhett Kovall for helping with thestructural analysis of FAF1

References

[1] G F Weber Molecular Mechanisms of Cancer Springer Dor-drecht The Netherlands 2007

[2] N G Sanerkin ldquoPrimary leiomyosarcoma of the bone and itscomparison with fibrosarcomardquoCancer vol 44 no 4 pp 1375ndash1387 1979

[3] J M Weaver and J A Abraham ldquoLeiomyosarcoma of the Boneand Soft Tissue A Reviewrdquo httpsarcomahelporglearningcenterleiomyosarcomahtml

[4] M A Depristo E Banks R Poplin et al ldquoA framework forvariation discovery and genotyping using next-generationDNAsequencing datardquo Nature Genetics vol 43 no 5 pp 491ndash5012011

[5] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[6] C W Menges D A Altomare and J R Testa ldquoFAS-associatedfactor 1 (FAF1) diverse functions and implications for oncoge-nesisrdquo Cell Cycle vol 8 no 16 pp 2528ndash2534 2009

[7] M Kim J H Lee S Y Lee E Kim and J Chung ldquoCaspar asuppressor of antibacterial immunity in Drosophilardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 44 pp 16358ndash16363 2006

[8] J Song K P Joon J-J Lee et al ldquoStructure and interaction ofubiquitin-associated domain of human Fas-associated factor 1rdquoProtein Science vol 18 no 11 pp 2265ndash2276 2009

[9] P H OrsquoFarrell ldquoHigh resolution two dimensional electrophore-sis of proteinsrdquo Journal of Biological Chemistry vol 250 no 10pp 4007ndash4021 1975

[10] A Burgess-Cassler J J Johansen D A Santek J R Ideand N C Kendrick ldquoComputerized quantitative analysis ofCoomassie-Blue-stained serum proteins separated by two-dimensional electrophoresisrdquo Clinical Chemistry vol 35 no 12pp 2297ndash2304 1989

[11] B R Oakley D R Kirsch and N RMorris ldquoA simplified ultra-sensitive silver stain for detecting proteins in polyacrylamidegelsrdquo Analytical Biochemistry vol 105 no 2 pp 361ndash363 1980

[12] K Chu X Niu and L T Williams ldquoA Fas-associated proteinfactor FAF1 potentiates Fas-mediated apoptosisrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 25 pp 11894ndash11898 1995

[13] B Rikhof S de Jong A J H Suurmeijer C Meijer and W TA van der Graaf ldquoThe insulin-like growth factor system andsarcomasrdquoThe Journal of Pathology vol 217 no 4 pp 469ndash4822009

[14] RGirling J F Partridge L R Bandara et al ldquoAnew componentof the transcription factor DRTF1E2Frdquo Nature vol 362 no6415 pp 83ndash87 1993

[15] F Mercurio and M Karin ldquoTranscription factors AP-3 andAP-2 interact with the SV40 enhancer in a mutually exclusivemannerrdquo EMBO Journal vol 8 no 5 pp 1455ndash1460 1989

[16] R Bassel-Duby M D Hernandez M A Gonzalez J KKrueger and R S Williams ldquoA 40-kilodalton protein bindsspecifically to an upstream sequence element essential for mus-cle-specific transcription of the human myoglobin promoterrdquoMolecular and Cellular Biology vol 12 no 11 pp 5024ndash50321992

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 11: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

Sarcoma 11

Bone Tumor

Muscle

(a)

Transcription factor Description Reference

E2F1 Transcriptional activation of cell cycle progression is selectively activated in response to specific cues key downstream target of RB1 can promote proliferation or apoptosis when activated [14]

AP-33 interact in a mutually exclusive manner [15]

CCAC CCAC box is an essential element for muscle-specific transcription from the myoglobin promoter [16]

LIII-BP The L-III transcriptional regulatory element (a carbohydrate-response element) is in the pyruvate kinase L gene necessary for cell type-specific expression and transcriptional stimulation by carbohydrates [17]

ADD-1 (SREBP-1) activates transcription from sterol-regulated elements and from an E-box sequence present in the promoter of fatty acid synthase [18]

PAX6 Member of the paired box gene family transcriptional regulator involved in developmental processes [19]

48kD transcription factor activator protein-3 binds to the core element and recognizes the SV40 enhancer and AP-2 and AP-

(b)

Figure 3 Transcription factor activation Nuclear extracts from tumor muscle and bone were tested for DNA binding activity on a protein-DNA array (a) Transcription factor binding that was high in all 3 tissues and is therefore considered constitutively active is circled in yellow(left to right top to bottom AhRAmt GATA1 GATA2 GATA12 HIF1 and HOXD8910) Transcription factors that show high binding inthe cancer but not in muscle or bone are circled in red (left to right top to bottom E2F1 AP3 LIII-BP PAX6 ADD-1 and CCAC) (b) Thefunctions of transcription factors that display high DNA binding in the cancer but not in muscle or bone are described

the lack of ABCB1 overexpression is favorable for avoidingdrug resistance

37 Population Analysis The above-described results indi-cated that a FAF1 mutation which replaces serine in position181 thus preventing FAF1 phosphorylation and activationmay be a driver for leiomyosarcomagenesis A custom Taq-Man assay confirmed the presence of the somatic mutationin the patient To assess whether this single nucleotidereplacement is common in this type of cancer we analyzedDNA from 29 leiomyosarcomas and 1 blood sample from aleiomyosarcoma patient For comparison to nonsarcomatoustumors 34 breast cancers served as a reference None of themdisplayed amutation in the same locus By contrast there wasa distribution across all leiomyosarcomas in the osteopontin

promoter position minus443 (used as a reference) with 12 CC 10TC and 9 TT

4 Discussion

TheFAF1mutation identified as the likely cause for the cancerunder study gives room for an explanation of the sarcomatoustransformation (Figure 5) DNA damage to mesenchymalcells occurs persistently in an oxidizing environment at 37∘CThese insults are rarely transforming and such an occurrencewould trigger the initiation of programmed cell death inapoptosis-competent cells Intact FAF1 associates with FASand enhances apoptosis mediated through this receptor [12]A loss of function in FAF1 could lead to transformation viaantiapoptosis Whereas the mutation S181G is not expected

12 Sarcoma

Muscle

Bone

Tumor

220

94

6043

29

14

220

94

60

43

29

14

220

94

60

43

29

14

1

7

6

23

24

21

22

25

26

89 1011 12

1516 1719

pH 4 8 pH 4 8

8pH 4

(a)

SwissProt or NCBISpot number Protein identified accession Description

(Structural)6 Transgelin Q01995 Smooth muscle-specific cross-links actin24 Transgelin-2 P37802 Smooth muscle-specific cross-links actin

Vimentin P08670 Intermediate filament in mesenchymal tissue1 Filamin-A P21333 Actin-binding protein regulates the cytoskeleton 21 P06709 Cytoskeleton

(Calcium homeostasis)8 9 Calumenin O43852 Calcium-binding protein in the ER and golgi apparatus26 Protein S100-A11 P31949 Calcium-binding EF hand protein10 Reticulocalbin-3 Q96D15 Calcium-binding protein located in the ER12 P11021 Heat shock 70-kD protein 5 ER lumenal calcium-binding

(Protein processing)25 40S ribosomal protein S12 P25398 Core component of the ribosomal accuracy center7 Glycine-tRNA ligase P41250 Catalyzes the attachment of glycine to tRNA19 P01009 Protease inhibitor23 P01009 Protease inhibitor21 Proteasome activator complex subunit 2 Q9UL46 Proteasome activation10 P07900 Chaperone

(Other)7 Serum albumin P02768 Carrier protein in blood22 Protein disulfide isomerase (C-terminal fragment) P07237 Prolyl hydroxylation in collagen microsomal triglyceride transfer

120573 actin (C-terminal fragment)

78kD glucose-regulated protein

120572-1-antitrypsin120572-1-antitrypsin (C-terminal fragment)

Heat shock protein HSP 90120572 (N-terminal fragment)

11 15ndash17

(b)

Figure 4 Continued

Sarcoma 13

Tumor Muscle

OPN

CD44

STAT3

P-STAT3

Tubulin

75

75

50

15010075

10075

50

(c)

Tumor bone Tumor muscleGene ID Symbol Name Fold change Expression Fold change Expression6876 TAGLN Transgelin 3274 2455 3087 15088407 TAGLN2 Transgelin-2 2164 7338 6975 13037431 VIM Vimentin 1653 295010 4061 696042316 FLNA Filamin A alpha 1304 7791 9005 065160 ACTB Actin beta 0656 973187 5491 67368813 CALU Calumenin 7601 19328 12063 70596282 S100A11 S100 calcium binding protein A11 0931 158914 5071 1686357333 RCN3 Reticulocalbin 3 EF-hand calcium binding domain 2439 0604 6412 01223309 HSPA5 1068 92754 2607 220356696 SPP1 Secreted phosphoprotein 1 7572 46508 547929 0355960 CD44 CD44 molecule (Indian blood group) 2053 26708 15436 20566774 STAT3 Signal transducer and activator of transcription 3 0617 46534 0832 200165925 RB1 Retinoblastoma 1 0736 14772 0442 14268

Heat shock 70kDa protein 5 (glucose-regulated protein 78kDa)

(d)

Figure 4 Protein overexpression (a) 2D protein gel electrophoresis of lysates from muscle (upper left) tumor (upper right) and bone(bottom) in RIPA buffer Red circles indicate the spots that were identified as overexpressed and were further analyzed by mass spectrometry(b) Proteins identified in 2D gel electrophoresis as overexpressed in the tumor in comparison to muscle and bone were analyzed for theiridentity by mass spectrometry The left column indicates the spot number corresponding to the 2D gel The next column contains theprotein name followed by the accession number and a description of the protein functionThe protein functions are grouped into structuralcalcium homeostasis and various others (c) Western blot 10 120583g lysates of tumor muscle and bone in RIPA buffer were loaded per laneand electrophoresed on 10 SDS-polyacrylamide minigels with reducing denaturing sample buffer After transfer to PVDF membranesthey were probed for markers of cancer progression including osteopontin CD44 STAT3 and phospho-STAT3 Antitubulin served as aloading control (d) RNA levels corresponding to the proteins found to be affected in the sarcoma With four exceptions in the tumor-bonecomparison (gray font) upregulated proteins are associated with increased RNA levels STAT3 is not increased on the protein or RNA levelThe reduced level of RB1 expression is consistent with the elevated DNA binding activity of E2F1

to disrupt the structure of the protein this site does scorehigh as a possible phosphorylation site for a number ofkinases involved in DNA damage repair supporting thehypothesis that the cancer cells containing thismutation havelost their ability to respond to transforming DNA damagewith programmed cell death FAF1 antagonizes WNT signal-ing by promoting 120573-catenin degradation in the proteasome[21] a function that may be lost after the point mutationThe elevated DNA binding activity of the protooncogenictranscription factor E2F1 (see Figure 3) could be caused byits interaction with LEF-1 [20] consecutive to persistent

WNT signaling The RNA level of IGF2BP1 (IMP-1) a stress-responsive regulator of mRNA stability is highly upregulatedin the tumor compared to muscle as well as bone (seeTable 2(c)) IGF2BP1 is a transcriptional target of the WNTpathway that regulates NF-120581B activity (see Table 2(e)) andis antiapoptotic [22 23] IGF2BP1 may be upregulated asa consequence of mutated FAF1 not being able to suppressWNT signaling in this specific cancerOf noteWNTpathwayoveractivity may not be required for transformation ratherthe persistence of a WNT pathway signal due to the lack of aFAF1-mediated termination signal may suffice

14 Sarcoma

WNTFrizzled

Axin Dvl

igf2bp1

FAF1

P65 P50

IKK

FAS

120573-Catenin

LEF + E2F1

I120581B

Figure 5 Possible transforming pathway The point mutation inFAF1 is believed to cause a loss of function (crossed out in red)This may lead to an overactivity of the WNT signaling pathwayConsistently E2F1 (activated by LEF1) and igf2bp1 (a transcriptionaltarget of the WNT pathway) have been identified to be upregulatedin the molecular analysis In contrast to the negative regulator FAF1IGF2BP1 is a positive regulator of NF-120581B activityThe overactivity ofthe NF-120581B pathway and the reduced efficacy of FAS signaling can betransforming

The role ofWNT signaling in sarcoma has been subject todebate (eg [24]) In metastatic leiomyosarcoma 120573-Cateninmay accumulate in the nucleus despite a relatively weakexpression of WNT [25] This may be due to WNT signalactivation via noncanonical ligands [26] or to 120573-Cateninbinding the nuclear receptor NR4A2 and releasing it from thecorepressor protein LEF-1 [27] Of note in the cancer understudy here the mRNA level of NR4A2 was overexpressed10-fold compared to bone and 3-fold compared to muscleThe results from this study are consistent with the possibilitythat a lack of termination in the WNT signal rather than itsoveractivation could contribute to sarcomatous transforma-tion Such a mechanism may be reflected in an upregulationof downstream targets even though overexpression of WNTpathway components is not detectable

Other mutations beside FAF1 are less likely to becausative for the cancer EPHA3 was revealed as mutated inthis cancer by DNA exome sequencing and RNASeq EPHA3is a receptor tyrosine kinase that is frequentlymutated in lungcancer Tumor-suppressive effects of wild-type EPHA3 can beoverridden by dominant negative EPHA3 somatic mutations[28]Thismechanism is unlikely to play a role in this sarcomaas the detected mutation is located far N-terminally on theextracellular Ephrin binding domain not on the intracellularkinase domain

FAF1 has been described to act as a tumor suppressor gene[29] Its depletion due to chromosome breakage can affectprognosis in glioblastoma patients [30 31] Single nucleotidepolymorphisms in FAF1 are associated with a risk for gastriccancer [32] While numerous FAF1 mutations are associatedwith various cancers none of these genetic changes in theTCGA database affects the amino acid position 181 (Table 4)

The major upregulations identified in this tumor com-prise muscle-specific gene products (transcription factors

CAAC binding proteomics transgelin transgelin-2 RNAanoctamin-4 and immunohistochemistry smooth mus-cle actin) and calcium-regulating molecules (proteomicscalumenin S100-A11 reticulocalbin-3 and 78 kD glucose-regulated protein) The muscle-specific gene products con-firm this recurrent sarcoma as a leiomyosarcoma (the firsttumor distal to the site of the recurring one had beendiagnosed as an osteosarcoma) Calcium is one of the majorsecond messengers in smooth muscle cells Its uptake isregulated by potential-sensitive ion channels in the cellmembrane and by the activities of various receptors Calciumis stored in the sarcoplasmic reticulum from where it canbe released to facilitate actin-myosin interaction and tensiongeneration Phosphorylation of the myosin light chain by acalmodulin-regulated enzyme is important for contractionThe upregulation of gene products associated with migrationand invasion (osteopontin MMP1 vimentin filamin-A and120573-actin) and gene products for extracellularmatrixmoleculesand their modulators (fibronectin collagen ITGBL1 andMXRA5) reflects the invasive nature of this cancer

The recurrence of a sarcoma after 14 years has twoprobable explanations either it is due to a cancer pre-disposition syndrome based on a germ-line mutation (theage of the patient weakens this hypothesis) or the secondtumor is a metastatic colony of the first that was reactivatedafter dormancy The location of the sarcoma in the sameextremity and proximal to a preceding mesenchymal cancer(and therefore in its natural path of dissemination) impliedthe probability that this was a relapse in a metastatic siteThe different histologic assessment as osteosarcoma in thefirst occurrence and leiomyosarcoma as the second cancerdoes not necessarily negate that Mixed histology [33 34]and transdifferentiation [35ndash37] have been described forsarcomatous tumors Of note however in this scenarioosteosarcoma seems to more commonly follow leiomyosar-coma than precede it Material from the first cancer of thispatient was not accessible to us It is very plausible that thiscould have been a mineralized leiomyosarcoma In thosetumors the differential diagnosis from osteosarcoma can bedifficult [38] The extracompartmental location of the firsttumor supports this interpretation

On the molecular pathology level sarcomas fall intotwo groups comprising tumors with simple karyotypes(with pathogenetic translocations or specific genetic muta-tions) and tumors with very complex karyotypes (overtchromosome and genomic instability with numerous gainsand losses) [39] Some molecular alterations that lead tocarcinogenesis can be defined in absolute termsThey includegain-of-function mutations or chromosome translocationsthat transformprotooncogenes to oncogenes However otherchanges are relative to the normal tissue of origin suchas pathway overactivity or overexpression on the proteinor RNA levels We have combined the analysis of absolutechanges (DNA exome sequence RNA sequence) with theanalysis of relative changes using skeletal muscle and bone asreference organs (protein-DNA array 2D gel electrophoresisand RNA expression levels) This choice was determined inpart by tissue availability after surgery andwas intended to aidin the distinction of osteosarcoma from myosarcoma While

Sarcoma 15

Table 3 Deregulation of genes for drug disposition Analysis of RNASeq for at least twofold overexpression (italic font) or underexpression(bold font) of genes for (a) transport and (b) metabolism in tumor compared to muscle and bone For the export transporters (ABCtransporters) only overexpression is considered relevant for drug resistance Not shown in the table are the underexpressed genes (ABCA7ABCA8 ABCA13 ABCB6 ABCB10 ABCC6 ABCC6P1 ABCC6P2 ABCC8 ABCC11 ABCD2 ABCG2 and ABCG5)

(a) Transport

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

650655 ABCA17P ATP-binding cassette subfamily A (ABC1) member 17 pseudogene 1360 211624 ABCA4 ATP-binding cassette subfamily A (ABC1) member 4 2038 2802

6555 SLC10A2 Solute carrier family 10 (sodiumbile acid cotransporter family)member 2 minus1962 minus2112

345274 SLC10A6 Solute carrier family 10 (sodiumbile acid cotransporter family)member 6 2623 3240

6563 SLC14A1 Solute carrier family 14 (urea transporter) member 1 (Kidd bloodgroup) minus3074 minus3152

6565 SLC15A2 Solute carrier family 15 (H+peptide transporter) member 2 minus2027 minus2152

6566 SLC16A1 Solute carrier family 16 member 1 (monocarboxylic acid transporter1) 1401 2170

117247 SLC16A10 Solute carrier family 16 member 10 (aromatic amino acid transporter) 2113 2848

6567 SLC16A2 Solute carrier family 16 member 2 (monocarboxylic acid transporter8) 3242 3848

10786 SLC17A3 Solute carrier family 17 (sodium phosphate) member 3 minus2868 minus29596571 SLC18A2 Solute carrier family 18 (vesicular monoamine) member 2 minus2087 minus21946573 SLC19A1 Solute carrier family 19 (folate transporter) member 1 minus2099 minus220310560 SLC19A2 Solute carrier family 19 (thiamine transporter) member 2 1820 254880704 SLC19A3 Solute carrier family 19 member 3 minus3331 minus3478387775 SLC22A10 Solute carrier family 22 member 10 5711 60859390 SLC22A13 Solute carrier family 22 (organic anion transporter) member 13 2623 3217

85413 SLC22A16 Solute carrier family 22 (organic cationcarnitine transporter)member 16 minus8101 minus7299

51310 SLC22A17 Solute carrier family 22 member 17 1475 21755002 SLC22A18 Solute carrier family 22 member 18 2038 27226582 SLC22A2 Solute carrier family 22 (organic cation transporter) member 2 4793 5216

6581 SLC22A3 Solute carrier family 22 (extraneuronal monoamine transporter)member 3 2554 3182

6583 SLC22A4 Solute carrier family 22 (organic cationergothioneine transporter)member 4 minus3603 minus3776

151295 SLC23A3 Solute carrier family 23 (nucleobase transporters) member 3 2109 284810478 SLC25A17 Solute carrier family 25 (mitochondrial carrier) member 17 1311 207783733 SLC25A18 Solute carrier family 25 (mitochondrial carrier) member 18 1846 2570

788 SLC25A20 Solute carrier family 25 (carnitineacylcarnitine translocase) member20 minus2105 minus2207

89874 SLC25A21 Solute carrier family 25 (mitochondrial oxodicarboxylate carrier)member 21 minus2962 minus3105

51312 SLC25A37 Solute carrier family 25 member 37 minus4633 minus486651629 SLC25A39 Solute carrier family 25 member 39 minus2585 minus2737203427 SLC25A43 Solute carrier family 25 member 43 1325 208865012 SLC26A10 Solute carrier family 26 member 10 3896 4472115111 SLC26A7 Solute carrier family 26 member 7 3431 4018116369 SLC26A8 Solute carrier family 26 member 8 minus5354 minus5536115019 SLC26A9 Solute carrier family 26 member 9 1623 241911001 SLC27A2 Solute carrier family 27 (fatty acid transporter) member 2 minus4278 minus4487

16 Sarcoma

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

64078 SLC28A3 Solute carrier family 28 (sodium-coupled nucleoside transporter)member 3 minus4543 minus4812

222962 SLC29A4 Solute carrier family 29 (nucleoside transporters) member 4 minus1962 minus204581031 SLC2A10 Solute carrier family 2 (facilitated glucose transporter) member 10 2532 3170

6518 SLC2A5 Solute carrier family 2 (facilitated glucosefructose transporter)member 5 minus3647 minus3821

55532 SLC30A10 Solute carrier family 30 member 10 minus3547 minus37257782 SLC30A4 Solute carrier family 30 (zinc transporter) member 4 2832 33726569 SLC34A1 Solute carrier family 34 (sodium phosphate) member 1 minus4716 minus4941340146 SLC35D3 Solute carrier family 35 member D3 minus5662 minus590654733 SLC35F2 Solute carrier family 35 member F2 1433 2170206358 SLC36A1 Solute carrier family 36 (protonamino acid symporter) member 1 minus2265 minus2345285641 SLC36A3 Solute carrier family 36 (protonamino acid symporter) member 3 minus2284 minus239354020 SLC37A1 Solute carrier family 37 (glycerol-3-phosphate transporter) member 1 minus2112 minus22122542 SLC37A4 Solute carrier family 37 (glucose-6-phosphate transporter) member 4 minus2117 minus2219151258 SLC38A11 Solute carrier family 38 member 11 2410 308555089 SLC38A4 Solute carrier family 38 member 4 1837 254892745 SLC38A5 Solute carrier family 38 member 5 minus1994 minus215291252 SLC39A13 Solute carrier family 39 (zinc transporter) member 13 1301 207023516 SLC39A14 Solute carrier family 39 (zinc transporter) member 14 2569 318829985 SLC39A3 Solute carrier family 39 (zinc transporter) member 3 minus2032 minus2152283375 SLC39A5 Solute carrier family 39 (metal ion transporter) member 5 1623 23707922 SLC39A7 Solute carrier family 39 (zinc transporter) member 7 1273 205830061 SLC40A1 Solute carrier family 40 (iron-regulated transporter) member 1 minus3612 minus379684102 SLC41A2 Solute carrier family 41 member 2 1293 20708501 SLC43A1 Solute carrier family 43 member 1 minus2013 minus215257153 SLC44A2 Solute carrier family 44 member 2 minus2047 minus215250651 SLC45A1 Solute carrier family 45 member 1 2038 2722146802 SLC47A2 Solute carrier family 47 member 2 minus1962 minus20266521 SLC4A1 Solute carrier family 4 anion exchanger member 1 minus6513 minus645357282 SLC4A10 Solute carrier family 4 sodium bicarbonate transporter member 10 minus2640 minus275383959 SLC4A11 Solute carrier family 4 sodium borate transporter member 11 1846 25696508 SLC4A3 Solute carrier family 4 anion exchanger member 3 1261 20458671 SLC4A4 Solute carrier family 4 sodium bicarbonate cotransporter member 4 2445 31139497 SLC4A7 Solute carrier family 4 sodium bicarbonate cotransporter member 7 2717 33076523 SLC5A1 Solute carrier family 5 (sodiumglucose cotransporter) member 1 minus2547 minus2705159963 SLC5A12 Solute carrier family 5 (sodiumglucose cotransporter) member 12 4753 52066527 SLC5A4 Solute carrier family 5 (low affinity glucose cotransporter) member 4 minus3969 minus4249

