ATAD2 Overexpression Identifies Colorectal Cancer Patients with

Research ArticleATAD2 Overexpression Identifies Colorectal Cancer Patientswith Poor Prognosis and Drives Proliferation of Cancer Cells

Yang Luo Guang-Yao Ye Shao-Lan Qin Min-Hao Yu Yi-Fei Mu and Ming Zhong

Department of Gastrointestinal Surgery Renji Hospital School of Medicine Shanghai Jiao Tong University Shanghai 200127 China

Correspondence should be addressed to Ming Zhong drzhongminghotmailcom

Received 10 May 2015 Accepted 20 September 2015

Academic Editor Miroslav Zavoral

Copyright copy 2015 Yang Luo et alThis is an open access article distributed under the Creative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

ATPase family AAA domain-containing 2 (ATAD2) has been identified as a critical modulator involved in cell proliferationand invasion The purpose of this study was to explore the expression of ATAD2 in CRC tissues as well as its relationship withdegree of malignancy Data containing three independent investigations from Oncomine database demonstrated that ATAD2 isoverexpressed in CRC compared with normal tissue and similar result was also found in 32 pairs of CRC tissues by qPCR Theprotein expression of ATAD2 was examined in six CRC cell lines and 300 CRC specimensThe results showed that high expressionof ATAD2 was significantly correlated with tumor size (119875 lt 0001) serum CEA (119875 = 0012) lymph node metastasis (119875 = 0018)liver metastasis (119875 = 0025) and clinical stage (119875 = 0004) Kaplan-Meier method suggested that higher ATAD2 protein expressionsignificantly associated with the overall survival (OS) of CRC patients (119875 lt 0001) and was an independent predictor of poor OSFunctional studies showed that suppression of ATAD2 expression with siRNA could significantly inhibit the growth in SW480 andHCT116 cells These results indicated that ATAD2 could serve as a prognostic marker and a therapeutic target for CRC

1 Introduction

Colorectal cancer (CRC) is one of the most common lethalmalignancies in terms of both incidence and mortality [1]Although the diagnosis and treatment of CRC have beenimproved the efficacy of surgery and chemotherapy remainsunsatisfactory due to late diagnosis [2] Therefore newdiagnostic and treatment strategies are urgently needed forthis malignancy

ATPase family AAA domain-containing 2 (ATAD2)also known as ANCCA (AAA+ nuclear coregulator cancerassociated) is a novel member of the AAA+ ATPase family[3 4] ATAD2 contains both a bromodomain and an ATPasedomain and also maps to chromosome 8q24 that is the mostcommonly amplified region in many types of cancer [5]The especial structure of ATAD2 indicates that it is asso-ciated with genome regulation including cell proliferationdivision apoptosis and differentiation [6ndash10] Recently it isreported that aberrant expression of ATAD2 contributes tohepatocellular carcinoma proliferation andmetastasis [11 12]Studies have revealed that ATAD2 is highly expressed in

several types of tumors such as breast cancer lung cancerand gastric cancer [13ndash15]Thus ATAD2manifests oncogenicfunction and plays a significant role in cancer developmentHowever the expression of the ATAD2 protein in CRC andits significance remain uncertain

2 Methods

21 In Silico Analysis Using the Oncomine Database Todetermine the expression pattern of ATAD2 in CRC threedatasets (Kaiser Colon Hong Colorectal and Hong Colorec-tal) in Oncomine database (httpswwwoncomineorg) wereused We compared ATAD2 gene expression in CRC tissueswith normal colorectal tissues according to the standardprocedures as previously described [16]

22 Cell Culture and Transfection Five human CRC celllines (HCT116 SW480 LoVo T84 and HT29) and a normalcontrol colon cell line (HCoEpiC) were all preserved inShanghai Cancer Institute All of these cells lines werecultured in DMEMmedium (Invitrogen) supplemented with

Hindawi Publishing CorporationGastroenterology Research and PracticeVolume 2015 Article ID 936564 8 pageshttpdxdoiorg1011552015936564

2 Gastroenterology Research and Practice

10 (vv) fetal bovine serum (FBS) and 1 antibiotics at 37∘Cin a humidified incubator under 5 CO

2condition

The transfections were performed using Lipofectamine2000 (Invitrogen USA) Small interfering RNAs (siRNA)targeting ATAD2 and a negative control were obtained fromGenePharma Technology (Shanghai China) The transfec-tionwas performed according to themanufacturerrsquos protocol