6540 SLC6A13 Solute carrier family 6 (neurotransmitter transporter GABA)member 13 1328 2092

55117 SLC6A15 Solute carrier family 6 (neutral amino acid transporter) member 15 1623 2333388662 SLC6A17 Solute carrier family 6 member 17 2038 272854716 SLC6A20 Solute carrier family 6 (proline IMINO transporter) member 20 1697 2433

6532 SLC6A4 Solute carrier family 6 (neurotransmitter transporter serotonin)member 4 minus2512 minus2611

6534 SLC6A7 Solute carrier family 6 (neurotransmitter transporter L-proline)member 7 2623 3228

56301 SLC7A10 Solute carrier family 7 (neutral amino acid transporter y+ system)member 10 minus3421 minus3603

Sarcoma 17

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

6547 SLC8A3 Solute carrier family 8 (sodiumcalcium exchanger) member 3 minus2204 minus2309285335 SLC9A10 Solute carrier family 9 member 10 1208 20006549 SLC9A2 Solute carrier family 9 (sodiumhydrogen exchanger) member 2 2038 2706

9368 SLC9A3R1 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 1 minus2760 minus2846

9351 SLC9A3R2 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 2 1889 2619

84679 SLC9A7 Solute carrier family 9 (sodiumhydrogen exchanger) member 7 3347 393510599 SLCO1B1 Solute carrier organic anion transporter family member 1B1 3360 397728234 SLCO1B3 Solute carrier organic anion transporter family member 1B3 9760 95696578 SLCO2A1 Solute carrier organic anion transporter family member 2A1 2432 309628232 SLCO3A1 Solute carrier organic anion transporter family member 3A1 minus2032 minus2152353189 SLCO4C1 Solute carrier organic anion transporter family member 4C1 minus6850 minus6611

(b) Metabolism

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold Cchange

1583 CYP11A1 Cytochrome P450 family 11 subfamily A polypeptide 1 1623 23961589 CYP21A2 Cytochrome P450 family 21 subfamily A polypeptide 2 minus1962 minus20361591 CYP24A1 Cytochrome P450 family 24 subfamily A polypeptide 1 5208 55771592 CYP26A1 Cytochrome P450 family 26 subfamily A polypeptide 1 2623 32571594 CYP27B1 Cytochrome P450 family 27 subfamily B polypeptide 1 1846 2585339761 CYP27C1 Cytochrome P450 family 27 subfamily C polypeptide 1 3585 41781553 CYP2A13 Cytochrome P450 family 2 subfamily A polypeptide 13 minus1962 minus20351580 CYP4B1 Cytochrome P450 family 4 subfamily B polypeptide 1 minus4820 minus506566002 CYP4F12 Cytochrome P450 family 4 subfamily F polypeptide 12 minus6070 minus61958529 CYP4F2 Cytochrome P450 family 4 subfamily F polypeptide 2 minus7769 minus70604051 CYP4F3 Cytochrome P450 family 4 subfamily F polypeptide 3 minus8244 minus749111283 CYP4F8 Cytochrome P450 family 4 subfamily F polypeptide 8 minus5006 minus5270260293 CYP4X1 Cytochrome P450 family 4 subfamily X polypeptide 1 minus2476 minus25809420 CYP7B1 Cytochrome P450 family 7 subfamily B polypeptide 1 2502 31702326 FMO1 Flavin containing monooxygenase 1 4360 48592327 FMO2 Flavin containing monooxygenase 2 (nonfunctional) minus4074 minus43372328 FMO3 Flavin containing monooxygenase 3 minus4036 minus4309388714 FMO6P Flavin containing monooxygenase 6 pseudogene minus2569 minus2737493869 GPX8 Glutathione peroxidase 8 (putative) 2814 33712938 GSTA1 Glutathione S-transferase alpha 1 1623 23632939 GSTA2 Glutathione S-transferase alpha 2 2038 27412941 GSTA4 Glutathione S-transferase alpha 4 1492 21882953 GSTT2 Glutathione S-transferase theta 2 4682 5170653689 GSTT2B Glutathione S-transferase theta 2B (genepseudogene) 3739 430784779 NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439 79969027 NAT8 N-acetyltransferase 8 (GCN5-related putative) minus2769 minus29207358 UGDH UDP-glucose 6-dehydrogenase 2333 301855757 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 1604 227110720 UGT2B11 UDP glucuronosyltransferase 2 family polypeptide B11 minus3586 minus37687367 UGT2B17 UDP glucuronosyltransferase 2 family polypeptide B17 minus2836 minus295954490 UGT2B28 UDP glucuronosyltransferase 2 family polypeptide B28 minus3132 minus3284167127 UGT3A2 UDP glycosyltransferase 3 family polypeptide A2 minus4716 minus4907

18 Sarcoma

Table 4 FAF1 mutations in various cancers FAF1 mutations listed in the TCGA data base were identified without restriction to any type ofcancer For the location of the affected domains on the protein compare Figure 2

AA Mutation Cancer DomainN4S Missense Cutaneous melanomaI10S Missense Stomach adenocarcinoma UBAE21lowast Nonsense Cutaneous melanoma UBAE25K Missense Uterine endometrioid carcinoma UBAV38 splice Splice Stomach adenocarcinoma UBAP86fs FS del Colorectal adenocarcinomaG123 splice Splice Uterine endometrioid carcinoma UB1P136H Missense Colorectal adenocarcinoma UB1T147M Missense Brain lower grade glioma UB1D149Y Missense Uterine endometrioid carcinoma UB1L159V Missense Lung adenocarcinoma UB1K163N Missense Uterine endometrioid carcinoma UB1L170F Missense Cutaneous melanomaG184 splice Splice Colorectal cancerQ187H Missense Stomach adenocarcinomaS214N Missense Colorectal adenocarcinoma UB2R222I Missense Uterine endometrioid carcinoma UB2E238D Missense Lung adenocarcinoma UB2P241S Missense Uterine endometrioid carcinoma UB2T245A Missense Renal clear cell carcinoma UB2M249V Missense Uterine endometrioid carcinoma UB2E280K Missense Brain lower grade gliomaG293lowast Nonsense Colorectal cancerT300I Missense Colorectal adenocarcinomaD305H Missense Lung adenocarcinomaE308Q Missense Lung adenocarcinomaA316V Missense Stomach adenocarcinomaK319fs FS ins Head and neck squamous cell carcinomaR344G Missense Stomach adenocarcinoma UASF353I Missense Cutaneous melanoma UASL379V Missense Breast invasive carcinoma UASC396F Missense Cutaneous melanoma UASS459lowast Nonsense Uterine endometrioid carcinoma UASG469 splice Splice Glioblastoma multiforme UASR509G Missense Lung squamous cell carcinomaE510fs FS del Cutaneous melanomaR516C Missense Uterine endometrioid carcinomaA534V Missense Stomach adenocarcinomaF537L Missense Stomach adenocarcinomaE551lowast Nonsense Lung adenocarcinomaR554W Missense Colorectal cancerS582I Missense Lung adenocarcinoma UBXF585L Missense Uterine endometrioid carcinoma UBXE587lowast Nonsense Lung adenocarcinoma UBXA592V Missense Stomach adenocarcinoma UBXW610lowast Nonsense Breast invasive carcinoma UBXD611Y Missense Lung adenocarcinoma UBXE635fs FS del Brain lower grade glioma UBXP640fs FS del Cutaneous melanoma UBX

Sarcoma 19

a more accurate reference point for a leiomyosarcoma wouldhave been smooth muscle we believe that the comparison tostriated muscle is sufficient to allow the assessment of tumorspecific changes We have measured RNA DNA and proteinwith various assays Similar assessments in the future shouldalso include chromosome analysis for possible translocations

In the first-line defense against cancer the gradualreplacement of conventional chemotherapy with molecularlytargeted agents has opened the possibility to tailor drug treat-ments to particular tumors This transition necessitates thecharacterization of the molecular lesions that are causativefor the transformation of healthy cells into cancerous cellsbecause drugs need to be matched with the underlying carci-nogenic defect to be effective An additional caveat causedby unique genetic changes in the primary tumor can affectdrug transport and metabolism and needs to be taken intoconsideration In this case the RNA levels for genes that regu-late transport andmetabolismwere extensively skewed in thetumor tissue as compared to muscle and bone (see Table 3)implying potential challenges to chemotherapy An advancedmolecular treatment strategy for cancer will rely on themolecular definition of drug target drug transport and drugmetabolism pharmacogenetics in the primary tumor Con-secutive to cancer dissemination it will also require adjust-ments to account for the genetic changes in the metastases

The cost of health care has been under increasing scrutinyThe treatment of cancer patients is expensive in particularwhen hospitalization is required and further in cases ofend-of-life care Avoidable expenditures are generated bysuboptimal treatment decisions that result in low efficacy orhigh toxicity of anticancer regimens Molecular medicine hasthe potential to preempt those problems and reduce wastefulspending Yet it requires the upfront cost of molecular cancerexamination The analysis performed in this study requiredabout $11000- in nonsalary expenses to perform While ithas not identified a drug target it has specified possibleconfines for drug treatment The costs for molecular analysisneed to be weighed against the societal cost derived fromlost productivity in the workforce disrupted lives of familiesand premature deaths While currently limited drug optionsconstitute the major constraint to the approach taken herethe foreseeable future will bring an increasing spectrum ofmolecularly targeted drugs along with faster and cheapertechnologies for the molecular assessment of cancers Theywill facilitate the clinical translation of our approach

Consent

The patient provided informed consent

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by DOD Grant PR094070under University of Cincinnati IRB protocol 04-01-29-01The

author is grateful to Dr Rhett Kovall for helping with thestructural analysis of FAF1

References

[1] G F Weber Molecular Mechanisms of Cancer Springer Dor-drecht The Netherlands 2007

[2] N G Sanerkin ldquoPrimary leiomyosarcoma of the bone and itscomparison with fibrosarcomardquoCancer vol 44 no 4 pp 1375ndash1387 1979

[3] J M Weaver and J A Abraham ldquoLeiomyosarcoma of the Boneand Soft Tissue A Reviewrdquo httpsarcomahelporglearningcenterleiomyosarcomahtml

[4] M A Depristo E Banks R Poplin et al ldquoA framework forvariation discovery and genotyping using next-generationDNAsequencing datardquo Nature Genetics vol 43 no 5 pp 491ndash5012011

[5] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[6] C W Menges D A Altomare and J R Testa ldquoFAS-associatedfactor 1 (FAF1) diverse functions and implications for oncoge-nesisrdquo Cell Cycle vol 8 no 16 pp 2528ndash2534 2009

[7] M Kim J H Lee S Y Lee E Kim and J Chung ldquoCaspar asuppressor of antibacterial immunity in Drosophilardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 44 pp 16358ndash16363 2006

[8] J Song K P Joon J-J Lee et al ldquoStructure and interaction ofubiquitin-associated domain of human Fas-associated factor 1rdquoProtein Science vol 18 no 11 pp 2265ndash2276 2009

[9] P H OrsquoFarrell ldquoHigh resolution two dimensional electrophore-sis of proteinsrdquo Journal of Biological Chemistry vol 250 no 10pp 4007ndash4021 1975

[10] A Burgess-Cassler J J Johansen D A Santek J R Ideand N C Kendrick ldquoComputerized quantitative analysis ofCoomassie-Blue-stained serum proteins separated by two-dimensional electrophoresisrdquo Clinical Chemistry vol 35 no 12pp 2297ndash2304 1989

[11] B R Oakley D R Kirsch and N RMorris ldquoA simplified ultra-sensitive silver stain for detecting proteins in polyacrylamidegelsrdquo Analytical Biochemistry vol 105 no 2 pp 361ndash363 1980

[12] K Chu X Niu and L T Williams ldquoA Fas-associated proteinfactor FAF1 potentiates Fas-mediated apoptosisrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 25 pp 11894ndash11898 1995

[13] B Rikhof S de Jong A J H Suurmeijer C Meijer and W TA van der Graaf ldquoThe insulin-like growth factor system andsarcomasrdquoThe Journal of Pathology vol 217 no 4 pp 469ndash4822009

[14] RGirling J F Partridge L R Bandara et al ldquoAnew componentof the transcription factor DRTF1E2Frdquo Nature vol 362 no6415 pp 83ndash87 1993

[15] F Mercurio and M Karin ldquoTranscription factors AP-3 andAP-2 interact with the SV40 enhancer in a mutually exclusivemannerrdquo EMBO Journal vol 8 no 5 pp 1455ndash1460 1989

[16] R Bassel-Duby M D Hernandez M A Gonzalez J KKrueger and R S Williams ldquoA 40-kilodalton protein bindsspecifically to an upstream sequence element essential for mus-cle-specific transcription of the human myoglobin promoterrdquoMolecular and Cellular Biology vol 12 no 11 pp 5024ndash50321992

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Computational and Mathematical Methods in Medicine

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 12: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

12 Sarcoma

Muscle

Bone

Tumor

220

94

6043

29

14

220

94

60

43

29

14

220

94

60

43

29

14

1

7

6

23

24

21

22

25

26

89 1011 12

1516 1719

pH 4 8 pH 4 8

8pH 4

(a)

SwissProt or NCBISpot number Protein identified accession Description

(Structural)6 Transgelin Q01995 Smooth muscle-specific cross-links actin24 Transgelin-2 P37802 Smooth muscle-specific cross-links actin

Vimentin P08670 Intermediate filament in mesenchymal tissue1 Filamin-A P21333 Actin-binding protein regulates the cytoskeleton 21 P06709 Cytoskeleton

(Calcium homeostasis)8 9 Calumenin O43852 Calcium-binding protein in the ER and golgi apparatus26 Protein S100-A11 P31949 Calcium-binding EF hand protein10 Reticulocalbin-3 Q96D15 Calcium-binding protein located in the ER12 P11021 Heat shock 70-kD protein 5 ER lumenal calcium-binding

(Protein processing)25 40S ribosomal protein S12 P25398 Core component of the ribosomal accuracy center7 Glycine-tRNA ligase P41250 Catalyzes the attachment of glycine to tRNA19 P01009 Protease inhibitor23 P01009 Protease inhibitor21 Proteasome activator complex subunit 2 Q9UL46 Proteasome activation10 P07900 Chaperone

(Other)7 Serum albumin P02768 Carrier protein in blood22 Protein disulfide isomerase (C-terminal fragment) P07237 Prolyl hydroxylation in collagen microsomal triglyceride transfer

120573 actin (C-terminal fragment)

78kD glucose-regulated protein

120572-1-antitrypsin120572-1-antitrypsin (C-terminal fragment)

Heat shock protein HSP 90120572 (N-terminal fragment)

11 15ndash17

(b)

Figure 4 Continued

Sarcoma 13

Tumor Muscle

OPN

CD44

STAT3

P-STAT3

Tubulin

75

75

50

15010075

10075

50

(c)

Tumor bone Tumor muscleGene ID Symbol Name Fold change Expression Fold change Expression6876 TAGLN Transgelin 3274 2455 3087 15088407 TAGLN2 Transgelin-2 2164 7338 6975 13037431 VIM Vimentin 1653 295010 4061 696042316 FLNA Filamin A alpha 1304 7791 9005 065160 ACTB Actin beta 0656 973187 5491 67368813 CALU Calumenin 7601 19328 12063 70596282 S100A11 S100 calcium binding protein A11 0931 158914 5071 1686357333 RCN3 Reticulocalbin 3 EF-hand calcium binding domain 2439 0604 6412 01223309 HSPA5 1068 92754 2607 220356696 SPP1 Secreted phosphoprotein 1 7572 46508 547929 0355960 CD44 CD44 molecule (Indian blood group) 2053 26708 15436 20566774 STAT3 Signal transducer and activator of transcription 3 0617 46534 0832 200165925 RB1 Retinoblastoma 1 0736 14772 0442 14268

Heat shock 70kDa protein 5 (glucose-regulated protein 78kDa)

(d)

Figure 4 Protein overexpression (a) 2D protein gel electrophoresis of lysates from muscle (upper left) tumor (upper right) and bone(bottom) in RIPA buffer Red circles indicate the spots that were identified as overexpressed and were further analyzed by mass spectrometry(b) Proteins identified in 2D gel electrophoresis as overexpressed in the tumor in comparison to muscle and bone were analyzed for theiridentity by mass spectrometry The left column indicates the spot number corresponding to the 2D gel The next column contains theprotein name followed by the accession number and a description of the protein functionThe protein functions are grouped into structuralcalcium homeostasis and various others (c) Western blot 10 120583g lysates of tumor muscle and bone in RIPA buffer were loaded per laneand electrophoresed on 10 SDS-polyacrylamide minigels with reducing denaturing sample buffer After transfer to PVDF membranesthey were probed for markers of cancer progression including osteopontin CD44 STAT3 and phospho-STAT3 Antitubulin served as aloading control (d) RNA levels corresponding to the proteins found to be affected in the sarcoma With four exceptions in the tumor-bonecomparison (gray font) upregulated proteins are associated with increased RNA levels STAT3 is not increased on the protein or RNA levelThe reduced level of RB1 expression is consistent with the elevated DNA binding activity of E2F1

to disrupt the structure of the protein this site does scorehigh as a possible phosphorylation site for a number ofkinases involved in DNA damage repair supporting thehypothesis that the cancer cells containing thismutation havelost their ability to respond to transforming DNA damagewith programmed cell death FAF1 antagonizes WNT signal-ing by promoting 120573-catenin degradation in the proteasome[21] a function that may be lost after the point mutationThe elevated DNA binding activity of the protooncogenictranscription factor E2F1 (see Figure 3) could be caused byits interaction with LEF-1 [20] consecutive to persistent

WNT signaling The RNA level of IGF2BP1 (IMP-1) a stress-responsive regulator of mRNA stability is highly upregulatedin the tumor compared to muscle as well as bone (seeTable 2(c)) IGF2BP1 is a transcriptional target of the WNTpathway that regulates NF-120581B activity (see Table 2(e)) andis antiapoptotic [22 23] IGF2BP1 may be upregulated asa consequence of mutated FAF1 not being able to suppressWNT signaling in this specific cancerOf noteWNTpathwayoveractivity may not be required for transformation ratherthe persistence of a WNT pathway signal due to the lack of aFAF1-mediated termination signal may suffice

14 Sarcoma

WNTFrizzled

Axin Dvl

igf2bp1

FAF1

P65 P50

IKK

FAS

120573-Catenin

LEF + E2F1

I120581B

Figure 5 Possible transforming pathway The point mutation inFAF1 is believed to cause a loss of function (crossed out in red)This may lead to an overactivity of the WNT signaling pathwayConsistently E2F1 (activated by LEF1) and igf2bp1 (a transcriptionaltarget of the WNT pathway) have been identified to be upregulatedin the molecular analysis In contrast to the negative regulator FAF1IGF2BP1 is a positive regulator of NF-120581B activityThe overactivity ofthe NF-120581B pathway and the reduced efficacy of FAS signaling can betransforming

The role ofWNT signaling in sarcoma has been subject todebate (eg [24]) In metastatic leiomyosarcoma 120573-Cateninmay accumulate in the nucleus despite a relatively weakexpression of WNT [25] This may be due to WNT signalactivation via noncanonical ligands [26] or to 120573-Cateninbinding the nuclear receptor NR4A2 and releasing it from thecorepressor protein LEF-1 [27] Of note in the cancer understudy here the mRNA level of NR4A2 was overexpressed10-fold compared to bone and 3-fold compared to muscleThe results from this study are consistent with the possibilitythat a lack of termination in the WNT signal rather than itsoveractivation could contribute to sarcomatous transforma-tion Such a mechanism may be reflected in an upregulationof downstream targets even though overexpression of WNTpathway components is not detectable

Other mutations beside FAF1 are less likely to becausative for the cancer EPHA3 was revealed as mutated inthis cancer by DNA exome sequencing and RNASeq EPHA3is a receptor tyrosine kinase that is frequentlymutated in lungcancer Tumor-suppressive effects of wild-type EPHA3 can beoverridden by dominant negative EPHA3 somatic mutations[28]Thismechanism is unlikely to play a role in this sarcomaas the detected mutation is located far N-terminally on theextracellular Ephrin binding domain not on the intracellularkinase domain

FAF1 has been described to act as a tumor suppressor gene[29] Its depletion due to chromosome breakage can affectprognosis in glioblastoma patients [30 31] Single nucleotidepolymorphisms in FAF1 are associated with a risk for gastriccancer [32] While numerous FAF1 mutations are associatedwith various cancers none of these genetic changes in theTCGA database affects the amino acid position 181 (Table 4)

The major upregulations identified in this tumor com-prise muscle-specific gene products (transcription factors

CAAC binding proteomics transgelin transgelin-2 RNAanoctamin-4 and immunohistochemistry smooth mus-cle actin) and calcium-regulating molecules (proteomicscalumenin S100-A11 reticulocalbin-3 and 78 kD glucose-regulated protein) The muscle-specific gene products con-firm this recurrent sarcoma as a leiomyosarcoma (the firsttumor distal to the site of the recurring one had beendiagnosed as an osteosarcoma) Calcium is one of the majorsecond messengers in smooth muscle cells Its uptake isregulated by potential-sensitive ion channels in the cellmembrane and by the activities of various receptors Calciumis stored in the sarcoplasmic reticulum from where it canbe released to facilitate actin-myosin interaction and tensiongeneration Phosphorylation of the myosin light chain by acalmodulin-regulated enzyme is important for contractionThe upregulation of gene products associated with migrationand invasion (osteopontin MMP1 vimentin filamin-A and120573-actin) and gene products for extracellularmatrixmoleculesand their modulators (fibronectin collagen ITGBL1 andMXRA5) reflects the invasive nature of this cancer

The recurrence of a sarcoma after 14 years has twoprobable explanations either it is due to a cancer pre-disposition syndrome based on a germ-line mutation (theage of the patient weakens this hypothesis) or the secondtumor is a metastatic colony of the first that was reactivatedafter dormancy The location of the sarcoma in the sameextremity and proximal to a preceding mesenchymal cancer(and therefore in its natural path of dissemination) impliedthe probability that this was a relapse in a metastatic siteThe different histologic assessment as osteosarcoma in thefirst occurrence and leiomyosarcoma as the second cancerdoes not necessarily negate that Mixed histology [33 34]and transdifferentiation [35ndash37] have been described forsarcomatous tumors Of note however in this scenarioosteosarcoma seems to more commonly follow leiomyosar-coma than precede it Material from the first cancer of thispatient was not accessible to us It is very plausible that thiscould have been a mineralized leiomyosarcoma In thosetumors the differential diagnosis from osteosarcoma can bedifficult [38] The extracompartmental location of the firsttumor supports this interpretation

On the molecular pathology level sarcomas fall intotwo groups comprising tumors with simple karyotypes(with pathogenetic translocations or specific genetic muta-tions) and tumors with very complex karyotypes (overtchromosome and genomic instability with numerous gainsand losses) [39] Some molecular alterations that lead tocarcinogenesis can be defined in absolute termsThey includegain-of-function mutations or chromosome translocationsthat transformprotooncogenes to oncogenes However otherchanges are relative to the normal tissue of origin suchas pathway overactivity or overexpression on the proteinor RNA levels We have combined the analysis of absolutechanges (DNA exome sequence RNA sequence) with theanalysis of relative changes using skeletal muscle and bone asreference organs (protein-DNA array 2D gel electrophoresisand RNA expression levels) This choice was determined inpart by tissue availability after surgery andwas intended to aidin the distinction of osteosarcoma from myosarcoma While

Sarcoma 15

Table 3 Deregulation of genes for drug disposition Analysis of RNASeq for at least twofold overexpression (italic font) or underexpression(bold font) of genes for (a) transport and (b) metabolism in tumor compared to muscle and bone For the export transporters (ABCtransporters) only overexpression is considered relevant for drug resistance Not shown in the table are the underexpressed genes (ABCA7ABCA8 ABCA13 ABCB6 ABCB10 ABCC6 ABCC6P1 ABCC6P2 ABCC8 ABCC11 ABCD2 ABCG2 and ABCG5)

(a) Transport

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

650655 ABCA17P ATP-binding cassette subfamily A (ABC1) member 17 pseudogene 1360 211624 ABCA4 ATP-binding cassette subfamily A (ABC1) member 4 2038 2802

6555 SLC10A2 Solute carrier family 10 (sodiumbile acid cotransporter family)member 2 minus1962 minus2112

345274 SLC10A6 Solute carrier family 10 (sodiumbile acid cotransporter family)member 6 2623 3240

6563 SLC14A1 Solute carrier family 14 (urea transporter) member 1 (Kidd bloodgroup) minus3074 minus3152

6565 SLC15A2 Solute carrier family 15 (H+peptide transporter) member 2 minus2027 minus2152

6566 SLC16A1 Solute carrier family 16 member 1 (monocarboxylic acid transporter1) 1401 2170

117247 SLC16A10 Solute carrier family 16 member 10 (aromatic amino acid transporter) 2113 2848

6567 SLC16A2 Solute carrier family 16 member 2 (monocarboxylic acid transporter8) 3242 3848

10786 SLC17A3 Solute carrier family 17 (sodium phosphate) member 3 minus2868 minus29596571 SLC18A2 Solute carrier family 18 (vesicular monoamine) member 2 minus2087 minus21946573 SLC19A1 Solute carrier family 19 (folate transporter) member 1 minus2099 minus220310560 SLC19A2 Solute carrier family 19 (thiamine transporter) member 2 1820 254880704 SLC19A3 Solute carrier family 19 member 3 minus3331 minus3478387775 SLC22A10 Solute carrier family 22 member 10 5711 60859390 SLC22A13 Solute carrier family 22 (organic anion transporter) member 13 2623 3217

85413 SLC22A16 Solute carrier family 22 (organic cationcarnitine transporter)member 16 minus8101 minus7299

51310 SLC22A17 Solute carrier family 22 member 17 1475 21755002 SLC22A18 Solute carrier family 22 member 18 2038 27226582 SLC22A2 Solute carrier family 22 (organic cation transporter) member 2 4793 5216

6581 SLC22A3 Solute carrier family 22 (extraneuronal monoamine transporter)member 3 2554 3182

6583 SLC22A4 Solute carrier family 22 (organic cationergothioneine transporter)member 4 minus3603 minus3776

151295 SLC23A3 Solute carrier family 23 (nucleobase transporters) member 3 2109 284810478 SLC25A17 Solute carrier family 25 (mitochondrial carrier) member 17 1311 207783733 SLC25A18 Solute carrier family 25 (mitochondrial carrier) member 18 1846 2570

788 SLC25A20 Solute carrier family 25 (carnitineacylcarnitine translocase) member20 minus2105 minus2207

89874 SLC25A21 Solute carrier family 25 (mitochondrial oxodicarboxylate carrier)member 21 minus2962 minus3105

51312 SLC25A37 Solute carrier family 25 member 37 minus4633 minus486651629 SLC25A39 Solute carrier family 25 member 39 minus2585 minus2737203427 SLC25A43 Solute carrier family 25 member 43 1325 208865012 SLC26A10 Solute carrier family 26 member 10 3896 4472115111 SLC26A7 Solute carrier family 26 member 7 3431 4018116369 SLC26A8 Solute carrier family 26 member 8 minus5354 minus5536115019 SLC26A9 Solute carrier family 26 member 9 1623 241911001 SLC27A2 Solute carrier family 27 (fatty acid transporter) member 2 minus4278 minus4487

16 Sarcoma

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

64078 SLC28A3 Solute carrier family 28 (sodium-coupled nucleoside transporter)member 3 minus4543 minus4812

222962 SLC29A4 Solute carrier family 29 (nucleoside transporters) member 4 minus1962 minus204581031 SLC2A10 Solute carrier family 2 (facilitated glucose transporter) member 10 2532 3170

6518 SLC2A5 Solute carrier family 2 (facilitated glucosefructose transporter)member 5 minus3647 minus3821

55532 SLC30A10 Solute carrier family 30 member 10 minus3547 minus37257782 SLC30A4 Solute carrier family 30 (zinc transporter) member 4 2832 33726569 SLC34A1 Solute carrier family 34 (sodium phosphate) member 1 minus4716 minus4941340146 SLC35D3 Solute carrier family 35 member D3 minus5662 minus590654733 SLC35F2 Solute carrier family 35 member F2 1433 2170206358 SLC36A1 Solute carrier family 36 (protonamino acid symporter) member 1 minus2265 minus2345285641 SLC36A3 Solute carrier family 36 (protonamino acid symporter) member 3 minus2284 minus239354020 SLC37A1 Solute carrier family 37 (glycerol-3-phosphate transporter) member 1 minus2112 minus22122542 SLC37A4 Solute carrier family 37 (glucose-6-phosphate transporter) member 4 minus2117 minus2219151258 SLC38A11 Solute carrier family 38 member 11 2410 308555089 SLC38A4 Solute carrier family 38 member 4 1837 254892745 SLC38A5 Solute carrier family 38 member 5 minus1994 minus215291252 SLC39A13 Solute carrier family 39 (zinc transporter) member 13 1301 207023516 SLC39A14 Solute carrier family 39 (zinc transporter) member 14 2569 318829985 SLC39A3 Solute carrier family 39 (zinc transporter) member 3 minus2032 minus2152283375 SLC39A5 Solute carrier family 39 (metal ion transporter) member 5 1623 23707922 SLC39A7 Solute carrier family 39 (zinc transporter) member 7 1273 205830061 SLC40A1 Solute carrier family 40 (iron-regulated transporter) member 1 minus3612 minus379684102 SLC41A2 Solute carrier family 41 member 2 1293 20708501 SLC43A1 Solute carrier family 43 member 1 minus2013 minus215257153 SLC44A2 Solute carrier family 44 member 2 minus2047 minus215250651 SLC45A1 Solute carrier family 45 member 1 2038 2722146802 SLC47A2 Solute carrier family 47 member 2 minus1962 minus20266521 SLC4A1 Solute carrier family 4 anion exchanger member 1 minus6513 minus645357282 SLC4A10 Solute carrier family 4 sodium bicarbonate transporter member 10 minus2640 minus275383959 SLC4A11 Solute carrier family 4 sodium borate transporter member 11 1846 25696508 SLC4A3 Solute carrier family 4 anion exchanger member 3 1261 20458671 SLC4A4 Solute carrier family 4 sodium bicarbonate cotransporter member 4 2445 31139497 SLC4A7 Solute carrier family 4 sodium bicarbonate cotransporter member 7 2717 33076523 SLC5A1 Solute carrier family 5 (sodiumglucose cotransporter) member 1 minus2547 minus2705159963 SLC5A12 Solute carrier family 5 (sodiumglucose cotransporter) member 12 4753 52066527 SLC5A4 Solute carrier family 5 (low affinity glucose cotransporter) member 4 minus3969 minus4249

6540 SLC6A13 Solute carrier family 6 (neurotransmitter transporter GABA)member 13 1328 2092

55117 SLC6A15 Solute carrier family 6 (neutral amino acid transporter) member 15 1623 2333388662 SLC6A17 Solute carrier family 6 member 17 2038 272854716 SLC6A20 Solute carrier family 6 (proline IMINO transporter) member 20 1697 2433

6532 SLC6A4 Solute carrier family 6 (neurotransmitter transporter serotonin)member 4 minus2512 minus2611

6534 SLC6A7 Solute carrier family 6 (neurotransmitter transporter L-proline)member 7 2623 3228

56301 SLC7A10 Solute carrier family 7 (neutral amino acid transporter y+ system)member 10 minus3421 minus3603

Sarcoma 17

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

6547 SLC8A3 Solute carrier family 8 (sodiumcalcium exchanger) member 3 minus2204 minus2309285335 SLC9A10 Solute carrier family 9 member 10 1208 20006549 SLC9A2 Solute carrier family 9 (sodiumhydrogen exchanger) member 2 2038 2706

9368 SLC9A3R1 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 1 minus2760 minus2846

9351 SLC9A3R2 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 2 1889 2619

84679 SLC9A7 Solute carrier family 9 (sodiumhydrogen exchanger) member 7 3347 393510599 SLCO1B1 Solute carrier organic anion transporter family member 1B1 3360 397728234 SLCO1B3 Solute carrier organic anion transporter family member 1B3 9760 95696578 SLCO2A1 Solute carrier organic anion transporter family member 2A1 2432 309628232 SLCO3A1 Solute carrier organic anion transporter family member 3A1 minus2032 minus2152353189 SLCO4C1 Solute carrier organic anion transporter family member 4C1 minus6850 minus6611

(b) Metabolism

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold Cchange

1583 CYP11A1 Cytochrome P450 family 11 subfamily A polypeptide 1 1623 23961589 CYP21A2 Cytochrome P450 family 21 subfamily A polypeptide 2 minus1962 minus20361591 CYP24A1 Cytochrome P450 family 24 subfamily A polypeptide 1 5208 55771592 CYP26A1 Cytochrome P450 family 26 subfamily A polypeptide 1 2623 32571594 CYP27B1 Cytochrome P450 family 27 subfamily B polypeptide 1 1846 2585339761 CYP27C1 Cytochrome P450 family 27 subfamily C polypeptide 1 3585 41781553 CYP2A13 Cytochrome P450 family 2 subfamily A polypeptide 13 minus1962 minus20351580 CYP4B1 Cytochrome P450 family 4 subfamily B polypeptide 1 minus4820 minus506566002 CYP4F12 Cytochrome P450 family 4 subfamily F polypeptide 12 minus6070 minus61958529 CYP4F2 Cytochrome P450 family 4 subfamily F polypeptide 2 minus7769 minus70604051 CYP4F3 Cytochrome P450 family 4 subfamily F polypeptide 3 minus8244 minus749111283 CYP4F8 Cytochrome P450 family 4 subfamily F polypeptide 8 minus5006 minus5270260293 CYP4X1 Cytochrome P450 family 4 subfamily X polypeptide 1 minus2476 minus25809420 CYP7B1 Cytochrome P450 family 7 subfamily B polypeptide 1 2502 31702326 FMO1 Flavin containing monooxygenase 1 4360 48592327 FMO2 Flavin containing monooxygenase 2 (nonfunctional) minus4074 minus43372328 FMO3 Flavin containing monooxygenase 3 minus4036 minus4309388714 FMO6P Flavin containing monooxygenase 6 pseudogene minus2569 minus2737493869 GPX8 Glutathione peroxidase 8 (putative) 2814 33712938 GSTA1 Glutathione S-transferase alpha 1 1623 23632939 GSTA2 Glutathione S-transferase alpha 2 2038 27412941 GSTA4 Glutathione S-transferase alpha 4 1492 21882953 GSTT2 Glutathione S-transferase theta 2 4682 5170653689 GSTT2B Glutathione S-transferase theta 2B (genepseudogene) 3739 430784779 NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439 79969027 NAT8 N-acetyltransferase 8 (GCN5-related putative) minus2769 minus29207358 UGDH UDP-glucose 6-dehydrogenase 2333 301855757 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 1604 227110720 UGT2B11 UDP glucuronosyltransferase 2 family polypeptide B11 minus3586 minus37687367 UGT2B17 UDP glucuronosyltransferase 2 family polypeptide B17 minus2836 minus295954490 UGT2B28 UDP glucuronosyltransferase 2 family polypeptide B28 minus3132 minus3284167127 UGT3A2 UDP glycosyltransferase 3 family polypeptide A2 minus4716 minus4907

18 Sarcoma

Table 4 FAF1 mutations in various cancers FAF1 mutations listed in the TCGA data base were identified without restriction to any type ofcancer For the location of the affected domains on the protein compare Figure 2

AA Mutation Cancer DomainN4S Missense Cutaneous melanomaI10S Missense Stomach adenocarcinoma UBAE21lowast Nonsense Cutaneous melanoma UBAE25K Missense Uterine endometrioid carcinoma UBAV38 splice Splice Stomach adenocarcinoma UBAP86fs FS del Colorectal adenocarcinomaG123 splice Splice Uterine endometrioid carcinoma UB1P136H Missense Colorectal adenocarcinoma UB1T147M Missense Brain lower grade glioma UB1D149Y Missense Uterine endometrioid carcinoma UB1L159V Missense Lung adenocarcinoma UB1K163N Missense Uterine endometrioid carcinoma UB1L170F Missense Cutaneous melanomaG184 splice Splice Colorectal cancerQ187H Missense Stomach adenocarcinomaS214N Missense Colorectal adenocarcinoma UB2R222I Missense Uterine endometrioid carcinoma UB2E238D Missense Lung adenocarcinoma UB2P241S Missense Uterine endometrioid carcinoma UB2T245A Missense Renal clear cell carcinoma UB2M249V Missense Uterine endometrioid carcinoma UB2E280K Missense Brain lower grade gliomaG293lowast Nonsense Colorectal cancerT300I Missense Colorectal adenocarcinomaD305H Missense Lung adenocarcinomaE308Q Missense Lung adenocarcinomaA316V Missense Stomach adenocarcinomaK319fs FS ins Head and neck squamous cell carcinomaR344G Missense Stomach adenocarcinoma UASF353I Missense Cutaneous melanoma UASL379V Missense Breast invasive carcinoma UASC396F Missense Cutaneous melanoma UASS459lowast Nonsense Uterine endometrioid carcinoma UASG469 splice Splice Glioblastoma multiforme UASR509G Missense Lung squamous cell carcinomaE510fs FS del Cutaneous melanomaR516C Missense Uterine endometrioid carcinomaA534V Missense Stomach adenocarcinomaF537L Missense Stomach adenocarcinomaE551lowast Nonsense Lung adenocarcinomaR554W Missense Colorectal cancerS582I Missense Lung adenocarcinoma UBXF585L Missense Uterine endometrioid carcinoma UBXE587lowast Nonsense Lung adenocarcinoma UBXA592V Missense Stomach adenocarcinoma UBXW610lowast Nonsense Breast invasive carcinoma UBXD611Y Missense Lung adenocarcinoma UBXE635fs FS del Brain lower grade glioma UBXP640fs FS del Cutaneous melanoma UBX

Sarcoma 19

a more accurate reference point for a leiomyosarcoma wouldhave been smooth muscle we believe that the comparison tostriated muscle is sufficient to allow the assessment of tumorspecific changes We have measured RNA DNA and proteinwith various assays Similar assessments in the future shouldalso include chromosome analysis for possible translocations

In the first-line defense against cancer the gradualreplacement of conventional chemotherapy with molecularlytargeted agents has opened the possibility to tailor drug treat-ments to particular tumors This transition necessitates thecharacterization of the molecular lesions that are causativefor the transformation of healthy cells into cancerous cellsbecause drugs need to be matched with the underlying carci-nogenic defect to be effective An additional caveat causedby unique genetic changes in the primary tumor can affectdrug transport and metabolism and needs to be taken intoconsideration In this case the RNA levels for genes that regu-late transport andmetabolismwere extensively skewed in thetumor tissue as compared to muscle and bone (see Table 3)implying potential challenges to chemotherapy An advancedmolecular treatment strategy for cancer will rely on themolecular definition of drug target drug transport and drugmetabolism pharmacogenetics in the primary tumor Con-secutive to cancer dissemination it will also require adjust-ments to account for the genetic changes in the metastases

The cost of health care has been under increasing scrutinyThe treatment of cancer patients is expensive in particularwhen hospitalization is required and further in cases ofend-of-life care Avoidable expenditures are generated bysuboptimal treatment decisions that result in low efficacy orhigh toxicity of anticancer regimens Molecular medicine hasthe potential to preempt those problems and reduce wastefulspending Yet it requires the upfront cost of molecular cancerexamination The analysis performed in this study requiredabout $11000- in nonsalary expenses to perform While ithas not identified a drug target it has specified possibleconfines for drug treatment The costs for molecular analysisneed to be weighed against the societal cost derived fromlost productivity in the workforce disrupted lives of familiesand premature deaths While currently limited drug optionsconstitute the major constraint to the approach taken herethe foreseeable future will bring an increasing spectrum ofmolecularly targeted drugs along with faster and cheapertechnologies for the molecular assessment of cancers Theywill facilitate the clinical translation of our approach

Consent

The patient provided informed consent

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by DOD Grant PR094070under University of Cincinnati IRB protocol 04-01-29-01The

author is grateful to Dr Rhett Kovall for helping with thestructural analysis of FAF1

References

[1] G F Weber Molecular Mechanisms of Cancer Springer Dor-drecht The Netherlands 2007

[2] N G Sanerkin ldquoPrimary leiomyosarcoma of the bone and itscomparison with fibrosarcomardquoCancer vol 44 no 4 pp 1375ndash1387 1979

[3] J M Weaver and J A Abraham ldquoLeiomyosarcoma of the Boneand Soft Tissue A Reviewrdquo httpsarcomahelporglearningcenterleiomyosarcomahtml

[4] M A Depristo E Banks R Poplin et al ldquoA framework forvariation discovery and genotyping using next-generationDNAsequencing datardquo Nature Genetics vol 43 no 5 pp 491ndash5012011

[5] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[6] C W Menges D A Altomare and J R Testa ldquoFAS-associatedfactor 1 (FAF1) diverse functions and implications for oncoge-nesisrdquo Cell Cycle vol 8 no 16 pp 2528ndash2534 2009

[7] M Kim J H Lee S Y Lee E Kim and J Chung ldquoCaspar asuppressor of antibacterial immunity in Drosophilardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 44 pp 16358ndash16363 2006

[8] J Song K P Joon J-J Lee et al ldquoStructure and interaction ofubiquitin-associated domain of human Fas-associated factor 1rdquoProtein Science vol 18 no 11 pp 2265ndash2276 2009

[9] P H OrsquoFarrell ldquoHigh resolution two dimensional electrophore-sis of proteinsrdquo Journal of Biological Chemistry vol 250 no 10pp 4007ndash4021 1975

[10] A Burgess-Cassler J J Johansen D A Santek J R Ideand N C Kendrick ldquoComputerized quantitative analysis ofCoomassie-Blue-stained serum proteins separated by two-dimensional electrophoresisrdquo Clinical Chemistry vol 35 no 12pp 2297ndash2304 1989

[11] B R Oakley D R Kirsch and N RMorris ldquoA simplified ultra-sensitive silver stain for detecting proteins in polyacrylamidegelsrdquo Analytical Biochemistry vol 105 no 2 pp 361ndash363 1980

[12] K Chu X Niu and L T Williams ldquoA Fas-associated proteinfactor FAF1 potentiates Fas-mediated apoptosisrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 25 pp 11894ndash11898 1995

[13] B Rikhof S de Jong A J H Suurmeijer C Meijer and W TA van der Graaf ldquoThe insulin-like growth factor system andsarcomasrdquoThe Journal of Pathology vol 217 no 4 pp 469ndash4822009

[14] RGirling J F Partridge L R Bandara et al ldquoAnew componentof the transcription factor DRTF1E2Frdquo Nature vol 362 no6415 pp 83ndash87 1993

[15] F Mercurio and M Karin ldquoTranscription factors AP-3 andAP-2 interact with the SV40 enhancer in a mutually exclusivemannerrdquo EMBO Journal vol 8 no 5 pp 1455ndash1460 1989

[16] R Bassel-Duby M D Hernandez M A Gonzalez J KKrueger and R S Williams ldquoA 40-kilodalton protein bindsspecifically to an upstream sequence element essential for mus-cle-specific transcription of the human myoglobin promoterrdquoMolecular and Cellular Biology vol 12 no 11 pp 5024ndash50321992

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Computational and Mathematical Methods in Medicine

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 13: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

Sarcoma 13

Tumor Muscle

OPN

CD44

STAT3

P-STAT3

Tubulin

75

75

50

15010075

10075

50

(c)

Tumor bone Tumor muscleGene ID Symbol Name Fold change Expression Fold change Expression6876 TAGLN Transgelin 3274 2455 3087 15088407 TAGLN2 Transgelin-2 2164 7338 6975 13037431 VIM Vimentin 1653 295010 4061 696042316 FLNA Filamin A alpha 1304 7791 9005 065160 ACTB Actin beta 0656 973187 5491 67368813 CALU Calumenin 7601 19328 12063 70596282 S100A11 S100 calcium binding protein A11 0931 158914 5071 1686357333 RCN3 Reticulocalbin 3 EF-hand calcium binding domain 2439 0604 6412 01223309 HSPA5 1068 92754 2607 220356696 SPP1 Secreted phosphoprotein 1 7572 46508 547929 0355960 CD44 CD44 molecule (Indian blood group) 2053 26708 15436 20566774 STAT3 Signal transducer and activator of transcription 3 0617 46534 0832 200165925 RB1 Retinoblastoma 1 0736 14772 0442 14268

Heat shock 70kDa protein 5 (glucose-regulated protein 78kDa)

(d)

Figure 4 Protein overexpression (a) 2D protein gel electrophoresis of lysates from muscle (upper left) tumor (upper right) and bone(bottom) in RIPA buffer Red circles indicate the spots that were identified as overexpressed and were further analyzed by mass spectrometry(b) Proteins identified in 2D gel electrophoresis as overexpressed in the tumor in comparison to muscle and bone were analyzed for theiridentity by mass spectrometry The left column indicates the spot number corresponding to the 2D gel The next column contains theprotein name followed by the accession number and a description of the protein functionThe protein functions are grouped into structuralcalcium homeostasis and various others (c) Western blot 10 120583g lysates of tumor muscle and bone in RIPA buffer were loaded per laneand electrophoresed on 10 SDS-polyacrylamide minigels with reducing denaturing sample buffer After transfer to PVDF membranesthey were probed for markers of cancer progression including osteopontin CD44 STAT3 and phospho-STAT3 Antitubulin served as aloading control (d) RNA levels corresponding to the proteins found to be affected in the sarcoma With four exceptions in the tumor-bonecomparison (gray font) upregulated proteins are associated with increased RNA levels STAT3 is not increased on the protein or RNA levelThe reduced level of RB1 expression is consistent with the elevated DNA binding activity of E2F1

to disrupt the structure of the protein this site does scorehigh as a possible phosphorylation site for a number ofkinases involved in DNA damage repair supporting thehypothesis that the cancer cells containing thismutation havelost their ability to respond to transforming DNA damagewith programmed cell death FAF1 antagonizes WNT signal-ing by promoting 120573-catenin degradation in the proteasome[21] a function that may be lost after the point mutationThe elevated DNA binding activity of the protooncogenictranscription factor E2F1 (see Figure 3) could be caused byits interaction with LEF-1 [20] consecutive to persistent

WNT signaling The RNA level of IGF2BP1 (IMP-1) a stress-responsive regulator of mRNA stability is highly upregulatedin the tumor compared to muscle as well as bone (seeTable 2(c)) IGF2BP1 is a transcriptional target of the WNTpathway that regulates NF-120581B activity (see Table 2(e)) andis antiapoptotic [22 23] IGF2BP1 may be upregulated asa consequence of mutated FAF1 not being able to suppressWNT signaling in this specific cancerOf noteWNTpathwayoveractivity may not be required for transformation ratherthe persistence of a WNT pathway signal due to the lack of aFAF1-mediated termination signal may suffice

14 Sarcoma

WNTFrizzled

Axin Dvl

igf2bp1

FAF1

P65 P50

IKK

FAS

120573-Catenin

LEF + E2F1

I120581B

Figure 5 Possible transforming pathway The point mutation inFAF1 is believed to cause a loss of function (crossed out in red)This may lead to an overactivity of the WNT signaling pathwayConsistently E2F1 (activated by LEF1) and igf2bp1 (a transcriptionaltarget of the WNT pathway) have been identified to be upregulatedin the molecular analysis In contrast to the negative regulator FAF1IGF2BP1 is a positive regulator of NF-120581B activityThe overactivity ofthe NF-120581B pathway and the reduced efficacy of FAS signaling can betransforming

The role ofWNT signaling in sarcoma has been subject todebate (eg [24]) In metastatic leiomyosarcoma 120573-Cateninmay accumulate in the nucleus despite a relatively weakexpression of WNT [25] This may be due to WNT signalactivation via noncanonical ligands [26] or to 120573-Cateninbinding the nuclear receptor NR4A2 and releasing it from thecorepressor protein LEF-1 [27] Of note in the cancer understudy here the mRNA level of NR4A2 was overexpressed10-fold compared to bone and 3-fold compared to muscleThe results from this study are consistent with the possibilitythat a lack of termination in the WNT signal rather than itsoveractivation could contribute to sarcomatous transforma-tion Such a mechanism may be reflected in an upregulationof downstream targets even though overexpression of WNTpathway components is not detectable