23 Patients and Tissue Samples A total of 300 formalin-fixed paraffin-embedded CRC tissues were collected to per-form immunohistochemical staining from January 2005 toNovember 2014 at the Renji Hospital Shanghai Jiao TongUniversity School of Medicine China Moreover additional32 snap-frozen CRC tissues and corresponding adjacent non-cancerous tissues to isolate RNA which were also obtainedfrom Renji Hospital were enrolled in this study simultane-ously Inclusion criteria were histologically confirmed CRCand curative resection of tumor without preoperative orpostoperative adjuvant therapy Important clinical data suchas tumor location serum CEA level and clinical stage werecollected from each patientrsquos medical records The follow-uptime was calculated from the date of surgery to the date ofdeath or the last known follow-up All CRC tissue samplesin this study were obtained with patientsrsquo written informedconsent and all experiments have been approved by the ethicscommittee at local hospital

24 Real-Time Quantitative PCR Total RNA from pri-mary tumor and adjacent noncancerous tissue samples wasextracted using Trizol reagent (Takara Japan) and accord-ing to the manufacturerrsquos instructions reverse transcriptionwas performed by PrimeScript RT-PCR kit (Takara Japan)Real-time quantitative PCR (qPCR) was performed usinga 7500 real-time PCR system (Applied Biosystems IncUSA) The primers for ATAD2 were as follows forward51015840-GGAATCCCAAACCACTGGACA-31015840 reverse 51015840-GGT-AGCGTCGTCGTAAAGCACA-31015840 GAPDH mRNA wasused to standardize the relative expression of ATAD2 Theprimers for GAPDH were as follows forward 51015840-GCATTG-CCCTCAACGACCAC-31015840 reverse 51015840-CCACCACCCTGT-TGCTGTAG-31015840

25 Immunohistochemical Staining Four-micrometer-thicktissue sections were subjected to immunohistochemicalstaining with avidin-biotin-peroxidase complex systemwhich was performed as previously described [17] Tissuesections were incubated by anti-ATAD2 antibody (1 400Abcam Cambridge UK) at 4∘C overnight Immunohisto-chemical staining was scored by two independentpathologists according to intensity and percentage ofpositive cells simultaneously Staining intensity was scoredas follows 0 negative 1 weak staining 2 moderate staining3 strong staining and the percentage of positive cells wasscored on a scale of 0ndash4 (0 lt5 1 5ndash30 2 30ndash50 351ndash75 4 gt75) And the final score was designated aslow or high expression group using the percent of positivecell score times staining intensity score as follows low expressionwas defined as a total score lt 6 and high expression with atotal score ge 6

26 Western Blot Western blot was performed as previ-ously described [18] Cells were harvested after 48-hourtransfection and 40120583g of protein samples was resolvedby SDS-PAGE and transferred to PVDF membranes Themembranes were probed with the same primary antibodyused in immunostaining overnight at 4∘C and incubatedfor 1 hour with secondary antibody (Boston MA USA)The Western blotting analysis was repeated at least threetimes

27 Cell Proliferation Assay Cell viability was assessed bythe Cell Counting Kit 8 (CCK-8 Dojindo) Briefly controland treated SW480 and HCT116 cells were seeded into 96-well plates at an initial density of 3000 cellswell At eachtime point 10 120583L of CCK-8 solution was added to each welland incubated for 2 hours The absorbance was measured byscanning with amicroplate reader at 450 nmThe experimentwas repeated at least three times

28 Statistical Analysis Statistical analyses were performedby SPSS 190 (SPSS Inc Chicago USA) The expressionof ATAD2 mRNA in CRC tissues and corresponding non-cancerous tissues was analyzed with the Studentrsquos 119905-test Thedifferences of the relative absorbance value of CCK8 assaysalso were determined using Studentrsquos 119905-test The Chi-squaretest was used to analyze the relationship between ATAD2expression and clinicopathological features Survival rate wasevaluated by Kaplan-Meier method and differences betweensurvival curves were tested by the log-rank test 119875 values lessthan 005 were considered to be statistically significant

3 Results

31 ATAD2 Is Overexpressed in CRC at mRNA Level Toroundly investigate ATAD2 expression in CRC we ana-lyzed three independentmicroarray datasets fromOncominedatabase [19ndash21] The results showed that the mRNA expres-sion levels of ATAD2 were upregulated in most of CRCtissues compared with normal tissue (Figures 1(a)ndash1(c))Then 32 pairs of CRC and matched normal tissues weresubjected to qPCR Consistent with the data fromOncominedatabase ATAD2 was also overexpressed in 6875 (2232)of CRC patients at mRNA level (Figure 1(d))

32 ATAD2 Is Expressed Diversely in CRC at Protein Level Tofurther investigate the expression of ATAD2 at the proteinlevel we measured ATAD2 level in CRC cell lines andtissues The expression of ATAD2 protein was increasedin all five CRC cell lines compared with the nonmalig-nant HCoEpiC cells Moreover we found that the higherexpression of ATAD2 protein is in low differentiated CRCcell lines (SW480 HCT116 and LoVo) compared with well-differentiated cell lines (HT29 T84) (Figures 1(e)-1(f))Thenwe tested 300 CRC tissue samples by using the method ofimmunohistochemical staining and found that ATAD2 waslow expressed in the 124 (4133) of the total 300 CRCsamples while the remaining 176 (5867) samples remainedat a high expression level (Figures 2(a)ndash2(c))