Other mutations beside FAF1 are less likely to becausative for the cancer EPHA3 was revealed as mutated inthis cancer by DNA exome sequencing and RNASeq EPHA3is a receptor tyrosine kinase that is frequentlymutated in lungcancer Tumor-suppressive effects of wild-type EPHA3 can beoverridden by dominant negative EPHA3 somatic mutations[28]Thismechanism is unlikely to play a role in this sarcomaas the detected mutation is located far N-terminally on theextracellular Ephrin binding domain not on the intracellularkinase domain

FAF1 has been described to act as a tumor suppressor gene[29] Its depletion due to chromosome breakage can affectprognosis in glioblastoma patients [30 31] Single nucleotidepolymorphisms in FAF1 are associated with a risk for gastriccancer [32] While numerous FAF1 mutations are associatedwith various cancers none of these genetic changes in theTCGA database affects the amino acid position 181 (Table 4)

The major upregulations identified in this tumor com-prise muscle-specific gene products (transcription factors

CAAC binding proteomics transgelin transgelin-2 RNAanoctamin-4 and immunohistochemistry smooth mus-cle actin) and calcium-regulating molecules (proteomicscalumenin S100-A11 reticulocalbin-3 and 78 kD glucose-regulated protein) The muscle-specific gene products con-firm this recurrent sarcoma as a leiomyosarcoma (the firsttumor distal to the site of the recurring one had beendiagnosed as an osteosarcoma) Calcium is one of the majorsecond messengers in smooth muscle cells Its uptake isregulated by potential-sensitive ion channels in the cellmembrane and by the activities of various receptors Calciumis stored in the sarcoplasmic reticulum from where it canbe released to facilitate actin-myosin interaction and tensiongeneration Phosphorylation of the myosin light chain by acalmodulin-regulated enzyme is important for contractionThe upregulation of gene products associated with migrationand invasion (osteopontin MMP1 vimentin filamin-A and120573-actin) and gene products for extracellularmatrixmoleculesand their modulators (fibronectin collagen ITGBL1 andMXRA5) reflects the invasive nature of this cancer

The recurrence of a sarcoma after 14 years has twoprobable explanations either it is due to a cancer pre-disposition syndrome based on a germ-line mutation (theage of the patient weakens this hypothesis) or the secondtumor is a metastatic colony of the first that was reactivatedafter dormancy The location of the sarcoma in the sameextremity and proximal to a preceding mesenchymal cancer(and therefore in its natural path of dissemination) impliedthe probability that this was a relapse in a metastatic siteThe different histologic assessment as osteosarcoma in thefirst occurrence and leiomyosarcoma as the second cancerdoes not necessarily negate that Mixed histology [33 34]and transdifferentiation [35ndash37] have been described forsarcomatous tumors Of note however in this scenarioosteosarcoma seems to more commonly follow leiomyosar-coma than precede it Material from the first cancer of thispatient was not accessible to us It is very plausible that thiscould have been a mineralized leiomyosarcoma In thosetumors the differential diagnosis from osteosarcoma can bedifficult [38] The extracompartmental location of the firsttumor supports this interpretation

On the molecular pathology level sarcomas fall intotwo groups comprising tumors with simple karyotypes(with pathogenetic translocations or specific genetic muta-tions) and tumors with very complex karyotypes (overtchromosome and genomic instability with numerous gainsand losses) [39] Some molecular alterations that lead tocarcinogenesis can be defined in absolute termsThey includegain-of-function mutations or chromosome translocationsthat transformprotooncogenes to oncogenes However otherchanges are relative to the normal tissue of origin suchas pathway overactivity or overexpression on the proteinor RNA levels We have combined the analysis of absolutechanges (DNA exome sequence RNA sequence) with theanalysis of relative changes using skeletal muscle and bone asreference organs (protein-DNA array 2D gel electrophoresisand RNA expression levels) This choice was determined inpart by tissue availability after surgery andwas intended to aidin the distinction of osteosarcoma from myosarcoma While

Sarcoma 15

Table 3 Deregulation of genes for drug disposition Analysis of RNASeq for at least twofold overexpression (italic font) or underexpression(bold font) of genes for (a) transport and (b) metabolism in tumor compared to muscle and bone For the export transporters (ABCtransporters) only overexpression is considered relevant for drug resistance Not shown in the table are the underexpressed genes (ABCA7ABCA8 ABCA13 ABCB6 ABCB10 ABCC6 ABCC6P1 ABCC6P2 ABCC8 ABCC11 ABCD2 ABCG2 and ABCG5)

(a) Transport

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

650655 ABCA17P ATP-binding cassette subfamily A (ABC1) member 17 pseudogene 1360 211624 ABCA4 ATP-binding cassette subfamily A (ABC1) member 4 2038 2802

6555 SLC10A2 Solute carrier family 10 (sodiumbile acid cotransporter family)member 2 minus1962 minus2112

345274 SLC10A6 Solute carrier family 10 (sodiumbile acid cotransporter family)member 6 2623 3240

6563 SLC14A1 Solute carrier family 14 (urea transporter) member 1 (Kidd bloodgroup) minus3074 minus3152

6565 SLC15A2 Solute carrier family 15 (H+peptide transporter) member 2 minus2027 minus2152

6566 SLC16A1 Solute carrier family 16 member 1 (monocarboxylic acid transporter1) 1401 2170

117247 SLC16A10 Solute carrier family 16 member 10 (aromatic amino acid transporter) 2113 2848

6567 SLC16A2 Solute carrier family 16 member 2 (monocarboxylic acid transporter8) 3242 3848

10786 SLC17A3 Solute carrier family 17 (sodium phosphate) member 3 minus2868 minus29596571 SLC18A2 Solute carrier family 18 (vesicular monoamine) member 2 minus2087 minus21946573 SLC19A1 Solute carrier family 19 (folate transporter) member 1 minus2099 minus220310560 SLC19A2 Solute carrier family 19 (thiamine transporter) member 2 1820 254880704 SLC19A3 Solute carrier family 19 member 3 minus3331 minus3478387775 SLC22A10 Solute carrier family 22 member 10 5711 60859390 SLC22A13 Solute carrier family 22 (organic anion transporter) member 13 2623 3217

85413 SLC22A16 Solute carrier family 22 (organic cationcarnitine transporter)member 16 minus8101 minus7299

51310 SLC22A17 Solute carrier family 22 member 17 1475 21755002 SLC22A18 Solute carrier family 22 member 18 2038 27226582 SLC22A2 Solute carrier family 22 (organic cation transporter) member 2 4793 5216

6581 SLC22A3 Solute carrier family 22 (extraneuronal monoamine transporter)member 3 2554 3182

6583 SLC22A4 Solute carrier family 22 (organic cationergothioneine transporter)member 4 minus3603 minus3776

151295 SLC23A3 Solute carrier family 23 (nucleobase transporters) member 3 2109 284810478 SLC25A17 Solute carrier family 25 (mitochondrial carrier) member 17 1311 207783733 SLC25A18 Solute carrier family 25 (mitochondrial carrier) member 18 1846 2570

788 SLC25A20 Solute carrier family 25 (carnitineacylcarnitine translocase) member20 minus2105 minus2207

89874 SLC25A21 Solute carrier family 25 (mitochondrial oxodicarboxylate carrier)member 21 minus2962 minus3105

51312 SLC25A37 Solute carrier family 25 member 37 minus4633 minus486651629 SLC25A39 Solute carrier family 25 member 39 minus2585 minus2737203427 SLC25A43 Solute carrier family 25 member 43 1325 208865012 SLC26A10 Solute carrier family 26 member 10 3896 4472115111 SLC26A7 Solute carrier family 26 member 7 3431 4018116369 SLC26A8 Solute carrier family 26 member 8 minus5354 minus5536115019 SLC26A9 Solute carrier family 26 member 9 1623 241911001 SLC27A2 Solute carrier family 27 (fatty acid transporter) member 2 minus4278 minus4487

16 Sarcoma

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

64078 SLC28A3 Solute carrier family 28 (sodium-coupled nucleoside transporter)member 3 minus4543 minus4812

222962 SLC29A4 Solute carrier family 29 (nucleoside transporters) member 4 minus1962 minus204581031 SLC2A10 Solute carrier family 2 (facilitated glucose transporter) member 10 2532 3170

6518 SLC2A5 Solute carrier family 2 (facilitated glucosefructose transporter)member 5 minus3647 minus3821

55532 SLC30A10 Solute carrier family 30 member 10 minus3547 minus37257782 SLC30A4 Solute carrier family 30 (zinc transporter) member 4 2832 33726569 SLC34A1 Solute carrier family 34 (sodium phosphate) member 1 minus4716 minus4941340146 SLC35D3 Solute carrier family 35 member D3 minus5662 minus590654733 SLC35F2 Solute carrier family 35 member F2 1433 2170206358 SLC36A1 Solute carrier family 36 (protonamino acid symporter) member 1 minus2265 minus2345285641 SLC36A3 Solute carrier family 36 (protonamino acid symporter) member 3 minus2284 minus239354020 SLC37A1 Solute carrier family 37 (glycerol-3-phosphate transporter) member 1 minus2112 minus22122542 SLC37A4 Solute carrier family 37 (glucose-6-phosphate transporter) member 4 minus2117 minus2219151258 SLC38A11 Solute carrier family 38 member 11 2410 308555089 SLC38A4 Solute carrier family 38 member 4 1837 254892745 SLC38A5 Solute carrier family 38 member 5 minus1994 minus215291252 SLC39A13 Solute carrier family 39 (zinc transporter) member 13 1301 207023516 SLC39A14 Solute carrier family 39 (zinc transporter) member 14 2569 318829985 SLC39A3 Solute carrier family 39 (zinc transporter) member 3 minus2032 minus2152283375 SLC39A5 Solute carrier family 39 (metal ion transporter) member 5 1623 23707922 SLC39A7 Solute carrier family 39 (zinc transporter) member 7 1273 205830061 SLC40A1 Solute carrier family 40 (iron-regulated transporter) member 1 minus3612 minus379684102 SLC41A2 Solute carrier family 41 member 2 1293 20708501 SLC43A1 Solute carrier family 43 member 1 minus2013 minus215257153 SLC44A2 Solute carrier family 44 member 2 minus2047 minus215250651 SLC45A1 Solute carrier family 45 member 1 2038 2722146802 SLC47A2 Solute carrier family 47 member 2 minus1962 minus20266521 SLC4A1 Solute carrier family 4 anion exchanger member 1 minus6513 minus645357282 SLC4A10 Solute carrier family 4 sodium bicarbonate transporter member 10 minus2640 minus275383959 SLC4A11 Solute carrier family 4 sodium borate transporter member 11 1846 25696508 SLC4A3 Solute carrier family 4 anion exchanger member 3 1261 20458671 SLC4A4 Solute carrier family 4 sodium bicarbonate cotransporter member 4 2445 31139497 SLC4A7 Solute carrier family 4 sodium bicarbonate cotransporter member 7 2717 33076523 SLC5A1 Solute carrier family 5 (sodiumglucose cotransporter) member 1 minus2547 minus2705159963 SLC5A12 Solute carrier family 5 (sodiumglucose cotransporter) member 12 4753 52066527 SLC5A4 Solute carrier family 5 (low affinity glucose cotransporter) member 4 minus3969 minus4249

6540 SLC6A13 Solute carrier family 6 (neurotransmitter transporter GABA)member 13 1328 2092

55117 SLC6A15 Solute carrier family 6 (neutral amino acid transporter) member 15 1623 2333388662 SLC6A17 Solute carrier family 6 member 17 2038 272854716 SLC6A20 Solute carrier family 6 (proline IMINO transporter) member 20 1697 2433

6532 SLC6A4 Solute carrier family 6 (neurotransmitter transporter serotonin)member 4 minus2512 minus2611

6534 SLC6A7 Solute carrier family 6 (neurotransmitter transporter L-proline)member 7 2623 3228

56301 SLC7A10 Solute carrier family 7 (neutral amino acid transporter y+ system)member 10 minus3421 minus3603

Sarcoma 17

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

6547 SLC8A3 Solute carrier family 8 (sodiumcalcium exchanger) member 3 minus2204 minus2309285335 SLC9A10 Solute carrier family 9 member 10 1208 20006549 SLC9A2 Solute carrier family 9 (sodiumhydrogen exchanger) member 2 2038 2706

9368 SLC9A3R1 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 1 minus2760 minus2846

9351 SLC9A3R2 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 2 1889 2619

84679 SLC9A7 Solute carrier family 9 (sodiumhydrogen exchanger) member 7 3347 393510599 SLCO1B1 Solute carrier organic anion transporter family member 1B1 3360 397728234 SLCO1B3 Solute carrier organic anion transporter family member 1B3 9760 95696578 SLCO2A1 Solute carrier organic anion transporter family member 2A1 2432 309628232 SLCO3A1 Solute carrier organic anion transporter family member 3A1 minus2032 minus2152353189 SLCO4C1 Solute carrier organic anion transporter family member 4C1 minus6850 minus6611

(b) Metabolism

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold Cchange

1583 CYP11A1 Cytochrome P450 family 11 subfamily A polypeptide 1 1623 23961589 CYP21A2 Cytochrome P450 family 21 subfamily A polypeptide 2 minus1962 minus20361591 CYP24A1 Cytochrome P450 family 24 subfamily A polypeptide 1 5208 55771592 CYP26A1 Cytochrome P450 family 26 subfamily A polypeptide 1 2623 32571594 CYP27B1 Cytochrome P450 family 27 subfamily B polypeptide 1 1846 2585339761 CYP27C1 Cytochrome P450 family 27 subfamily C polypeptide 1 3585 41781553 CYP2A13 Cytochrome P450 family 2 subfamily A polypeptide 13 minus1962 minus20351580 CYP4B1 Cytochrome P450 family 4 subfamily B polypeptide 1 minus4820 minus506566002 CYP4F12 Cytochrome P450 family 4 subfamily F polypeptide 12 minus6070 minus61958529 CYP4F2 Cytochrome P450 family 4 subfamily F polypeptide 2 minus7769 minus70604051 CYP4F3 Cytochrome P450 family 4 subfamily F polypeptide 3 minus8244 minus749111283 CYP4F8 Cytochrome P450 family 4 subfamily F polypeptide 8 minus5006 minus5270260293 CYP4X1 Cytochrome P450 family 4 subfamily X polypeptide 1 minus2476 minus25809420 CYP7B1 Cytochrome P450 family 7 subfamily B polypeptide 1 2502 31702326 FMO1 Flavin containing monooxygenase 1 4360 48592327 FMO2 Flavin containing monooxygenase 2 (nonfunctional) minus4074 minus43372328 FMO3 Flavin containing monooxygenase 3 minus4036 minus4309388714 FMO6P Flavin containing monooxygenase 6 pseudogene minus2569 minus2737493869 GPX8 Glutathione peroxidase 8 (putative) 2814 33712938 GSTA1 Glutathione S-transferase alpha 1 1623 23632939 GSTA2 Glutathione S-transferase alpha 2 2038 27412941 GSTA4 Glutathione S-transferase alpha 4 1492 21882953 GSTT2 Glutathione S-transferase theta 2 4682 5170653689 GSTT2B Glutathione S-transferase theta 2B (genepseudogene) 3739 430784779 NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439 79969027 NAT8 N-acetyltransferase 8 (GCN5-related putative) minus2769 minus29207358 UGDH UDP-glucose 6-dehydrogenase 2333 301855757 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 1604 227110720 UGT2B11 UDP glucuronosyltransferase 2 family polypeptide B11 minus3586 minus37687367 UGT2B17 UDP glucuronosyltransferase 2 family polypeptide B17 minus2836 minus295954490 UGT2B28 UDP glucuronosyltransferase 2 family polypeptide B28 minus3132 minus3284167127 UGT3A2 UDP glycosyltransferase 3 family polypeptide A2 minus4716 minus4907

18 Sarcoma

Table 4 FAF1 mutations in various cancers FAF1 mutations listed in the TCGA data base were identified without restriction to any type ofcancer For the location of the affected domains on the protein compare Figure 2

AA Mutation Cancer DomainN4S Missense Cutaneous melanomaI10S Missense Stomach adenocarcinoma UBAE21lowast Nonsense Cutaneous melanoma UBAE25K Missense Uterine endometrioid carcinoma UBAV38 splice Splice Stomach adenocarcinoma UBAP86fs FS del Colorectal adenocarcinomaG123 splice Splice Uterine endometrioid carcinoma UB1P136H Missense Colorectal adenocarcinoma UB1T147M Missense Brain lower grade glioma UB1D149Y Missense Uterine endometrioid carcinoma UB1L159V Missense Lung adenocarcinoma UB1K163N Missense Uterine endometrioid carcinoma UB1L170F Missense Cutaneous melanomaG184 splice Splice Colorectal cancerQ187H Missense Stomach adenocarcinomaS214N Missense Colorectal adenocarcinoma UB2R222I Missense Uterine endometrioid carcinoma UB2E238D Missense Lung adenocarcinoma UB2P241S Missense Uterine endometrioid carcinoma UB2T245A Missense Renal clear cell carcinoma UB2M249V Missense Uterine endometrioid carcinoma UB2E280K Missense Brain lower grade gliomaG293lowast Nonsense Colorectal cancerT300I Missense Colorectal adenocarcinomaD305H Missense Lung adenocarcinomaE308Q Missense Lung adenocarcinomaA316V Missense Stomach adenocarcinomaK319fs FS ins Head and neck squamous cell carcinomaR344G Missense Stomach adenocarcinoma UASF353I Missense Cutaneous melanoma UASL379V Missense Breast invasive carcinoma UASC396F Missense Cutaneous melanoma UASS459lowast Nonsense Uterine endometrioid carcinoma UASG469 splice Splice Glioblastoma multiforme UASR509G Missense Lung squamous cell carcinomaE510fs FS del Cutaneous melanomaR516C Missense Uterine endometrioid carcinomaA534V Missense Stomach adenocarcinomaF537L Missense Stomach adenocarcinomaE551lowast Nonsense Lung adenocarcinomaR554W Missense Colorectal cancerS582I Missense Lung adenocarcinoma UBXF585L Missense Uterine endometrioid carcinoma UBXE587lowast Nonsense Lung adenocarcinoma UBXA592V Missense Stomach adenocarcinoma UBXW610lowast Nonsense Breast invasive carcinoma UBXD611Y Missense Lung adenocarcinoma UBXE635fs FS del Brain lower grade glioma UBXP640fs FS del Cutaneous melanoma UBX

Sarcoma 19

a more accurate reference point for a leiomyosarcoma wouldhave been smooth muscle we believe that the comparison tostriated muscle is sufficient to allow the assessment of tumorspecific changes We have measured RNA DNA and proteinwith various assays Similar assessments in the future shouldalso include chromosome analysis for possible translocations

In the first-line defense against cancer the gradualreplacement of conventional chemotherapy with molecularlytargeted agents has opened the possibility to tailor drug treat-ments to particular tumors This transition necessitates thecharacterization of the molecular lesions that are causativefor the transformation of healthy cells into cancerous cellsbecause drugs need to be matched with the underlying carci-nogenic defect to be effective An additional caveat causedby unique genetic changes in the primary tumor can affectdrug transport and metabolism and needs to be taken intoconsideration In this case the RNA levels for genes that regu-late transport andmetabolismwere extensively skewed in thetumor tissue as compared to muscle and bone (see Table 3)implying potential challenges to chemotherapy An advancedmolecular treatment strategy for cancer will rely on themolecular definition of drug target drug transport and drugmetabolism pharmacogenetics in the primary tumor Con-secutive to cancer dissemination it will also require adjust-ments to account for the genetic changes in the metastases

The cost of health care has been under increasing scrutinyThe treatment of cancer patients is expensive in particularwhen hospitalization is required and further in cases ofend-of-life care Avoidable expenditures are generated bysuboptimal treatment decisions that result in low efficacy orhigh toxicity of anticancer regimens Molecular medicine hasthe potential to preempt those problems and reduce wastefulspending Yet it requires the upfront cost of molecular cancerexamination The analysis performed in this study requiredabout $11000- in nonsalary expenses to perform While ithas not identified a drug target it has specified possibleconfines for drug treatment The costs for molecular analysisneed to be weighed against the societal cost derived fromlost productivity in the workforce disrupted lives of familiesand premature deaths While currently limited drug optionsconstitute the major constraint to the approach taken herethe foreseeable future will bring an increasing spectrum ofmolecularly targeted drugs along with faster and cheapertechnologies for the molecular assessment of cancers Theywill facilitate the clinical translation of our approach

Consent

The patient provided informed consent

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by DOD Grant PR094070under University of Cincinnati IRB protocol 04-01-29-01The

author is grateful to Dr Rhett Kovall for helping with thestructural analysis of FAF1

References

[1] G F Weber Molecular Mechanisms of Cancer Springer Dor-drecht The Netherlands 2007

[2] N G Sanerkin ldquoPrimary leiomyosarcoma of the bone and itscomparison with fibrosarcomardquoCancer vol 44 no 4 pp 1375ndash1387 1979

[3] J M Weaver and J A Abraham ldquoLeiomyosarcoma of the Boneand Soft Tissue A Reviewrdquo httpsarcomahelporglearningcenterleiomyosarcomahtml

[4] M A Depristo E Banks R Poplin et al ldquoA framework forvariation discovery and genotyping using next-generationDNAsequencing datardquo Nature Genetics vol 43 no 5 pp 491ndash5012011

[5] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[6] C W Menges D A Altomare and J R Testa ldquoFAS-associatedfactor 1 (FAF1) diverse functions and implications for oncoge-nesisrdquo Cell Cycle vol 8 no 16 pp 2528ndash2534 2009

[7] M Kim J H Lee S Y Lee E Kim and J Chung ldquoCaspar asuppressor of antibacterial immunity in Drosophilardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 44 pp 16358ndash16363 2006

[8] J Song K P Joon J-J Lee et al ldquoStructure and interaction ofubiquitin-associated domain of human Fas-associated factor 1rdquoProtein Science vol 18 no 11 pp 2265ndash2276 2009

[9] P H OrsquoFarrell ldquoHigh resolution two dimensional electrophore-sis of proteinsrdquo Journal of Biological Chemistry vol 250 no 10pp 4007ndash4021 1975

[10] A Burgess-Cassler J J Johansen D A Santek J R Ideand N C Kendrick ldquoComputerized quantitative analysis ofCoomassie-Blue-stained serum proteins separated by two-dimensional electrophoresisrdquo Clinical Chemistry vol 35 no 12pp 2297ndash2304 1989

[11] B R Oakley D R Kirsch and N RMorris ldquoA simplified ultra-sensitive silver stain for detecting proteins in polyacrylamidegelsrdquo Analytical Biochemistry vol 105 no 2 pp 361ndash363 1980

[12] K Chu X Niu and L T Williams ldquoA Fas-associated proteinfactor FAF1 potentiates Fas-mediated apoptosisrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 25 pp 11894ndash11898 1995

[13] B Rikhof S de Jong A J H Suurmeijer C Meijer and W TA van der Graaf ldquoThe insulin-like growth factor system andsarcomasrdquoThe Journal of Pathology vol 217 no 4 pp 469ndash4822009

[14] RGirling J F Partridge L R Bandara et al ldquoAnew componentof the transcription factor DRTF1E2Frdquo Nature vol 362 no6415 pp 83ndash87 1993

[15] F Mercurio and M Karin ldquoTranscription factors AP-3 andAP-2 interact with the SV40 enhancer in a mutually exclusivemannerrdquo EMBO Journal vol 8 no 5 pp 1455ndash1460 1989

[16] R Bassel-Duby M D Hernandez M A Gonzalez J KKrueger and R S Williams ldquoA 40-kilodalton protein bindsspecifically to an upstream sequence element essential for mus-cle-specific transcription of the human myoglobin promoterrdquoMolecular and Cellular Biology vol 12 no 11 pp 5024ndash50321992

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 14: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

14 Sarcoma

WNTFrizzled

Axin Dvl

igf2bp1

FAF1

P65 P50

IKK

FAS

120573-Catenin

LEF + E2F1

I120581B

Figure 5 Possible transforming pathway The point mutation inFAF1 is believed to cause a loss of function (crossed out in red)This may lead to an overactivity of the WNT signaling pathwayConsistently E2F1 (activated by LEF1) and igf2bp1 (a transcriptionaltarget of the WNT pathway) have been identified to be upregulatedin the molecular analysis In contrast to the negative regulator FAF1IGF2BP1 is a positive regulator of NF-120581B activityThe overactivity ofthe NF-120581B pathway and the reduced efficacy of FAS signaling can betransforming

The role ofWNT signaling in sarcoma has been subject todebate (eg [24]) In metastatic leiomyosarcoma 120573-Cateninmay accumulate in the nucleus despite a relatively weakexpression of WNT [25] This may be due to WNT signalactivation via noncanonical ligands [26] or to 120573-Cateninbinding the nuclear receptor NR4A2 and releasing it from thecorepressor protein LEF-1 [27] Of note in the cancer understudy here the mRNA level of NR4A2 was overexpressed10-fold compared to bone and 3-fold compared to muscleThe results from this study are consistent with the possibilitythat a lack of termination in the WNT signal rather than itsoveractivation could contribute to sarcomatous transforma-tion Such a mechanism may be reflected in an upregulationof downstream targets even though overexpression of WNTpathway components is not detectable

Other mutations beside FAF1 are less likely to becausative for the cancer EPHA3 was revealed as mutated inthis cancer by DNA exome sequencing and RNASeq EPHA3is a receptor tyrosine kinase that is frequentlymutated in lungcancer Tumor-suppressive effects of wild-type EPHA3 can beoverridden by dominant negative EPHA3 somatic mutations[28]Thismechanism is unlikely to play a role in this sarcomaas the detected mutation is located far N-terminally on theextracellular Ephrin binding domain not on the intracellularkinase domain

FAF1 has been described to act as a tumor suppressor gene[29] Its depletion due to chromosome breakage can affectprognosis in glioblastoma patients [30 31] Single nucleotidepolymorphisms in FAF1 are associated with a risk for gastriccancer [32] While numerous FAF1 mutations are associatedwith various cancers none of these genetic changes in theTCGA database affects the amino acid position 181 (Table 4)

The major upregulations identified in this tumor com-prise muscle-specific gene products (transcription factors

CAAC binding proteomics transgelin transgelin-2 RNAanoctamin-4 and immunohistochemistry smooth mus-cle actin) and calcium-regulating molecules (proteomicscalumenin S100-A11 reticulocalbin-3 and 78 kD glucose-regulated protein) The muscle-specific gene products con-firm this recurrent sarcoma as a leiomyosarcoma (the firsttumor distal to the site of the recurring one had beendiagnosed as an osteosarcoma) Calcium is one of the majorsecond messengers in smooth muscle cells Its uptake isregulated by potential-sensitive ion channels in the cellmembrane and by the activities of various receptors Calciumis stored in the sarcoplasmic reticulum from where it canbe released to facilitate actin-myosin interaction and tensiongeneration Phosphorylation of the myosin light chain by acalmodulin-regulated enzyme is important for contractionThe upregulation of gene products associated with migrationand invasion (osteopontin MMP1 vimentin filamin-A and120573-actin) and gene products for extracellularmatrixmoleculesand their modulators (fibronectin collagen ITGBL1 andMXRA5) reflects the invasive nature of this cancer

The recurrence of a sarcoma after 14 years has twoprobable explanations either it is due to a cancer pre-disposition syndrome based on a germ-line mutation (theage of the patient weakens this hypothesis) or the secondtumor is a metastatic colony of the first that was reactivatedafter dormancy The location of the sarcoma in the sameextremity and proximal to a preceding mesenchymal cancer(and therefore in its natural path of dissemination) impliedthe probability that this was a relapse in a metastatic siteThe different histologic assessment as osteosarcoma in thefirst occurrence and leiomyosarcoma as the second cancerdoes not necessarily negate that Mixed histology [33 34]and transdifferentiation [35ndash37] have been described forsarcomatous tumors Of note however in this scenarioosteosarcoma seems to more commonly follow leiomyosar-coma than precede it Material from the first cancer of thispatient was not accessible to us It is very plausible that thiscould have been a mineralized leiomyosarcoma In thosetumors the differential diagnosis from osteosarcoma can bedifficult [38] The extracompartmental location of the firsttumor supports this interpretation

On the molecular pathology level sarcomas fall intotwo groups comprising tumors with simple karyotypes(with pathogenetic translocations or specific genetic muta-tions) and tumors with very complex karyotypes (overtchromosome and genomic instability with numerous gainsand losses) [39] Some molecular alterations that lead tocarcinogenesis can be defined in absolute termsThey includegain-of-function mutations or chromosome translocationsthat transformprotooncogenes to oncogenes However otherchanges are relative to the normal tissue of origin suchas pathway overactivity or overexpression on the proteinor RNA levels We have combined the analysis of absolutechanges (DNA exome sequence RNA sequence) with theanalysis of relative changes using skeletal muscle and bone asreference organs (protein-DNA array 2D gel electrophoresisand RNA expression levels) This choice was determined inpart by tissue availability after surgery andwas intended to aidin the distinction of osteosarcoma from myosarcoma While

Sarcoma 15

Table 3 Deregulation of genes for drug disposition Analysis of RNASeq for at least twofold overexpression (italic font) or underexpression(bold font) of genes for (a) transport and (b) metabolism in tumor compared to muscle and bone For the export transporters (ABCtransporters) only overexpression is considered relevant for drug resistance Not shown in the table are the underexpressed genes (ABCA7ABCA8 ABCA13 ABCB6 ABCB10 ABCC6 ABCC6P1 ABCC6P2 ABCC8 ABCC11 ABCD2 ABCG2 and ABCG5)

(a) Transport

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

650655 ABCA17P ATP-binding cassette subfamily A (ABC1) member 17 pseudogene 1360 211624 ABCA4 ATP-binding cassette subfamily A (ABC1) member 4 2038 2802

6555 SLC10A2 Solute carrier family 10 (sodiumbile acid cotransporter family)member 2 minus1962 minus2112

345274 SLC10A6 Solute carrier family 10 (sodiumbile acid cotransporter family)member 6 2623 3240

6563 SLC14A1 Solute carrier family 14 (urea transporter) member 1 (Kidd bloodgroup) minus3074 minus3152

6565 SLC15A2 Solute carrier family 15 (H+peptide transporter) member 2 minus2027 minus2152

6566 SLC16A1 Solute carrier family 16 member 1 (monocarboxylic acid transporter1) 1401 2170

117247 SLC16A10 Solute carrier family 16 member 10 (aromatic amino acid transporter) 2113 2848

6567 SLC16A2 Solute carrier family 16 member 2 (monocarboxylic acid transporter8) 3242 3848

10786 SLC17A3 Solute carrier family 17 (sodium phosphate) member 3 minus2868 minus29596571 SLC18A2 Solute carrier family 18 (vesicular monoamine) member 2 minus2087 minus21946573 SLC19A1 Solute carrier family 19 (folate transporter) member 1 minus2099 minus220310560 SLC19A2 Solute carrier family 19 (thiamine transporter) member 2 1820 254880704 SLC19A3 Solute carrier family 19 member 3 minus3331 minus3478387775 SLC22A10 Solute carrier family 22 member 10 5711 60859390 SLC22A13 Solute carrier family 22 (organic anion transporter) member 13 2623 3217

85413 SLC22A16 Solute carrier family 22 (organic cationcarnitine transporter)member 16 minus8101 minus7299

51310 SLC22A17 Solute carrier family 22 member 17 1475 21755002 SLC22A18 Solute carrier family 22 member 18 2038 27226582 SLC22A2 Solute carrier family 22 (organic cation transporter) member 2 4793 5216

6581 SLC22A3 Solute carrier family 22 (extraneuronal monoamine transporter)member 3 2554 3182

6583 SLC22A4 Solute carrier family 22 (organic cationergothioneine transporter)member 4 minus3603 minus3776

151295 SLC23A3 Solute carrier family 23 (nucleobase transporters) member 3 2109 284810478 SLC25A17 Solute carrier family 25 (mitochondrial carrier) member 17 1311 207783733 SLC25A18 Solute carrier family 25 (mitochondrial carrier) member 18 1846 2570

788 SLC25A20 Solute carrier family 25 (carnitineacylcarnitine translocase) member20 minus2105 minus2207

89874 SLC25A21 Solute carrier family 25 (mitochondrial oxodicarboxylate carrier)member 21 minus2962 minus3105

51312 SLC25A37 Solute carrier family 25 member 37 minus4633 minus486651629 SLC25A39 Solute carrier family 25 member 39 minus2585 minus2737203427 SLC25A43 Solute carrier family 25 member 43 1325 208865012 SLC26A10 Solute carrier family 26 member 10 3896 4472115111 SLC26A7 Solute carrier family 26 member 7 3431 4018116369 SLC26A8 Solute carrier family 26 member 8 minus5354 minus5536115019 SLC26A9 Solute carrier family 26 member 9 1623 241911001 SLC27A2 Solute carrier family 27 (fatty acid transporter) member 2 minus4278 minus4487

16 Sarcoma

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

64078 SLC28A3 Solute carrier family 28 (sodium-coupled nucleoside transporter)member 3 minus4543 minus4812

222962 SLC29A4 Solute carrier family 29 (nucleoside transporters) member 4 minus1962 minus204581031 SLC2A10 Solute carrier family 2 (facilitated glucose transporter) member 10 2532 3170

6518 SLC2A5 Solute carrier family 2 (facilitated glucosefructose transporter)member 5 minus3647 minus3821

55532 SLC30A10 Solute carrier family 30 member 10 minus3547 minus37257782 SLC30A4 Solute carrier family 30 (zinc transporter) member 4 2832 33726569 SLC34A1 Solute carrier family 34 (sodium phosphate) member 1 minus4716 minus4941340146 SLC35D3 Solute carrier family 35 member D3 minus5662 minus590654733 SLC35F2 Solute carrier family 35 member F2 1433 2170206358 SLC36A1 Solute carrier family 36 (protonamino acid symporter) member 1 minus2265 minus2345285641 SLC36A3 Solute carrier family 36 (protonamino acid symporter) member 3 minus2284 minus239354020 SLC37A1 Solute carrier family 37 (glycerol-3-phosphate transporter) member 1 minus2112 minus22122542 SLC37A4 Solute carrier family 37 (glucose-6-phosphate transporter) member 4 minus2117 minus2219151258 SLC38A11 Solute carrier family 38 member 11 2410 308555089 SLC38A4 Solute carrier family 38 member 4 1837 254892745 SLC38A5 Solute carrier family 38 member 5 minus1994 minus215291252 SLC39A13 Solute carrier family 39 (zinc transporter) member 13 1301 207023516 SLC39A14 Solute carrier family 39 (zinc transporter) member 14 2569 318829985 SLC39A3 Solute carrier family 39 (zinc transporter) member 3 minus2032 minus2152283375 SLC39A5 Solute carrier family 39 (metal ion transporter) member 5 1623 23707922 SLC39A7 Solute carrier family 39 (zinc transporter) member 7 1273 205830061 SLC40A1 Solute carrier family 40 (iron-regulated transporter) member 1 minus3612 minus379684102 SLC41A2 Solute carrier family 41 member 2 1293 20708501 SLC43A1 Solute carrier family 43 member 1 minus2013 minus215257153 SLC44A2 Solute carrier family 44 member 2 minus2047 minus215250651 SLC45A1 Solute carrier family 45 member 1 2038 2722146802 SLC47A2 Solute carrier family 47 member 2 minus1962 minus20266521 SLC4A1 Solute carrier family 4 anion exchanger member 1 minus6513 minus645357282 SLC4A10 Solute carrier family 4 sodium bicarbonate transporter member 10 minus2640 minus275383959 SLC4A11 Solute carrier family 4 sodium borate transporter member 11 1846 25696508 SLC4A3 Solute carrier family 4 anion exchanger member 3 1261 20458671 SLC4A4 Solute carrier family 4 sodium bicarbonate cotransporter member 4 2445 31139497 SLC4A7 Solute carrier family 4 sodium bicarbonate cotransporter member 7 2717 33076523 SLC5A1 Solute carrier family 5 (sodiumglucose cotransporter) member 1 minus2547 minus2705159963 SLC5A12 Solute carrier family 5 (sodiumglucose cotransporter) member 12 4753 52066527 SLC5A4 Solute carrier family 5 (low affinity glucose cotransporter) member 4 minus3969 minus4249

6540 SLC6A13 Solute carrier family 6 (neurotransmitter transporter GABA)member 13 1328 2092

55117 SLC6A15 Solute carrier family 6 (neutral amino acid transporter) member 15 1623 2333388662 SLC6A17 Solute carrier family 6 member 17 2038 272854716 SLC6A20 Solute carrier family 6 (proline IMINO transporter) member 20 1697 2433

6532 SLC6A4 Solute carrier family 6 (neurotransmitter transporter serotonin)member 4 minus2512 minus2611

6534 SLC6A7 Solute carrier family 6 (neurotransmitter transporter L-proline)member 7 2623 3228

56301 SLC7A10 Solute carrier family 7 (neutral amino acid transporter y+ system)member 10 minus3421 minus3603

Sarcoma 17

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

6547 SLC8A3 Solute carrier family 8 (sodiumcalcium exchanger) member 3 minus2204 minus2309285335 SLC9A10 Solute carrier family 9 member 10 1208 20006549 SLC9A2 Solute carrier family 9 (sodiumhydrogen exchanger) member 2 2038 2706

9368 SLC9A3R1 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 1 minus2760 minus2846

9351 SLC9A3R2 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 2 1889 2619

84679 SLC9A7 Solute carrier family 9 (sodiumhydrogen exchanger) member 7 3347 393510599 SLCO1B1 Solute carrier organic anion transporter family member 1B1 3360 397728234 SLCO1B3 Solute carrier organic anion transporter family member 1B3 9760 95696578 SLCO2A1 Solute carrier organic anion transporter family member 2A1 2432 309628232 SLCO3A1 Solute carrier organic anion transporter family member 3A1 minus2032 minus2152353189 SLCO4C1 Solute carrier organic anion transporter family member 4C1 minus6850 minus6611

(b) Metabolism

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold Cchange

1583 CYP11A1 Cytochrome P450 family 11 subfamily A polypeptide 1 1623 23961589 CYP21A2 Cytochrome P450 family 21 subfamily A polypeptide 2 minus1962 minus20361591 CYP24A1 Cytochrome P450 family 24 subfamily A polypeptide 1 5208 55771592 CYP26A1 Cytochrome P450 family 26 subfamily A polypeptide 1 2623 32571594 CYP27B1 Cytochrome P450 family 27 subfamily B polypeptide 1 1846 2585339761 CYP27C1 Cytochrome P450 family 27 subfamily C polypeptide 1 3585 41781553 CYP2A13 Cytochrome P450 family 2 subfamily A polypeptide 13 minus1962 minus20351580 CYP4B1 Cytochrome P450 family 4 subfamily B polypeptide 1 minus4820 minus506566002 CYP4F12 Cytochrome P450 family 4 subfamily F polypeptide 12 minus6070 minus61958529 CYP4F2 Cytochrome P450 family 4 subfamily F polypeptide 2 minus7769 minus70604051 CYP4F3 Cytochrome P450 family 4 subfamily F polypeptide 3 minus8244 minus749111283 CYP4F8 Cytochrome P450 family 4 subfamily F polypeptide 8 minus5006 minus5270260293 CYP4X1 Cytochrome P450 family 4 subfamily X polypeptide 1 minus2476 minus25809420 CYP7B1 Cytochrome P450 family 7 subfamily B polypeptide 1 2502 31702326 FMO1 Flavin containing monooxygenase 1 4360 48592327 FMO2 Flavin containing monooxygenase 2 (nonfunctional) minus4074 minus43372328 FMO3 Flavin containing monooxygenase 3 minus4036 minus4309388714 FMO6P Flavin containing monooxygenase 6 pseudogene minus2569 minus2737493869 GPX8 Glutathione peroxidase 8 (putative) 2814 33712938 GSTA1 Glutathione S-transferase alpha 1 1623 23632939 GSTA2 Glutathione S-transferase alpha 2 2038 27412941 GSTA4 Glutathione S-transferase alpha 4 1492 21882953 GSTT2 Glutathione S-transferase theta 2 4682 5170653689 GSTT2B Glutathione S-transferase theta 2B (genepseudogene) 3739 430784779 NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439 79969027 NAT8 N-acetyltransferase 8 (GCN5-related putative) minus2769 minus29207358 UGDH UDP-glucose 6-dehydrogenase 2333 301855757 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 1604 227110720 UGT2B11 UDP glucuronosyltransferase 2 family polypeptide B11 minus3586 minus37687367 UGT2B17 UDP glucuronosyltransferase 2 family polypeptide B17 minus2836 minus295954490 UGT2B28 UDP glucuronosyltransferase 2 family polypeptide B28 minus3132 minus3284167127 UGT3A2 UDP glycosyltransferase 3 family polypeptide A2 minus4716 minus4907

18 Sarcoma

Table 4 FAF1 mutations in various cancers FAF1 mutations listed in the TCGA data base were identified without restriction to any type ofcancer For the location of the affected domains on the protein compare Figure 2

AA Mutation Cancer DomainN4S Missense Cutaneous melanomaI10S Missense Stomach adenocarcinoma UBAE21lowast Nonsense Cutaneous melanoma UBAE25K Missense Uterine endometrioid carcinoma UBAV38 splice Splice Stomach adenocarcinoma UBAP86fs FS del Colorectal adenocarcinomaG123 splice Splice Uterine endometrioid carcinoma UB1P136H Missense Colorectal adenocarcinoma UB1T147M Missense Brain lower grade glioma UB1D149Y Missense Uterine endometrioid carcinoma UB1L159V Missense Lung adenocarcinoma UB1K163N Missense Uterine endometrioid carcinoma UB1L170F Missense Cutaneous melanomaG184 splice Splice Colorectal cancerQ187H Missense Stomach adenocarcinomaS214N Missense Colorectal adenocarcinoma UB2R222I Missense Uterine endometrioid carcinoma UB2E238D Missense Lung adenocarcinoma UB2P241S Missense Uterine endometrioid carcinoma UB2T245A Missense Renal clear cell carcinoma UB2M249V Missense Uterine endometrioid carcinoma UB2E280K Missense Brain lower grade gliomaG293lowast Nonsense Colorectal cancerT300I Missense Colorectal adenocarcinomaD305H Missense Lung adenocarcinomaE308Q Missense Lung adenocarcinomaA316V Missense Stomach adenocarcinomaK319fs FS ins Head and neck squamous cell carcinomaR344G Missense Stomach adenocarcinoma UASF353I Missense Cutaneous melanoma UASL379V Missense Breast invasive carcinoma UASC396F Missense Cutaneous melanoma UASS459lowast Nonsense Uterine endometrioid carcinoma UASG469 splice Splice Glioblastoma multiforme UASR509G Missense Lung squamous cell carcinomaE510fs FS del Cutaneous melanomaR516C Missense Uterine endometrioid carcinomaA534V Missense Stomach adenocarcinomaF537L Missense Stomach adenocarcinomaE551lowast Nonsense Lung adenocarcinomaR554W Missense Colorectal cancerS582I Missense Lung adenocarcinoma UBXF585L Missense Uterine endometrioid carcinoma UBXE587lowast Nonsense Lung adenocarcinoma UBXA592V Missense Stomach adenocarcinoma UBXW610lowast Nonsense Breast invasive carcinoma UBXD611Y Missense Lung adenocarcinoma UBXE635fs FS del Brain lower grade glioma UBXP640fs FS del Cutaneous melanoma UBX

Sarcoma 19

a more accurate reference point for a leiomyosarcoma wouldhave been smooth muscle we believe that the comparison tostriated muscle is sufficient to allow the assessment of tumorspecific changes We have measured RNA DNA and proteinwith various assays Similar assessments in the future shouldalso include chromosome analysis for possible translocations

In the first-line defense against cancer the gradualreplacement of conventional chemotherapy with molecularlytargeted agents has opened the possibility to tailor drug treat-ments to particular tumors This transition necessitates thecharacterization of the molecular lesions that are causativefor the transformation of healthy cells into cancerous cellsbecause drugs need to be matched with the underlying carci-nogenic defect to be effective An additional caveat causedby unique genetic changes in the primary tumor can affectdrug transport and metabolism and needs to be taken intoconsideration In this case the RNA levels for genes that regu-late transport andmetabolismwere extensively skewed in thetumor tissue as compared to muscle and bone (see Table 3)implying potential challenges to chemotherapy An advancedmolecular treatment strategy for cancer will rely on themolecular definition of drug target drug transport and drugmetabolism pharmacogenetics in the primary tumor Con-secutive to cancer dissemination it will also require adjust-ments to account for the genetic changes in the metastases

The cost of health care has been under increasing scrutinyThe treatment of cancer patients is expensive in particularwhen hospitalization is required and further in cases ofend-of-life care Avoidable expenditures are generated bysuboptimal treatment decisions that result in low efficacy orhigh toxicity of anticancer regimens Molecular medicine hasthe potential to preempt those problems and reduce wastefulspending Yet it requires the upfront cost of molecular cancerexamination The analysis performed in this study requiredabout $11000- in nonsalary expenses to perform While ithas not identified a drug target it has specified possibleconfines for drug treatment The costs for molecular analysisneed to be weighed against the societal cost derived fromlost productivity in the workforce disrupted lives of familiesand premature deaths While currently limited drug optionsconstitute the major constraint to the approach taken herethe foreseeable future will bring an increasing spectrum ofmolecularly targeted drugs along with faster and cheapertechnologies for the molecular assessment of cancers Theywill facilitate the clinical translation of our approach

Consent

The patient provided informed consent

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by DOD Grant PR094070under University of Cincinnati IRB protocol 04-01-29-01The

author is grateful to Dr Rhett Kovall for helping with thestructural analysis of FAF1

References

[1] G F Weber Molecular Mechanisms of Cancer Springer Dor-drecht The Netherlands 2007

[2] N G Sanerkin ldquoPrimary leiomyosarcoma of the bone and itscomparison with fibrosarcomardquoCancer vol 44 no 4 pp 1375ndash1387 1979

[3] J M Weaver and J A Abraham ldquoLeiomyosarcoma of the Boneand Soft Tissue A Reviewrdquo httpsarcomahelporglearningcenterleiomyosarcomahtml

[4] M A Depristo E Banks R Poplin et al ldquoA framework forvariation discovery and genotyping using next-generationDNAsequencing datardquo Nature Genetics vol 43 no 5 pp 491ndash5012011

[5] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[6] C W Menges D A Altomare and J R Testa ldquoFAS-associatedfactor 1 (FAF1) diverse functions and implications for oncoge-nesisrdquo Cell Cycle vol 8 no 16 pp 2528ndash2534 2009

[7] M Kim J H Lee S Y Lee E Kim and J Chung ldquoCaspar asuppressor of antibacterial immunity in Drosophilardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 44 pp 16358ndash16363 2006

[8] J Song K P Joon J-J Lee et al ldquoStructure and interaction ofubiquitin-associated domain of human Fas-associated factor 1rdquoProtein Science vol 18 no 11 pp 2265ndash2276 2009

[9] P H OrsquoFarrell ldquoHigh resolution two dimensional electrophore-sis of proteinsrdquo Journal of Biological Chemistry vol 250 no 10pp 4007ndash4021 1975

[10] A Burgess-Cassler J J Johansen D A Santek J R Ideand N C Kendrick ldquoComputerized quantitative analysis ofCoomassie-Blue-stained serum proteins separated by two-dimensional electrophoresisrdquo Clinical Chemistry vol 35 no 12pp 2297ndash2304 1989

[11] B R Oakley D R Kirsch and N RMorris ldquoA simplified ultra-sensitive silver stain for detecting proteins in polyacrylamidegelsrdquo Analytical Biochemistry vol 105 no 2 pp 361ndash363 1980

[12] K Chu X Niu and L T Williams ldquoA Fas-associated proteinfactor FAF1 potentiates Fas-mediated apoptosisrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 25 pp 11894ndash11898 1995

[13] B Rikhof S de Jong A J H Suurmeijer C Meijer and W TA van der Graaf ldquoThe insulin-like growth factor system andsarcomasrdquoThe Journal of Pathology vol 217 no 4 pp 469ndash4822009

[14] RGirling J F Partridge L R Bandara et al ldquoAnew componentof the transcription factor DRTF1E2Frdquo Nature vol 362 no6415 pp 83ndash87 1993

[15] F Mercurio and M Karin ldquoTranscription factors AP-3 andAP-2 interact with the SV40 enhancer in a mutually exclusivemannerrdquo EMBO Journal vol 8 no 5 pp 1455ndash1460 1989

[16] R Bassel-Duby M D Hernandez M A Gonzalez J KKrueger and R S Williams ldquoA 40-kilodalton protein bindsspecifically to an upstream sequence element essential for mus-cle-specific transcription of the human myoglobin promoterrdquoMolecular and Cellular Biology vol 12 no 11 pp 5024ndash50321992

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

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Evidence-Based Complementary and Alternative Medicine

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Page 15: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

Sarcoma 15

Table 3 Deregulation of genes for drug disposition Analysis of RNASeq for at least twofold overexpression (italic font) or underexpression(bold font) of genes for (a) transport and (b) metabolism in tumor compared to muscle and bone For the export transporters (ABCtransporters) only overexpression is considered relevant for drug resistance Not shown in the table are the underexpressed genes (ABCA7ABCA8 ABCA13 ABCB6 ABCB10 ABCC6 ABCC6P1 ABCC6P2 ABCC8 ABCC11 ABCD2 ABCG2 and ABCG5)

(a) Transport

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

650655 ABCA17P ATP-binding cassette subfamily A (ABC1) member 17 pseudogene 1360 211624 ABCA4 ATP-binding cassette subfamily A (ABC1) member 4 2038 2802

6555 SLC10A2 Solute carrier family 10 (sodiumbile acid cotransporter family)member 2 minus1962 minus2112

345274 SLC10A6 Solute carrier family 10 (sodiumbile acid cotransporter family)member 6 2623 3240

6563 SLC14A1 Solute carrier family 14 (urea transporter) member 1 (Kidd bloodgroup) minus3074 minus3152

6565 SLC15A2 Solute carrier family 15 (H+peptide transporter) member 2 minus2027 minus2152

6566 SLC16A1 Solute carrier family 16 member 1 (monocarboxylic acid transporter1) 1401 2170

117247 SLC16A10 Solute carrier family 16 member 10 (aromatic amino acid transporter) 2113 2848

6567 SLC16A2 Solute carrier family 16 member 2 (monocarboxylic acid transporter8) 3242 3848

10786 SLC17A3 Solute carrier family 17 (sodium phosphate) member 3 minus2868 minus29596571 SLC18A2 Solute carrier family 18 (vesicular monoamine) member 2 minus2087 minus21946573 SLC19A1 Solute carrier family 19 (folate transporter) member 1 minus2099 minus220310560 SLC19A2 Solute carrier family 19 (thiamine transporter) member 2 1820 254880704 SLC19A3 Solute carrier family 19 member 3 minus3331 minus3478387775 SLC22A10 Solute carrier family 22 member 10 5711 60859390 SLC22A13 Solute carrier family 22 (organic anion transporter) member 13 2623 3217

85413 SLC22A16 Solute carrier family 22 (organic cationcarnitine transporter)member 16 minus8101 minus7299

51310 SLC22A17 Solute carrier family 22 member 17 1475 21755002 SLC22A18 Solute carrier family 22 member 18 2038 27226582 SLC22A2 Solute carrier family 22 (organic cation transporter) member 2 4793 5216

6581 SLC22A3 Solute carrier family 22 (extraneuronal monoamine transporter)member 3 2554 3182

6583 SLC22A4 Solute carrier family 22 (organic cationergothioneine transporter)member 4 minus3603 minus3776

151295 SLC23A3 Solute carrier family 23 (nucleobase transporters) member 3 2109 284810478 SLC25A17 Solute carrier family 25 (mitochondrial carrier) member 17 1311 207783733 SLC25A18 Solute carrier family 25 (mitochondrial carrier) member 18 1846 2570

788 SLC25A20 Solute carrier family 25 (carnitineacylcarnitine translocase) member20 minus2105 minus2207

89874 SLC25A21 Solute carrier family 25 (mitochondrial oxodicarboxylate carrier)member 21 minus2962 minus3105

51312 SLC25A37 Solute carrier family 25 member 37 minus4633 minus486651629 SLC25A39 Solute carrier family 25 member 39 minus2585 minus2737203427 SLC25A43 Solute carrier family 25 member 43 1325 208865012 SLC26A10 Solute carrier family 26 member 10 3896 4472115111 SLC26A7 Solute carrier family 26 member 7 3431 4018116369 SLC26A8 Solute carrier family 26 member 8 minus5354 minus5536115019 SLC26A9 Solute carrier family 26 member 9 1623 241911001 SLC27A2 Solute carrier family 27 (fatty acid transporter) member 2 minus4278 minus4487

16 Sarcoma

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

64078 SLC28A3 Solute carrier family 28 (sodium-coupled nucleoside transporter)member 3 minus4543 minus4812

222962 SLC29A4 Solute carrier family 29 (nucleoside transporters) member 4 minus1962 minus204581031 SLC2A10 Solute carrier family 2 (facilitated glucose transporter) member 10 2532 3170

6518 SLC2A5 Solute carrier family 2 (facilitated glucosefructose transporter)member 5 minus3647 minus3821

55532 SLC30A10 Solute carrier family 30 member 10 minus3547 minus37257782 SLC30A4 Solute carrier family 30 (zinc transporter) member 4 2832 33726569 SLC34A1 Solute carrier family 34 (sodium phosphate) member 1 minus4716 minus4941340146 SLC35D3 Solute carrier family 35 member D3 minus5662 minus590654733 SLC35F2 Solute carrier family 35 member F2 1433 2170206358 SLC36A1 Solute carrier family 36 (protonamino acid symporter) member 1 minus2265 minus2345285641 SLC36A3 Solute carrier family 36 (protonamino acid symporter) member 3 minus2284 minus239354020 SLC37A1 Solute carrier family 37 (glycerol-3-phosphate transporter) member 1 minus2112 minus22122542 SLC37A4 Solute carrier family 37 (glucose-6-phosphate transporter) member 4 minus2117 minus2219151258 SLC38A11 Solute carrier family 38 member 11 2410 308555089 SLC38A4 Solute carrier family 38 member 4 1837 254892745 SLC38A5 Solute carrier family 38 member 5 minus1994 minus215291252 SLC39A13 Solute carrier family 39 (zinc transporter) member 13 1301 207023516 SLC39A14 Solute carrier family 39 (zinc transporter) member 14 2569 318829985 SLC39A3 Solute carrier family 39 (zinc transporter) member 3 minus2032 minus2152283375 SLC39A5 Solute carrier family 39 (metal ion transporter) member 5 1623 23707922 SLC39A7 Solute carrier family 39 (zinc transporter) member 7 1273 205830061 SLC40A1 Solute carrier family 40 (iron-regulated transporter) member 1 minus3612 minus379684102 SLC41A2 Solute carrier family 41 member 2 1293 20708501 SLC43A1 Solute carrier family 43 member 1 minus2013 minus215257153 SLC44A2 Solute carrier family 44 member 2 minus2047 minus215250651 SLC45A1 Solute carrier family 45 member 1 2038 2722146802 SLC47A2 Solute carrier family 47 member 2 minus1962 minus20266521 SLC4A1 Solute carrier family 4 anion exchanger member 1 minus6513 minus645357282 SLC4A10 Solute carrier family 4 sodium bicarbonate transporter member 10 minus2640 minus275383959 SLC4A11 Solute carrier family 4 sodium borate transporter member 11 1846 25696508 SLC4A3 Solute carrier family 4 anion exchanger member 3 1261 20458671 SLC4A4 Solute carrier family 4 sodium bicarbonate cotransporter member 4 2445 31139497 SLC4A7 Solute carrier family 4 sodium bicarbonate cotransporter member 7 2717 33076523 SLC5A1 Solute carrier family 5 (sodiumglucose cotransporter) member 1 minus2547 minus2705159963 SLC5A12 Solute carrier family 5 (sodiumglucose cotransporter) member 12 4753 52066527 SLC5A4 Solute carrier family 5 (low affinity glucose cotransporter) member 4 minus3969 minus4249

6540 SLC6A13 Solute carrier family 6 (neurotransmitter transporter GABA)member 13 1328 2092

55117 SLC6A15 Solute carrier family 6 (neutral amino acid transporter) member 15 1623 2333388662 SLC6A17 Solute carrier family 6 member 17 2038 272854716 SLC6A20 Solute carrier family 6 (proline IMINO transporter) member 20 1697 2433

6532 SLC6A4 Solute carrier family 6 (neurotransmitter transporter serotonin)member 4 minus2512 minus2611

6534 SLC6A7 Solute carrier family 6 (neurotransmitter transporter L-proline)member 7 2623 3228

56301 SLC7A10 Solute carrier family 7 (neutral amino acid transporter y+ system)member 10 minus3421 minus3603

Sarcoma 17

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

6547 SLC8A3 Solute carrier family 8 (sodiumcalcium exchanger) member 3 minus2204 minus2309285335 SLC9A10 Solute carrier family 9 member 10 1208 20006549 SLC9A2 Solute carrier family 9 (sodiumhydrogen exchanger) member 2 2038 2706

9368 SLC9A3R1 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 1 minus2760 minus2846

9351 SLC9A3R2 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 2 1889 2619

84679 SLC9A7 Solute carrier family 9 (sodiumhydrogen exchanger) member 7 3347 393510599 SLCO1B1 Solute carrier organic anion transporter family member 1B1 3360 397728234 SLCO1B3 Solute carrier organic anion transporter family member 1B3 9760 95696578 SLCO2A1 Solute carrier organic anion transporter family member 2A1 2432 309628232 SLCO3A1 Solute carrier organic anion transporter family member 3A1 minus2032 minus2152353189 SLCO4C1 Solute carrier organic anion transporter family member 4C1 minus6850 minus6611

(b) Metabolism

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold Cchange

1583 CYP11A1 Cytochrome P450 family 11 subfamily A polypeptide 1 1623 23961589 CYP21A2 Cytochrome P450 family 21 subfamily A polypeptide 2 minus1962 minus20361591 CYP24A1 Cytochrome P450 family 24 subfamily A polypeptide 1 5208 55771592 CYP26A1 Cytochrome P450 family 26 subfamily A polypeptide 1 2623 32571594 CYP27B1 Cytochrome P450 family 27 subfamily B polypeptide 1 1846 2585339761 CYP27C1 Cytochrome P450 family 27 subfamily C polypeptide 1 3585 41781553 CYP2A13 Cytochrome P450 family 2 subfamily A polypeptide 13 minus1962 minus20351580 CYP4B1 Cytochrome P450 family 4 subfamily B polypeptide 1 minus4820 minus506566002 CYP4F12 Cytochrome P450 family 4 subfamily F polypeptide 12 minus6070 minus61958529 CYP4F2 Cytochrome P450 family 4 subfamily F polypeptide 2 minus7769 minus70604051 CYP4F3 Cytochrome P450 family 4 subfamily F polypeptide 3 minus8244 minus749111283 CYP4F8 Cytochrome P450 family 4 subfamily F polypeptide 8 minus5006 minus5270260293 CYP4X1 Cytochrome P450 family 4 subfamily X polypeptide 1 minus2476 minus25809420 CYP7B1 Cytochrome P450 family 7 subfamily B polypeptide 1 2502 31702326 FMO1 Flavin containing monooxygenase 1 4360 48592327 FMO2 Flavin containing monooxygenase 2 (nonfunctional) minus4074 minus43372328 FMO3 Flavin containing monooxygenase 3 minus4036 minus4309388714 FMO6P Flavin containing monooxygenase 6 pseudogene minus2569 minus2737493869 GPX8 Glutathione peroxidase 8 (putative) 2814 33712938 GSTA1 Glutathione S-transferase alpha 1 1623 23632939 GSTA2 Glutathione S-transferase alpha 2 2038 27412941 GSTA4 Glutathione S-transferase alpha 4 1492 21882953 GSTT2 Glutathione S-transferase theta 2 4682 5170653689 GSTT2B Glutathione S-transferase theta 2B (genepseudogene) 3739 430784779 NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439 79969027 NAT8 N-acetyltransferase 8 (GCN5-related putative) minus2769 minus29207358 UGDH UDP-glucose 6-dehydrogenase 2333 301855757 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 1604 227110720 UGT2B11 UDP glucuronosyltransferase 2 family polypeptide B11 minus3586 minus37687367 UGT2B17 UDP glucuronosyltransferase 2 family polypeptide B17 minus2836 minus295954490 UGT2B28 UDP glucuronosyltransferase 2 family polypeptide B28 minus3132 minus3284167127 UGT3A2 UDP glycosyltransferase 3 family polypeptide A2 minus4716 minus4907

18 Sarcoma

Table 4 FAF1 mutations in various cancers FAF1 mutations listed in the TCGA data base were identified without restriction to any type ofcancer For the location of the affected domains on the protein compare Figure 2

AA Mutation Cancer DomainN4S Missense Cutaneous melanomaI10S Missense Stomach adenocarcinoma UBAE21lowast Nonsense Cutaneous melanoma UBAE25K Missense Uterine endometrioid carcinoma UBAV38 splice Splice Stomach adenocarcinoma UBAP86fs FS del Colorectal adenocarcinomaG123 splice Splice Uterine endometrioid carcinoma UB1P136H Missense Colorectal adenocarcinoma UB1T147M Missense Brain lower grade glioma UB1D149Y Missense Uterine endometrioid carcinoma UB1L159V Missense Lung adenocarcinoma UB1K163N Missense Uterine endometrioid carcinoma UB1L170F Missense Cutaneous melanomaG184 splice Splice Colorectal cancerQ187H Missense Stomach adenocarcinomaS214N Missense Colorectal adenocarcinoma UB2R222I Missense Uterine endometrioid carcinoma UB2E238D Missense Lung adenocarcinoma UB2P241S Missense Uterine endometrioid carcinoma UB2T245A Missense Renal clear cell carcinoma UB2M249V Missense Uterine endometrioid carcinoma UB2E280K Missense Brain lower grade gliomaG293lowast Nonsense Colorectal cancerT300I Missense Colorectal adenocarcinomaD305H Missense Lung adenocarcinomaE308Q Missense Lung adenocarcinomaA316V Missense Stomach adenocarcinomaK319fs FS ins Head and neck squamous cell carcinomaR344G Missense Stomach adenocarcinoma UASF353I Missense Cutaneous melanoma UASL379V Missense Breast invasive carcinoma UASC396F Missense Cutaneous melanoma UASS459lowast Nonsense Uterine endometrioid carcinoma UASG469 splice Splice Glioblastoma multiforme UASR509G Missense Lung squamous cell carcinomaE510fs FS del Cutaneous melanomaR516C Missense Uterine endometrioid carcinomaA534V Missense Stomach adenocarcinomaF537L Missense Stomach adenocarcinomaE551lowast Nonsense Lung adenocarcinomaR554W Missense Colorectal cancerS582I Missense Lung adenocarcinoma UBXF585L Missense Uterine endometrioid carcinoma UBXE587lowast Nonsense Lung adenocarcinoma UBXA592V Missense Stomach adenocarcinoma UBXW610lowast Nonsense Breast invasive carcinoma UBXD611Y Missense Lung adenocarcinoma UBXE635fs FS del Brain lower grade glioma UBXP640fs FS del Cutaneous melanoma UBX

Sarcoma 19

a more accurate reference point for a leiomyosarcoma wouldhave been smooth muscle we believe that the comparison tostriated muscle is sufficient to allow the assessment of tumorspecific changes We have measured RNA DNA and proteinwith various assays Similar assessments in the future shouldalso include chromosome analysis for possible translocations

In the first-line defense against cancer the gradualreplacement of conventional chemotherapy with molecularlytargeted agents has opened the possibility to tailor drug treat-ments to particular tumors This transition necessitates thecharacterization of the molecular lesions that are causativefor the transformation of healthy cells into cancerous cellsbecause drugs need to be matched with the underlying carci-nogenic defect to be effective An additional caveat causedby unique genetic changes in the primary tumor can affectdrug transport and metabolism and needs to be taken intoconsideration In this case the RNA levels for genes that regu-late transport andmetabolismwere extensively skewed in thetumor tissue as compared to muscle and bone (see Table 3)implying potential challenges to chemotherapy An advancedmolecular treatment strategy for cancer will rely on themolecular definition of drug target drug transport and drugmetabolism pharmacogenetics in the primary tumor Con-secutive to cancer dissemination it will also require adjust-ments to account for the genetic changes in the metastases

The cost of health care has been under increasing scrutinyThe treatment of cancer patients is expensive in particularwhen hospitalization is required and further in cases ofend-of-life care Avoidable expenditures are generated bysuboptimal treatment decisions that result in low efficacy orhigh toxicity of anticancer regimens Molecular medicine hasthe potential to preempt those problems and reduce wastefulspending Yet it requires the upfront cost of molecular cancerexamination The analysis performed in this study requiredabout $11000- in nonsalary expenses to perform While ithas not identified a drug target it has specified possibleconfines for drug treatment The costs for molecular analysisneed to be weighed against the societal cost derived fromlost productivity in the workforce disrupted lives of familiesand premature deaths While currently limited drug optionsconstitute the major constraint to the approach taken herethe foreseeable future will bring an increasing spectrum ofmolecularly targeted drugs along with faster and cheapertechnologies for the molecular assessment of cancers Theywill facilitate the clinical translation of our approach

Consent

The patient provided informed consent

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by DOD Grant PR094070under University of Cincinnati IRB protocol 04-01-29-01The

author is grateful to Dr Rhett Kovall for helping with thestructural analysis of FAF1

References

[1] G F Weber Molecular Mechanisms of Cancer Springer Dor-drecht The Netherlands 2007

[2] N G Sanerkin ldquoPrimary leiomyosarcoma of the bone and itscomparison with fibrosarcomardquoCancer vol 44 no 4 pp 1375ndash1387 1979

[3] J M Weaver and J A Abraham ldquoLeiomyosarcoma of the Boneand Soft Tissue A Reviewrdquo httpsarcomahelporglearningcenterleiomyosarcomahtml

[4] M A Depristo E Banks R Poplin et al ldquoA framework forvariation discovery and genotyping using next-generationDNAsequencing datardquo Nature Genetics vol 43 no 5 pp 491ndash5012011

[5] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[6] C W Menges D A Altomare and J R Testa ldquoFAS-associatedfactor 1 (FAF1) diverse functions and implications for oncoge-nesisrdquo Cell Cycle vol 8 no 16 pp 2528ndash2534 2009

[7] M Kim J H Lee S Y Lee E Kim and J Chung ldquoCaspar asuppressor of antibacterial immunity in Drosophilardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 44 pp 16358ndash16363 2006

[8] J Song K P Joon J-J Lee et al ldquoStructure and interaction ofubiquitin-associated domain of human Fas-associated factor 1rdquoProtein Science vol 18 no 11 pp 2265ndash2276 2009

[9] P H OrsquoFarrell ldquoHigh resolution two dimensional electrophore-sis of proteinsrdquo Journal of Biological Chemistry vol 250 no 10pp 4007ndash4021 1975

[10] A Burgess-Cassler J J Johansen D A Santek J R Ideand N C Kendrick ldquoComputerized quantitative analysis ofCoomassie-Blue-stained serum proteins separated by two-dimensional electrophoresisrdquo Clinical Chemistry vol 35 no 12pp 2297ndash2304 1989

[11] B R Oakley D R Kirsch and N RMorris ldquoA simplified ultra-sensitive silver stain for detecting proteins in polyacrylamidegelsrdquo Analytical Biochemistry vol 105 no 2 pp 361ndash363 1980

[12] K Chu X Niu and L T Williams ldquoA Fas-associated proteinfactor FAF1 potentiates Fas-mediated apoptosisrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 25 pp 11894ndash11898 1995

[13] B Rikhof S de Jong A J H Suurmeijer C Meijer and W TA van der Graaf ldquoThe insulin-like growth factor system andsarcomasrdquoThe Journal of Pathology vol 217 no 4 pp 469ndash4822009

[14] RGirling J F Partridge L R Bandara et al ldquoAnew componentof the transcription factor DRTF1E2Frdquo Nature vol 362 no6415 pp 83ndash87 1993

[15] F Mercurio and M Karin ldquoTranscription factors AP-3 andAP-2 interact with the SV40 enhancer in a mutually exclusivemannerrdquo EMBO Journal vol 8 no 5 pp 1455ndash1460 1989

[16] R Bassel-Duby M D Hernandez M A Gonzalez J KKrueger and R S Williams ldquoA 40-kilodalton protein bindsspecifically to an upstream sequence element essential for mus-cle-specific transcription of the human myoglobin promoterrdquoMolecular and Cellular Biology vol 12 no 11 pp 5024ndash50321992

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 16: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

16 Sarcoma

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

64078 SLC28A3 Solute carrier family 28 (sodium-coupled nucleoside transporter)member 3 minus4543 minus4812

222962 SLC29A4 Solute carrier family 29 (nucleoside transporters) member 4 minus1962 minus204581031 SLC2A10 Solute carrier family 2 (facilitated glucose transporter) member 10 2532 3170

6518 SLC2A5 Solute carrier family 2 (facilitated glucosefructose transporter)member 5 minus3647 minus3821

55532 SLC30A10 Solute carrier family 30 member 10 minus3547 minus37257782 SLC30A4 Solute carrier family 30 (zinc transporter) member 4 2832 33726569 SLC34A1 Solute carrier family 34 (sodium phosphate) member 1 minus4716 minus4941340146 SLC35D3 Solute carrier family 35 member D3 minus5662 minus590654733 SLC35F2 Solute carrier family 35 member F2 1433 2170206358 SLC36A1 Solute carrier family 36 (protonamino acid symporter) member 1 minus2265 minus2345285641 SLC36A3 Solute carrier family 36 (protonamino acid symporter) member 3 minus2284 minus239354020 SLC37A1 Solute carrier family 37 (glycerol-3-phosphate transporter) member 1 minus2112 minus22122542 SLC37A4 Solute carrier family 37 (glucose-6-phosphate transporter) member 4 minus2117 minus2219151258 SLC38A11 Solute carrier family 38 member 11 2410 308555089 SLC38A4 Solute carrier family 38 member 4 1837 254892745 SLC38A5 Solute carrier family 38 member 5 minus1994 minus215291252 SLC39A13 Solute carrier family 39 (zinc transporter) member 13 1301 207023516 SLC39A14 Solute carrier family 39 (zinc transporter) member 14 2569 318829985 SLC39A3 Solute carrier family 39 (zinc transporter) member 3 minus2032 minus2152283375 SLC39A5 Solute carrier family 39 (metal ion transporter) member 5 1623 23707922 SLC39A7 Solute carrier family 39 (zinc transporter) member 7 1273 205830061 SLC40A1 Solute carrier family 40 (iron-regulated transporter) member 1 minus3612 minus379684102 SLC41A2 Solute carrier family 41 member 2 1293 20708501 SLC43A1 Solute carrier family 43 member 1 minus2013 minus215257153 SLC44A2 Solute carrier family 44 member 2 minus2047 minus215250651 SLC45A1 Solute carrier family 45 member 1 2038 2722146802 SLC47A2 Solute carrier family 47 member 2 minus1962 minus20266521 SLC4A1 Solute carrier family 4 anion exchanger member 1 minus6513 minus645357282 SLC4A10 Solute carrier family 4 sodium bicarbonate transporter member 10 minus2640 minus275383959 SLC4A11 Solute carrier family 4 sodium borate transporter member 11 1846 25696508 SLC4A3 Solute carrier family 4 anion exchanger member 3 1261 20458671 SLC4A4 Solute carrier family 4 sodium bicarbonate cotransporter member 4 2445 31139497 SLC4A7 Solute carrier family 4 sodium bicarbonate cotransporter member 7 2717 33076523 SLC5A1 Solute carrier family 5 (sodiumglucose cotransporter) member 1 minus2547 minus2705159963 SLC5A12 Solute carrier family 5 (sodiumglucose cotransporter) member 12 4753 52066527 SLC5A4 Solute carrier family 5 (low affinity glucose cotransporter) member 4 minus3969 minus4249

6540 SLC6A13 Solute carrier family 6 (neurotransmitter transporter GABA)member 13 1328 2092

55117 SLC6A15 Solute carrier family 6 (neutral amino acid transporter) member 15 1623 2333388662 SLC6A17 Solute carrier family 6 member 17 2038 272854716 SLC6A20 Solute carrier family 6 (proline IMINO transporter) member 20 1697 2433

6532 SLC6A4 Solute carrier family 6 (neurotransmitter transporter serotonin)member 4 minus2512 minus2611

6534 SLC6A7 Solute carrier family 6 (neurotransmitter transporter L-proline)member 7 2623 3228

56301 SLC7A10 Solute carrier family 7 (neutral amino acid transporter y+ system)member 10 minus3421 minus3603

Sarcoma 17

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

6547 SLC8A3 Solute carrier family 8 (sodiumcalcium exchanger) member 3 minus2204 minus2309285335 SLC9A10 Solute carrier family 9 member 10 1208 20006549 SLC9A2 Solute carrier family 9 (sodiumhydrogen exchanger) member 2 2038 2706

9368 SLC9A3R1 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 1 minus2760 minus2846

9351 SLC9A3R2 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 2 1889 2619

84679 SLC9A7 Solute carrier family 9 (sodiumhydrogen exchanger) member 7 3347 393510599 SLCO1B1 Solute carrier organic anion transporter family member 1B1 3360 397728234 SLCO1B3 Solute carrier organic anion transporter family member 1B3 9760 95696578 SLCO2A1 Solute carrier organic anion transporter family member 2A1 2432 309628232 SLCO3A1 Solute carrier organic anion transporter family member 3A1 minus2032 minus2152353189 SLCO4C1 Solute carrier organic anion transporter family member 4C1 minus6850 minus6611

(b) Metabolism

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold Cchange

1583 CYP11A1 Cytochrome P450 family 11 subfamily A polypeptide 1 1623 23961589 CYP21A2 Cytochrome P450 family 21 subfamily A polypeptide 2 minus1962 minus20361591 CYP24A1 Cytochrome P450 family 24 subfamily A polypeptide 1 5208 55771592 CYP26A1 Cytochrome P450 family 26 subfamily A polypeptide 1 2623 32571594 CYP27B1 Cytochrome P450 family 27 subfamily B polypeptide 1 1846 2585339761 CYP27C1 Cytochrome P450 family 27 subfamily C polypeptide 1 3585 41781553 CYP2A13 Cytochrome P450 family 2 subfamily A polypeptide 13 minus1962 minus20351580 CYP4B1 Cytochrome P450 family 4 subfamily B polypeptide 1 minus4820 minus506566002 CYP4F12 Cytochrome P450 family 4 subfamily F polypeptide 12 minus6070 minus61958529 CYP4F2 Cytochrome P450 family 4 subfamily F polypeptide 2 minus7769 minus70604051 CYP4F3 Cytochrome P450 family 4 subfamily F polypeptide 3 minus8244 minus749111283 CYP4F8 Cytochrome P450 family 4 subfamily F polypeptide 8 minus5006 minus5270260293 CYP4X1 Cytochrome P450 family 4 subfamily X polypeptide 1 minus2476 minus25809420 CYP7B1 Cytochrome P450 family 7 subfamily B polypeptide 1 2502 31702326 FMO1 Flavin containing monooxygenase 1 4360 48592327 FMO2 Flavin containing monooxygenase 2 (nonfunctional) minus4074 minus43372328 FMO3 Flavin containing monooxygenase 3 minus4036 minus4309388714 FMO6P Flavin containing monooxygenase 6 pseudogene minus2569 minus2737493869 GPX8 Glutathione peroxidase 8 (putative) 2814 33712938 GSTA1 Glutathione S-transferase alpha 1 1623 23632939 GSTA2 Glutathione S-transferase alpha 2 2038 27412941 GSTA4 Glutathione S-transferase alpha 4 1492 21882953 GSTT2 Glutathione S-transferase theta 2 4682 5170653689 GSTT2B Glutathione S-transferase theta 2B (genepseudogene) 3739 430784779 NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439 79969027 NAT8 N-acetyltransferase 8 (GCN5-related putative) minus2769 minus29207358 UGDH UDP-glucose 6-dehydrogenase 2333 301855757 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 1604 227110720 UGT2B11 UDP glucuronosyltransferase 2 family polypeptide B11 minus3586 minus37687367 UGT2B17 UDP glucuronosyltransferase 2 family polypeptide B17 minus2836 minus295954490 UGT2B28 UDP glucuronosyltransferase 2 family polypeptide B28 minus3132 minus3284167127 UGT3A2 UDP glycosyltransferase 3 family polypeptide A2 minus4716 minus4907

18 Sarcoma

Table 4 FAF1 mutations in various cancers FAF1 mutations listed in the TCGA data base were identified without restriction to any type ofcancer For the location of the affected domains on the protein compare Figure 2

AA Mutation Cancer DomainN4S Missense Cutaneous melanomaI10S Missense Stomach adenocarcinoma UBAE21lowast Nonsense Cutaneous melanoma UBAE25K Missense Uterine endometrioid carcinoma UBAV38 splice Splice Stomach adenocarcinoma UBAP86fs FS del Colorectal adenocarcinomaG123 splice Splice Uterine endometrioid carcinoma UB1P136H Missense Colorectal adenocarcinoma UB1T147M Missense Brain lower grade glioma UB1D149Y Missense Uterine endometrioid carcinoma UB1L159V Missense Lung adenocarcinoma UB1K163N Missense Uterine endometrioid carcinoma UB1L170F Missense Cutaneous melanomaG184 splice Splice Colorectal cancerQ187H Missense Stomach adenocarcinomaS214N Missense Colorectal adenocarcinoma UB2R222I Missense Uterine endometrioid carcinoma UB2E238D Missense Lung adenocarcinoma UB2P241S Missense Uterine endometrioid carcinoma UB2T245A Missense Renal clear cell carcinoma UB2M249V Missense Uterine endometrioid carcinoma UB2E280K Missense Brain lower grade gliomaG293lowast Nonsense Colorectal cancerT300I Missense Colorectal adenocarcinomaD305H Missense Lung adenocarcinomaE308Q Missense Lung adenocarcinomaA316V Missense Stomach adenocarcinomaK319fs FS ins Head and neck squamous cell carcinomaR344G Missense Stomach adenocarcinoma UASF353I Missense Cutaneous melanoma UASL379V Missense Breast invasive carcinoma UASC396F Missense Cutaneous melanoma UASS459lowast Nonsense Uterine endometrioid carcinoma UASG469 splice Splice Glioblastoma multiforme UASR509G Missense Lung squamous cell carcinomaE510fs FS del Cutaneous melanomaR516C Missense Uterine endometrioid carcinomaA534V Missense Stomach adenocarcinomaF537L Missense Stomach adenocarcinomaE551lowast Nonsense Lung adenocarcinomaR554W Missense Colorectal cancerS582I Missense Lung adenocarcinoma UBXF585L Missense Uterine endometrioid carcinoma UBXE587lowast Nonsense Lung adenocarcinoma UBXA592V Missense Stomach adenocarcinoma UBXW610lowast Nonsense Breast invasive carcinoma UBXD611Y Missense Lung adenocarcinoma UBXE635fs FS del Brain lower grade glioma UBXP640fs FS del Cutaneous melanoma UBX

Sarcoma 19

a more accurate reference point for a leiomyosarcoma wouldhave been smooth muscle we believe that the comparison tostriated muscle is sufficient to allow the assessment of tumorspecific changes We have measured RNA DNA and proteinwith various assays Similar assessments in the future shouldalso include chromosome analysis for possible translocations

In the first-line defense against cancer the gradualreplacement of conventional chemotherapy with molecularlytargeted agents has opened the possibility to tailor drug treat-ments to particular tumors This transition necessitates thecharacterization of the molecular lesions that are causativefor the transformation of healthy cells into cancerous cellsbecause drugs need to be matched with the underlying carci-nogenic defect to be effective An additional caveat causedby unique genetic changes in the primary tumor can affectdrug transport and metabolism and needs to be taken intoconsideration In this case the RNA levels for genes that regu-late transport andmetabolismwere extensively skewed in thetumor tissue as compared to muscle and bone (see Table 3)implying potential challenges to chemotherapy An advancedmolecular treatment strategy for cancer will rely on themolecular definition of drug target drug transport and drugmetabolism pharmacogenetics in the primary tumor Con-secutive to cancer dissemination it will also require adjust-ments to account for the genetic changes in the metastases

The cost of health care has been under increasing scrutinyThe treatment of cancer patients is expensive in particularwhen hospitalization is required and further in cases ofend-of-life care Avoidable expenditures are generated bysuboptimal treatment decisions that result in low efficacy orhigh toxicity of anticancer regimens Molecular medicine hasthe potential to preempt those problems and reduce wastefulspending Yet it requires the upfront cost of molecular cancerexamination The analysis performed in this study requiredabout $11000- in nonsalary expenses to perform While ithas not identified a drug target it has specified possibleconfines for drug treatment The costs for molecular analysisneed to be weighed against the societal cost derived fromlost productivity in the workforce disrupted lives of familiesand premature deaths While currently limited drug optionsconstitute the major constraint to the approach taken herethe foreseeable future will bring an increasing spectrum ofmolecularly targeted drugs along with faster and cheapertechnologies for the molecular assessment of cancers Theywill facilitate the clinical translation of our approach

Consent

The patient provided informed consent

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by DOD Grant PR094070under University of Cincinnati IRB protocol 04-01-29-01The

author is grateful to Dr Rhett Kovall for helping with thestructural analysis of FAF1

References

[1] G F Weber Molecular Mechanisms of Cancer Springer Dor-drecht The Netherlands 2007

[2] N G Sanerkin ldquoPrimary leiomyosarcoma of the bone and itscomparison with fibrosarcomardquoCancer vol 44 no 4 pp 1375ndash1387 1979

[3] J M Weaver and J A Abraham ldquoLeiomyosarcoma of the Boneand Soft Tissue A Reviewrdquo httpsarcomahelporglearningcenterleiomyosarcomahtml

[4] M A Depristo E Banks R Poplin et al ldquoA framework forvariation discovery and genotyping using next-generationDNAsequencing datardquo Nature Genetics vol 43 no 5 pp 491ndash5012011

[5] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[6] C W Menges D A Altomare and J R Testa ldquoFAS-associatedfactor 1 (FAF1) diverse functions and implications for oncoge-nesisrdquo Cell Cycle vol 8 no 16 pp 2528ndash2534 2009

[7] M Kim J H Lee S Y Lee E Kim and J Chung ldquoCaspar asuppressor of antibacterial immunity in Drosophilardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 44 pp 16358ndash16363 2006

[8] J Song K P Joon J-J Lee et al ldquoStructure and interaction ofubiquitin-associated domain of human Fas-associated factor 1rdquoProtein Science vol 18 no 11 pp 2265ndash2276 2009

[9] P H OrsquoFarrell ldquoHigh resolution two dimensional electrophore-sis of proteinsrdquo Journal of Biological Chemistry vol 250 no 10pp 4007ndash4021 1975

[10] A Burgess-Cassler J J Johansen D A Santek J R Ideand N C Kendrick ldquoComputerized quantitative analysis ofCoomassie-Blue-stained serum proteins separated by two-dimensional electrophoresisrdquo Clinical Chemistry vol 35 no 12pp 2297ndash2304 1989

[11] B R Oakley D R Kirsch and N RMorris ldquoA simplified ultra-sensitive silver stain for detecting proteins in polyacrylamidegelsrdquo Analytical Biochemistry vol 105 no 2 pp 361ndash363 1980

[12] K Chu X Niu and L T Williams ldquoA Fas-associated proteinfactor FAF1 potentiates Fas-mediated apoptosisrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 25 pp 11894ndash11898 1995

[13] B Rikhof S de Jong A J H Suurmeijer C Meijer and W TA van der Graaf ldquoThe insulin-like growth factor system andsarcomasrdquoThe Journal of Pathology vol 217 no 4 pp 469ndash4822009

[14] RGirling J F Partridge L R Bandara et al ldquoAnew componentof the transcription factor DRTF1E2Frdquo Nature vol 362 no6415 pp 83ndash87 1993

[15] F Mercurio and M Karin ldquoTranscription factors AP-3 andAP-2 interact with the SV40 enhancer in a mutually exclusivemannerrdquo EMBO Journal vol 8 no 5 pp 1455ndash1460 1989

[16] R Bassel-Duby M D Hernandez M A Gonzalez J KKrueger and R S Williams ldquoA 40-kilodalton protein bindsspecifically to an upstream sequence element essential for mus-cle-specific transcription of the human myoglobin promoterrdquoMolecular and Cellular Biology vol 12 no 11 pp 5024ndash50321992

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 17: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

Sarcoma 17

(a) Continued

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold change

6547 SLC8A3 Solute carrier family 8 (sodiumcalcium exchanger) member 3 minus2204 minus2309285335 SLC9A10 Solute carrier family 9 member 10 1208 20006549 SLC9A2 Solute carrier family 9 (sodiumhydrogen exchanger) member 2 2038 2706

9368 SLC9A3R1 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 1 minus2760 minus2846

9351 SLC9A3R2 Solute carrier family 9 (sodiumhydrogen exchanger) member 3regulator 2 1889 2619

84679 SLC9A7 Solute carrier family 9 (sodiumhydrogen exchanger) member 7 3347 393510599 SLCO1B1 Solute carrier organic anion transporter family member 1B1 3360 397728234 SLCO1B3 Solute carrier organic anion transporter family member 1B3 9760 95696578 SLCO2A1 Solute carrier organic anion transporter family member 2A1 2432 309628232 SLCO3A1 Solute carrier organic anion transporter family member 3A1 minus2032 minus2152353189 SLCO4C1 Solute carrier organic anion transporter family member 4C1 minus6850 minus6611

(b) Metabolism

Gene ID Symbol Name Tumor bone Tumor musclelog2-fold change log2-fold Cchange

1583 CYP11A1 Cytochrome P450 family 11 subfamily A polypeptide 1 1623 23961589 CYP21A2 Cytochrome P450 family 21 subfamily A polypeptide 2 minus1962 minus20361591 CYP24A1 Cytochrome P450 family 24 subfamily A polypeptide 1 5208 55771592 CYP26A1 Cytochrome P450 family 26 subfamily A polypeptide 1 2623 32571594 CYP27B1 Cytochrome P450 family 27 subfamily B polypeptide 1 1846 2585339761 CYP27C1 Cytochrome P450 family 27 subfamily C polypeptide 1 3585 41781553 CYP2A13 Cytochrome P450 family 2 subfamily A polypeptide 13 minus1962 minus20351580 CYP4B1 Cytochrome P450 family 4 subfamily B polypeptide 1 minus4820 minus506566002 CYP4F12 Cytochrome P450 family 4 subfamily F polypeptide 12 minus6070 minus61958529 CYP4F2 Cytochrome P450 family 4 subfamily F polypeptide 2 minus7769 minus70604051 CYP4F3 Cytochrome P450 family 4 subfamily F polypeptide 3 minus8244 minus749111283 CYP4F8 Cytochrome P450 family 4 subfamily F polypeptide 8 minus5006 minus5270260293 CYP4X1 Cytochrome P450 family 4 subfamily X polypeptide 1 minus2476 minus25809420 CYP7B1 Cytochrome P450 family 7 subfamily B polypeptide 1 2502 31702326 FMO1 Flavin containing monooxygenase 1 4360 48592327 FMO2 Flavin containing monooxygenase 2 (nonfunctional) minus4074 minus43372328 FMO3 Flavin containing monooxygenase 3 minus4036 minus4309388714 FMO6P Flavin containing monooxygenase 6 pseudogene minus2569 minus2737493869 GPX8 Glutathione peroxidase 8 (putative) 2814 33712938 GSTA1 Glutathione S-transferase alpha 1 1623 23632939 GSTA2 Glutathione S-transferase alpha 2 2038 27412941 GSTA4 Glutathione S-transferase alpha 4 1492 21882953 GSTT2 Glutathione S-transferase theta 2 4682 5170653689 GSTT2B Glutathione S-transferase theta 2B (genepseudogene) 3739 430784779 NAA11 N(alpha)-acetyltransferase 11 NatA catalytic subunit 7439 79969027 NAT8 N-acetyltransferase 8 (GCN5-related putative) minus2769 minus29207358 UGDH UDP-glucose 6-dehydrogenase 2333 301855757 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 1604 227110720 UGT2B11 UDP glucuronosyltransferase 2 family polypeptide B11 minus3586 minus37687367 UGT2B17 UDP glucuronosyltransferase 2 family polypeptide B17 minus2836 minus295954490 UGT2B28 UDP glucuronosyltransferase 2 family polypeptide B28 minus3132 minus3284167127 UGT3A2 UDP glycosyltransferase 3 family polypeptide A2 minus4716 minus4907

18 Sarcoma

Table 4 FAF1 mutations in various cancers FAF1 mutations listed in the TCGA data base were identified without restriction to any type ofcancer For the location of the affected domains on the protein compare Figure 2

AA Mutation Cancer DomainN4S Missense Cutaneous melanomaI10S Missense Stomach adenocarcinoma UBAE21lowast Nonsense Cutaneous melanoma UBAE25K Missense Uterine endometrioid carcinoma UBAV38 splice Splice Stomach adenocarcinoma UBAP86fs FS del Colorectal adenocarcinomaG123 splice Splice Uterine endometrioid carcinoma UB1P136H Missense Colorectal adenocarcinoma UB1T147M Missense Brain lower grade glioma UB1D149Y Missense Uterine endometrioid carcinoma UB1L159V Missense Lung adenocarcinoma UB1K163N Missense Uterine endometrioid carcinoma UB1L170F Missense Cutaneous melanomaG184 splice Splice Colorectal cancerQ187H Missense Stomach adenocarcinomaS214N Missense Colorectal adenocarcinoma UB2R222I Missense Uterine endometrioid carcinoma UB2E238D Missense Lung adenocarcinoma UB2P241S Missense Uterine endometrioid carcinoma UB2T245A Missense Renal clear cell carcinoma UB2M249V Missense Uterine endometrioid carcinoma UB2E280K Missense Brain lower grade gliomaG293lowast Nonsense Colorectal cancerT300I Missense Colorectal adenocarcinomaD305H Missense Lung adenocarcinomaE308Q Missense Lung adenocarcinomaA316V Missense Stomach adenocarcinomaK319fs FS ins Head and neck squamous cell carcinomaR344G Missense Stomach adenocarcinoma UASF353I Missense Cutaneous melanoma UASL379V Missense Breast invasive carcinoma UASC396F Missense Cutaneous melanoma UASS459lowast Nonsense Uterine endometrioid carcinoma UASG469 splice Splice Glioblastoma multiforme UASR509G Missense Lung squamous cell carcinomaE510fs FS del Cutaneous melanomaR516C Missense Uterine endometrioid carcinomaA534V Missense Stomach adenocarcinomaF537L Missense Stomach adenocarcinomaE551lowast Nonsense Lung adenocarcinomaR554W Missense Colorectal cancerS582I Missense Lung adenocarcinoma UBXF585L Missense Uterine endometrioid carcinoma UBXE587lowast Nonsense Lung adenocarcinoma UBXA592V Missense Stomach adenocarcinoma UBXW610lowast Nonsense Breast invasive carcinoma UBXD611Y Missense Lung adenocarcinoma UBXE635fs FS del Brain lower grade glioma UBXP640fs FS del Cutaneous melanoma UBX

Sarcoma 19

a more accurate reference point for a leiomyosarcoma wouldhave been smooth muscle we believe that the comparison tostriated muscle is sufficient to allow the assessment of tumorspecific changes We have measured RNA DNA and proteinwith various assays Similar assessments in the future shouldalso include chromosome analysis for possible translocations

In the first-line defense against cancer the gradualreplacement of conventional chemotherapy with molecularlytargeted agents has opened the possibility to tailor drug treat-ments to particular tumors This transition necessitates thecharacterization of the molecular lesions that are causativefor the transformation of healthy cells into cancerous cellsbecause drugs need to be matched with the underlying carci-nogenic defect to be effective An additional caveat causedby unique genetic changes in the primary tumor can affectdrug transport and metabolism and needs to be taken intoconsideration In this case the RNA levels for genes that regu-late transport andmetabolismwere extensively skewed in thetumor tissue as compared to muscle and bone (see Table 3)implying potential challenges to chemotherapy An advancedmolecular treatment strategy for cancer will rely on themolecular definition of drug target drug transport and drugmetabolism pharmacogenetics in the primary tumor Con-secutive to cancer dissemination it will also require adjust-ments to account for the genetic changes in the metastases

The cost of health care has been under increasing scrutinyThe treatment of cancer patients is expensive in particularwhen hospitalization is required and further in cases ofend-of-life care Avoidable expenditures are generated bysuboptimal treatment decisions that result in low efficacy orhigh toxicity of anticancer regimens Molecular medicine hasthe potential to preempt those problems and reduce wastefulspending Yet it requires the upfront cost of molecular cancerexamination The analysis performed in this study requiredabout $11000- in nonsalary expenses to perform While ithas not identified a drug target it has specified possibleconfines for drug treatment The costs for molecular analysisneed to be weighed against the societal cost derived fromlost productivity in the workforce disrupted lives of familiesand premature deaths While currently limited drug optionsconstitute the major constraint to the approach taken herethe foreseeable future will bring an increasing spectrum ofmolecularly targeted drugs along with faster and cheapertechnologies for the molecular assessment of cancers Theywill facilitate the clinical translation of our approach

Consent

The patient provided informed consent

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by DOD Grant PR094070under University of Cincinnati IRB protocol 04-01-29-01The

author is grateful to Dr Rhett Kovall for helping with thestructural analysis of FAF1

References

[1] G F Weber Molecular Mechanisms of Cancer Springer Dor-drecht The Netherlands 2007

[2] N G Sanerkin ldquoPrimary leiomyosarcoma of the bone and itscomparison with fibrosarcomardquoCancer vol 44 no 4 pp 1375ndash1387 1979

[3] J M Weaver and J A Abraham ldquoLeiomyosarcoma of the Boneand Soft Tissue A Reviewrdquo httpsarcomahelporglearningcenterleiomyosarcomahtml

[4] M A Depristo E Banks R Poplin et al ldquoA framework forvariation discovery and genotyping using next-generationDNAsequencing datardquo Nature Genetics vol 43 no 5 pp 491ndash5012011

[5] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[6] C W Menges D A Altomare and J R Testa ldquoFAS-associatedfactor 1 (FAF1) diverse functions and implications for oncoge-nesisrdquo Cell Cycle vol 8 no 16 pp 2528ndash2534 2009

[7] M Kim J H Lee S Y Lee E Kim and J Chung ldquoCaspar asuppressor of antibacterial immunity in Drosophilardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 44 pp 16358ndash16363 2006

[8] J Song K P Joon J-J Lee et al ldquoStructure and interaction ofubiquitin-associated domain of human Fas-associated factor 1rdquoProtein Science vol 18 no 11 pp 2265ndash2276 2009

[9] P H OrsquoFarrell ldquoHigh resolution two dimensional electrophore-sis of proteinsrdquo Journal of Biological Chemistry vol 250 no 10pp 4007ndash4021 1975

[10] A Burgess-Cassler J J Johansen D A Santek J R Ideand N C Kendrick ldquoComputerized quantitative analysis ofCoomassie-Blue-stained serum proteins separated by two-dimensional electrophoresisrdquo Clinical Chemistry vol 35 no 12pp 2297ndash2304 1989

[11] B R Oakley D R Kirsch and N RMorris ldquoA simplified ultra-sensitive silver stain for detecting proteins in polyacrylamidegelsrdquo Analytical Biochemistry vol 105 no 2 pp 361ndash363 1980

[12] K Chu X Niu and L T Williams ldquoA Fas-associated proteinfactor FAF1 potentiates Fas-mediated apoptosisrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 25 pp 11894ndash11898 1995

[13] B Rikhof S de Jong A J H Suurmeijer C Meijer and W TA van der Graaf ldquoThe insulin-like growth factor system andsarcomasrdquoThe Journal of Pathology vol 217 no 4 pp 469ndash4822009

[14] RGirling J F Partridge L R Bandara et al ldquoAnew componentof the transcription factor DRTF1E2Frdquo Nature vol 362 no6415 pp 83ndash87 1993

[15] F Mercurio and M Karin ldquoTranscription factors AP-3 andAP-2 interact with the SV40 enhancer in a mutually exclusivemannerrdquo EMBO Journal vol 8 no 5 pp 1455ndash1460 1989

[16] R Bassel-Duby M D Hernandez M A Gonzalez J KKrueger and R S Williams ldquoA 40-kilodalton protein bindsspecifically to an upstream sequence element essential for mus-cle-specific transcription of the human myoglobin promoterrdquoMolecular and Cellular Biology vol 12 no 11 pp 5024ndash50321992

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 18: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

18 Sarcoma

Table 4 FAF1 mutations in various cancers FAF1 mutations listed in the TCGA data base were identified without restriction to any type ofcancer For the location of the affected domains on the protein compare Figure 2

AA Mutation Cancer DomainN4S Missense Cutaneous melanomaI10S Missense Stomach adenocarcinoma UBAE21lowast Nonsense Cutaneous melanoma UBAE25K Missense Uterine endometrioid carcinoma UBAV38 splice Splice Stomach adenocarcinoma UBAP86fs FS del Colorectal adenocarcinomaG123 splice Splice Uterine endometrioid carcinoma UB1P136H Missense Colorectal adenocarcinoma UB1T147M Missense Brain lower grade glioma UB1D149Y Missense Uterine endometrioid carcinoma UB1L159V Missense Lung adenocarcinoma UB1K163N Missense Uterine endometrioid carcinoma UB1L170F Missense Cutaneous melanomaG184 splice Splice Colorectal cancerQ187H Missense Stomach adenocarcinomaS214N Missense Colorectal adenocarcinoma UB2R222I Missense Uterine endometrioid carcinoma UB2E238D Missense Lung adenocarcinoma UB2P241S Missense Uterine endometrioid carcinoma UB2T245A Missense Renal clear cell carcinoma UB2M249V Missense Uterine endometrioid carcinoma UB2E280K Missense Brain lower grade gliomaG293lowast Nonsense Colorectal cancerT300I Missense Colorectal adenocarcinomaD305H Missense Lung adenocarcinomaE308Q Missense Lung adenocarcinomaA316V Missense Stomach adenocarcinomaK319fs FS ins Head and neck squamous cell carcinomaR344G Missense Stomach adenocarcinoma UASF353I Missense Cutaneous melanoma UASL379V Missense Breast invasive carcinoma UASC396F Missense Cutaneous melanoma UASS459lowast Nonsense Uterine endometrioid carcinoma UASG469 splice Splice Glioblastoma multiforme UASR509G Missense Lung squamous cell carcinomaE510fs FS del Cutaneous melanomaR516C Missense Uterine endometrioid carcinomaA534V Missense Stomach adenocarcinomaF537L Missense Stomach adenocarcinomaE551lowast Nonsense Lung adenocarcinomaR554W Missense Colorectal cancerS582I Missense Lung adenocarcinoma UBXF585L Missense Uterine endometrioid carcinoma UBXE587lowast Nonsense Lung adenocarcinoma UBXA592V Missense Stomach adenocarcinoma UBXW610lowast Nonsense Breast invasive carcinoma UBXD611Y Missense Lung adenocarcinoma UBXE635fs FS del Brain lower grade glioma UBXP640fs FS del Cutaneous melanoma UBX

Sarcoma 19

a more accurate reference point for a leiomyosarcoma wouldhave been smooth muscle we believe that the comparison tostriated muscle is sufficient to allow the assessment of tumorspecific changes We have measured RNA DNA and proteinwith various assays Similar assessments in the future shouldalso include chromosome analysis for possible translocations

In the first-line defense against cancer the gradualreplacement of conventional chemotherapy with molecularlytargeted agents has opened the possibility to tailor drug treat-ments to particular tumors This transition necessitates thecharacterization of the molecular lesions that are causativefor the transformation of healthy cells into cancerous cellsbecause drugs need to be matched with the underlying carci-nogenic defect to be effective An additional caveat causedby unique genetic changes in the primary tumor can affectdrug transport and metabolism and needs to be taken intoconsideration In this case the RNA levels for genes that regu-late transport andmetabolismwere extensively skewed in thetumor tissue as compared to muscle and bone (see Table 3)implying potential challenges to chemotherapy An advancedmolecular treatment strategy for cancer will rely on themolecular definition of drug target drug transport and drugmetabolism pharmacogenetics in the primary tumor Con-secutive to cancer dissemination it will also require adjust-ments to account for the genetic changes in the metastases

The cost of health care has been under increasing scrutinyThe treatment of cancer patients is expensive in particularwhen hospitalization is required and further in cases ofend-of-life care Avoidable expenditures are generated bysuboptimal treatment decisions that result in low efficacy orhigh toxicity of anticancer regimens Molecular medicine hasthe potential to preempt those problems and reduce wastefulspending Yet it requires the upfront cost of molecular cancerexamination The analysis performed in this study requiredabout $11000- in nonsalary expenses to perform While ithas not identified a drug target it has specified possibleconfines for drug treatment The costs for molecular analysisneed to be weighed against the societal cost derived fromlost productivity in the workforce disrupted lives of familiesand premature deaths While currently limited drug optionsconstitute the major constraint to the approach taken herethe foreseeable future will bring an increasing spectrum ofmolecularly targeted drugs along with faster and cheapertechnologies for the molecular assessment of cancers Theywill facilitate the clinical translation of our approach

Consent

The patient provided informed consent

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by DOD Grant PR094070under University of Cincinnati IRB protocol 04-01-29-01The

author is grateful to Dr Rhett Kovall for helping with thestructural analysis of FAF1

References

[1] G F Weber Molecular Mechanisms of Cancer Springer Dor-drecht The Netherlands 2007

[2] N G Sanerkin ldquoPrimary leiomyosarcoma of the bone and itscomparison with fibrosarcomardquoCancer vol 44 no 4 pp 1375ndash1387 1979

[3] J M Weaver and J A Abraham ldquoLeiomyosarcoma of the Boneand Soft Tissue A Reviewrdquo httpsarcomahelporglearningcenterleiomyosarcomahtml

[4] M A Depristo E Banks R Poplin et al ldquoA framework forvariation discovery and genotyping using next-generationDNAsequencing datardquo Nature Genetics vol 43 no 5 pp 491ndash5012011

[5] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[6] C W Menges D A Altomare and J R Testa ldquoFAS-associatedfactor 1 (FAF1) diverse functions and implications for oncoge-nesisrdquo Cell Cycle vol 8 no 16 pp 2528ndash2534 2009

[7] M Kim J H Lee S Y Lee E Kim and J Chung ldquoCaspar asuppressor of antibacterial immunity in Drosophilardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 44 pp 16358ndash16363 2006

[8] J Song K P Joon J-J Lee et al ldquoStructure and interaction ofubiquitin-associated domain of human Fas-associated factor 1rdquoProtein Science vol 18 no 11 pp 2265ndash2276 2009

[9] P H OrsquoFarrell ldquoHigh resolution two dimensional electrophore-sis of proteinsrdquo Journal of Biological Chemistry vol 250 no 10pp 4007ndash4021 1975

[10] A Burgess-Cassler J J Johansen D A Santek J R Ideand N C Kendrick ldquoComputerized quantitative analysis ofCoomassie-Blue-stained serum proteins separated by two-dimensional electrophoresisrdquo Clinical Chemistry vol 35 no 12pp 2297ndash2304 1989

[11] B R Oakley D R Kirsch and N RMorris ldquoA simplified ultra-sensitive silver stain for detecting proteins in polyacrylamidegelsrdquo Analytical Biochemistry vol 105 no 2 pp 361ndash363 1980

[12] K Chu X Niu and L T Williams ldquoA Fas-associated proteinfactor FAF1 potentiates Fas-mediated apoptosisrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 25 pp 11894ndash11898 1995

[13] B Rikhof S de Jong A J H Suurmeijer C Meijer and W TA van der Graaf ldquoThe insulin-like growth factor system andsarcomasrdquoThe Journal of Pathology vol 217 no 4 pp 469ndash4822009

[14] RGirling J F Partridge L R Bandara et al ldquoAnew componentof the transcription factor DRTF1E2Frdquo Nature vol 362 no6415 pp 83ndash87 1993

[15] F Mercurio and M Karin ldquoTranscription factors AP-3 andAP-2 interact with the SV40 enhancer in a mutually exclusivemannerrdquo EMBO Journal vol 8 no 5 pp 1455ndash1460 1989

[16] R Bassel-Duby M D Hernandez M A Gonzalez J KKrueger and R S Williams ldquoA 40-kilodalton protein bindsspecifically to an upstream sequence element essential for mus-cle-specific transcription of the human myoglobin promoterrdquoMolecular and Cellular Biology vol 12 no 11 pp 5024ndash50321992

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 19: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

Sarcoma 19

a more accurate reference point for a leiomyosarcoma wouldhave been smooth muscle we believe that the comparison tostriated muscle is sufficient to allow the assessment of tumorspecific changes We have measured RNA DNA and proteinwith various assays Similar assessments in the future shouldalso include chromosome analysis for possible translocations

In the first-line defense against cancer the gradualreplacement of conventional chemotherapy with molecularlytargeted agents has opened the possibility to tailor drug treat-ments to particular tumors This transition necessitates thecharacterization of the molecular lesions that are causativefor the transformation of healthy cells into cancerous cellsbecause drugs need to be matched with the underlying carci-nogenic defect to be effective An additional caveat causedby unique genetic changes in the primary tumor can affectdrug transport and metabolism and needs to be taken intoconsideration In this case the RNA levels for genes that regu-late transport andmetabolismwere extensively skewed in thetumor tissue as compared to muscle and bone (see Table 3)implying potential challenges to chemotherapy An advancedmolecular treatment strategy for cancer will rely on themolecular definition of drug target drug transport and drugmetabolism pharmacogenetics in the primary tumor Con-secutive to cancer dissemination it will also require adjust-ments to account for the genetic changes in the metastases

The cost of health care has been under increasing scrutinyThe treatment of cancer patients is expensive in particularwhen hospitalization is required and further in cases ofend-of-life care Avoidable expenditures are generated bysuboptimal treatment decisions that result in low efficacy orhigh toxicity of anticancer regimens Molecular medicine hasthe potential to preempt those problems and reduce wastefulspending Yet it requires the upfront cost of molecular cancerexamination The analysis performed in this study requiredabout $11000- in nonsalary expenses to perform While ithas not identified a drug target it has specified possibleconfines for drug treatment The costs for molecular analysisneed to be weighed against the societal cost derived fromlost productivity in the workforce disrupted lives of familiesand premature deaths While currently limited drug optionsconstitute the major constraint to the approach taken herethe foreseeable future will bring an increasing spectrum ofmolecularly targeted drugs along with faster and cheapertechnologies for the molecular assessment of cancers Theywill facilitate the clinical translation of our approach

Consent

The patient provided informed consent

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by DOD Grant PR094070under University of Cincinnati IRB protocol 04-01-29-01The

author is grateful to Dr Rhett Kovall for helping with thestructural analysis of FAF1

References

[1] G F Weber Molecular Mechanisms of Cancer Springer Dor-drecht The Netherlands 2007

[2] N G Sanerkin ldquoPrimary leiomyosarcoma of the bone and itscomparison with fibrosarcomardquoCancer vol 44 no 4 pp 1375ndash1387 1979

[3] J M Weaver and J A Abraham ldquoLeiomyosarcoma of the Boneand Soft Tissue A Reviewrdquo httpsarcomahelporglearningcenterleiomyosarcomahtml

[4] M A Depristo E Banks R Poplin et al ldquoA framework forvariation discovery and genotyping using next-generationDNAsequencing datardquo Nature Genetics vol 43 no 5 pp 491ndash5012011

[5] H Li and R Durbin ldquoFast and accurate short read alignmentwith Burrows-Wheeler transformrdquo Bioinformatics vol 25 no14 pp 1754ndash1760 2009

[6] C W Menges D A Altomare and J R Testa ldquoFAS-associatedfactor 1 (FAF1) diverse functions and implications for oncoge-nesisrdquo Cell Cycle vol 8 no 16 pp 2528ndash2534 2009

[7] M Kim J H Lee S Y Lee E Kim and J Chung ldquoCaspar asuppressor of antibacterial immunity in Drosophilardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 44 pp 16358ndash16363 2006

[8] J Song K P Joon J-J Lee et al ldquoStructure and interaction ofubiquitin-associated domain of human Fas-associated factor 1rdquoProtein Science vol 18 no 11 pp 2265ndash2276 2009

[9] P H OrsquoFarrell ldquoHigh resolution two dimensional electrophore-sis of proteinsrdquo Journal of Biological Chemistry vol 250 no 10pp 4007ndash4021 1975

[10] A Burgess-Cassler J J Johansen D A Santek J R Ideand N C Kendrick ldquoComputerized quantitative analysis ofCoomassie-Blue-stained serum proteins separated by two-dimensional electrophoresisrdquo Clinical Chemistry vol 35 no 12pp 2297ndash2304 1989

[11] B R Oakley D R Kirsch and N RMorris ldquoA simplified ultra-sensitive silver stain for detecting proteins in polyacrylamidegelsrdquo Analytical Biochemistry vol 105 no 2 pp 361ndash363 1980

[12] K Chu X Niu and L T Williams ldquoA Fas-associated proteinfactor FAF1 potentiates Fas-mediated apoptosisrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 25 pp 11894ndash11898 1995

[13] B Rikhof S de Jong A J H Suurmeijer C Meijer and W TA van der Graaf ldquoThe insulin-like growth factor system andsarcomasrdquoThe Journal of Pathology vol 217 no 4 pp 469ndash4822009

[14] RGirling J F Partridge L R Bandara et al ldquoAnew componentof the transcription factor DRTF1E2Frdquo Nature vol 362 no6415 pp 83ndash87 1993

[15] F Mercurio and M Karin ldquoTranscription factors AP-3 andAP-2 interact with the SV40 enhancer in a mutually exclusivemannerrdquo EMBO Journal vol 8 no 5 pp 1455ndash1460 1989

[16] R Bassel-Duby M D Hernandez M A Gonzalez J KKrueger and R S Williams ldquoA 40-kilodalton protein bindsspecifically to an upstream sequence element essential for mus-cle-specific transcription of the human myoglobin promoterrdquoMolecular and Cellular Biology vol 12 no 11 pp 5024ndash50321992

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 20: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

20 Sarcoma

[17] K Yamada T Tanaka and T Noguchi ldquoCharacterization andpurification of carbohydrate response element-binding proteinof the rat L-type pyruvate kinase gene promoterrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp44ndash49 1999

[18] G Guan G Jiang R L Koch and I Shechter ldquoMolecularcloning and functional analysis of the promoter of the humansqualene synthase generdquo The Journal of Biological Chemistryvol 270 no 37 pp 21958ndash21965 1995

[19] T Jordan I Hanson D Zaletayev et al ldquoThe human PAX6 geneis mutated in two patients with aniridiardquoNature Genetics vol 1no 5 pp 328ndash332 1992

[20] F Zhou L Zhang K Gong et al ldquoLEF-1 activates the transcrip-tion of E2F1rdquo Biochemical and Biophysical Research Communi-cations vol 365 no 1 pp 149ndash153 2008

[21] L Zhang F Zhou T van Laar J Zhang H van Dam and PTen Dijke ldquoFas-associated factor 1 antagonizes Wnt signalingby promoting 120573-catenin degradationrdquo Molecular Biology of theCell vol 22 no 9 pp 1617ndash1624 2011

[22] F K Noubissi I Elcheva N Bhatia et al ldquoCRD-BP mediatesstabilization of 120573TrCP1 and c-myc mRNA in response to 120573-catenin signallingrdquoNature vol 441 no 7095 pp 898ndash901 2006

[23] I Elcheva R S Tarapore N Bhatia and V S SpiegelmanldquoOverexpression of mRNA-binding protein CRD-BP in malig-nant melanomasrdquo Oncogene vol 27 no 37 pp 5069ndash50742008

[24] C H Lin T Ji C F Chen and B H Hoang ldquoWnt signaling inosteosarcomardquo in Current Advances in Osteosarcoma vol 804of Advances in Experimental Medicine and Biology pp 33ndash45Springer 2014

[25] S M Kim H Myoung P H Choung M J Kim S K Leeand J H Lee ldquoMetastatic leiomyosarcoma in the oral cavitycase report with protein expression profilesrdquo Journal of Cranio-Maxillofacial Surgery vol 37 no 8 pp 454ndash460 2009

[26] A Hrzenjak M Dieber-Rotheneder F Moinfar E Petru andK Zatloukal ldquoMolecular mechanisms of endometrial stromalsarcoma and undifferentiated endometrial sarcoma as premisesfor new therapeutic strategiesrdquoCancer Letters vol 354 no 1 pp21ndash27 2014

[27] H M Mohan C M Aherne A C Rogers A W Baird DC Winter and E P Murphy ldquoMolecular pathways the roleof NR4A orphan nuclear receptors in cancerrdquo Clinical CancerResearch vol 18 no 12 pp 3223ndash3228 2012

[28] G Zhuang W Song K Amato et al ldquoEffects of cancer-asso-ciated EPHA3mutations on lung cancerrdquo Journal of theNationalCancer Institute vol 104 no 15 pp 1182ndash1197 2012

[29] J-J Lee YM Kim J Jeong D S Bae andK-J Lee ldquoUbiquitin-associated (UBA) domain in human fas associated factor 1inhibits tumor formation by promoting Hsp70 degradationrdquoPLoS ONE vol 7 no 8 Article ID e40361 2012

[30] S Zheng J Fu R Vegesna et al ldquoA survey of intragenic break-points in glioblastoma identifies a distinct subset associatedwith poor survivalrdquo Genes and Development vol 27 no 13 pp1462ndash1472 2013

[31] D Carvalho A Mackay L Bjerke et al ldquoThe prognostic roleof intragenic copy number breakpoints and identification ofnovel fusion genes in paediatric high grade gliomardquoActaNeuro-pathologica Communications vol 2 no 1 p 23 2014

[32] P L Hyland S-W Lin N Hu et al ldquoGenetic variants in fas sig-naling pathway genes and risk of gastric cancerrdquo InternationalJournal of Cancer vol 134 no 4 pp 822ndash831 2014

[33] Y-F Li C-P Yu S-TWuM-S Dai and H-S Lee ldquoMalignantmesenchymal tumor with leiomyosarcoma rhabdomyosar-coma chondrosarcoma and osteosarcoma differentiation casereport and literature reviewrdquoDiagnostic Pathology vol 6 article35 2011

[34] M Kefeli S Baris O Aydin L Yildiz S Yamak and BKandemir ldquoAn unusual case of an osteosarcoma arising in aleiomyoma of the uterusrdquo Annals of Saudi Medicine vol 32 no5 pp 544ndash546 2012

[35] C Feeley P Gallagher and D Lang ldquoOsteosarcoma arising ina metastatic leiomyosarcomardquoHistopathology vol 46 no 1 pp111ndash113 2005

[36] F Grabellus S-Y Sheu B Schmidt et al ldquoRecurrent high-grade leiomyosarcoma with heterologous osteosarcomatousdifferentiationrdquoVirchowsArchiv vol 448 no 1 pp 85ndash89 2006

[37] TAnhTran andRWHolloway ldquoMetastatic leiomyosarcomaofthe uterus with heterologous differentiation to malignant mes-enchymomardquo International Journal of Gynecological Pathologyvol 31 no 5 pp 453ndash457 2012

[38] C H Bush J D Reith and S S Spanier ldquoMineralization inmusculoskeletal leiomyosarcoma radiologic-pathologic corre-lationrdquo The American Journal of Roentgenology vol 180 no 1pp 109ndash113 2003

[39] D Osuna and E de Alava ldquoMolecular pathology of sarcomasrdquoReviews on Recent Clinical Trials vol 4 no 1 pp 12ndash26 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 21: Research Article Molecular Analysis of a Recurrent Sarcoma ...downloads.hindawi.com/journals/sarcoma/2015/839182.pdf · breast cancer DNA samples served as nonsarcoma control. e assay

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Sarcoma Uterino: Patología. Estadificación. … · Sarcoma Uterino: Patología. Estadificación. Tratamiento de la enfermedad localizada ^Sarcoma Uterino: Adyuvancia. Enfermedad

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Understanding retroperitoneal sarcoma · 2 • Understanding retroperitoneal sarcomas sarcomaorgu Understanding retroperitoneal sarcomas • 3 What is retroperitoneal sarcoma? Sarcoma

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Ewings sarcoma

Ewings sarcoma

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Extremity Sarcoma

Extremity Sarcoma

Health & Medicine
Ewing sarcoma

Ewing sarcoma

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Successful Salvage and Long-Term Survival after Recurrent ...downloads.hindawi.com › journals › sarcoma › 2007 › 053549.pdf · in a 43-year-old man who initially presented

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ATP-Based Chemotherapy Response Assay in Primary or … · 2019-09-04 · ATP-Based Chemotherapy Response Assay in Primary or . Recurrent Ovarian and Peritoneal Cancer. Maria Lee,

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Robert S. Benjamin, M.D. Department of Sarcoma Medical Oncology The SARCOMA Center Department of Sarcoma Medical Oncology The SARCOMA Center Texas Medical

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Programmed cell death-1 blockade in recurrent disseminated ......Ewing sarcoma (EWS) is a poorly differentiated, aggressive malignant small blue round cell tumour of the bone and soft

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Genetic risk in sarcoma INTERNATIONAL SARCOMA KINDRED STUDY

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Pre-operative Imatinib for metastatic, recurrent and locally advanced GISTs E. Efthimiou, S Mudan E. Efthimiou, S Mudan On behalf of the Sarcoma Group

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Implementation of a Sensitive Troponin I Assay and Risk of Recurrent Myocardial Infarction and Death in Patients With Suspected Acute Coronary Syndrome

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Sarcoma Uterino

Sarcoma Uterino

Health & Medicine
Retroperitoneal Sarcoma

Retroperitoneal Sarcoma

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Uterine sarcoma

Uterine sarcoma

Health & Medicine
Sarcoma Oncology

Sarcoma Oncology

Healthcare
An Unusual Association of Chronic Recurrent Multifocal ... · with chronic osteomyelitis. Skin biopsy was suggestive of PG. Her QuantiFERON Gold (Interferon Gamma assay), Mantoux,

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Kaposi's sarcoma Kaposi sarcoma 1872 Moritz Kaposi Kaposi

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Oncogenic osteomalacia as a harbinger of recurrent ...downloads.hindawi.com/journals/sarcoma/1999/232749.pdf · Sarcoma(1999)3,95±99 ORIGINAL ARTICLE Oncogenic osteomalacia as a

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Molecular Analysis of a Recurrent Sarcoma Identifies a ... · ResearchArticle Molecular Analysis of a Recurrent Sarcoma Identifies a Mutation in FAF1 GeorgF.Weber CollegeofPharmacy,UniversityofCincinnati,3225EdenAvenue,Cincinnati,OH45267-0004,USA

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Sarcoma Pleomorph

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Understanding bone sarcoma · • Understanding bone sarcoma sarcoma.org.uk Understanding bone sarcoma • 3 What is bone sarcoma? Bone sarcoma is a cancer that starts in the bone

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10 Sarcoma

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Archer® FusionPlex® Sarcoma kit flyer - Illumina...The FusionPlex Sarcoma Panel is a targeted sequencing assay that simultaneously detects and identifies fusions of 26 genes associated

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Ewing's sarcoma

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