Gastroenterology Research and Practice 3

10

05

00

minus05

minus10

minus15

minus20

minus25

(1) Colon (5)(2) Rectosigmoid adenocarcinoma (10)

1 2

log2

med

ian-

cent

ered

inte

nsity

(a)

55

50

45

40

35

30

25

20

15

10

05

00

(0) No value (12)(1) Colorectal carcinoma (70)

0 1

log2

med

ian-

cent

ered

inte

nsity

(b)

1 2

45

40

35

30

25

20

15

10

05

00

minus05

(1) Colorectal tissue (24)(2) Colorectal carcinoma (36)

log2

med

ian-

cent

ered

inte

nsity

(c)

80

60

40

20

0Rela

tive A

TAD2

mRN

A ex

pres

sion

leve

l

N T

P = 0001

(d)

Nonmalignantcells

Low differentiated cells

High differentiatedcells

ATAD2

GAPDH

HC

oEpi

C

SW480

HCT

116

LoVo

HT2

9

T84

(e)

HC

oEpi

C

SW480

HCT

116

LoVo

HT2

9

T84

Rela

tive A

TAD2

prot

ein

expr

essio

n le

vel 15

10

05

00

(f)

Figure 1 ATAD2 expression in CRC at mRNA and protein level (a)ndash(c) ATAD2 expression in Kaiser Colon (a) Hong Colorectal (b) andSkrzypczakColorectal (c) (d) IncreasedATAD2mRNAexpression in 32matched tumor (T) and nontumor tissues (N)was detected by qPCR(e)-(f) Western blots show the ATAD2 expression in five CRC cell lines and the nonmalignant HCoEpiC cells 119875 values were calculated bypaired 119905-test


200x 400x

ATAD2 negative expression

(a)

200x400x

ATAD2 low expression

(b)

ATAD2 high expression

200x 400x

(c)

Figure 2 ATAD2 expression in CRCwas determined by immunochemistry (a) Negative expression of ATAD2 (b) low expression of ATAD2and (c) high expression of ATAD2 Representative images are shown at 200x and 400x magnification respectively

33 Relationship between ATAD2 Expression and Correspond-ing Clinical Parameters To evaluate the clinical significanceof ATAD2 the Chi-square test was used to analyze correla-tions between ATAD2 protein expression and clinicopatho-logical parameters in CRC The results indicated that highexpression of ATAD2 inCRC tissues is closely correlatedwithtumor size (119875 lt 0001) serum CEA level (119875 = 0012) lymphnodemetastasis (119875 = 0018) livermetastasis (119875 = 0025) andclinical stage (119875 = 0004) However no statistically significantcorrelations were identified between ATAD2 expression and

other clinicopathologic characteristics including age genderand tumor location (Table 1)

34 Correlation between ATAD2 Expression and Prognosisin CRC Patients To investigate the prognostic influence ofATAD2 the overall survival (OS) rate of CRC patients wasanalyzed using Kaplan-Meier survival curves and the log-rank test The result revealed that high expression of ATAD2was inversely associated with OS for all 189 samples (119875 lt0001) (Figure 3(a)) In addition The OS of ATAD2 negative


100

80

60

40

20

0

0 20 40 60 80 100 120 140

Ove

rall

surv

ival

()

Months following tumor resection

Low expression ATAD2 (n = 89)High expression ATAD2 (n = 100)

P lt 0001P lt 0001

Overall survival rate of different in ATAD2 level

(a)

100

80

60

40

20

0

0 20 40 60 80 100 120 140

Ove

rall

surv

ival

()



P = 0002P = 00

Without lymph node metastasis

(b)

100

80

60

40

20

0

0 20 40 60 80 100 120

Ove

rall

surv

ival

()



P = 0037

With lymph node metastasis

(c)

100

80

60

40

20

0

0 20 40 60 80 100 120 140

Ove

rall

surv

ival

()



P = 0001

Without liver metastasis

(d)

100

80

60

40

20

0

0 20 40 60 80

Ove

rall

surv

ival

()



P = 0018

With liver metastasis

(e)

Figure 3 ATAD2 is correlated with overall survival rate in CRC patients (a) Kaplan-Meier survival curves show high expression level ofATAD2 is significantly correlatedwith poor survival of CRC (b)ndash(e) Correlation betweenATAD2 expression and patient survival in colorectalcancer is independent of lymphatic metastasis and liver metastasis 119875 values were calculated using the log-rank test


Table 1 Relationship betweenATAD2 expression and clinicopatho-logical features in 300 colorectal cancer patients

VariableATAD (119899)

Low High119875

119899 = 124 119899 = 176

Agele65 years 73 (2433) 97 (3233) 0518gt65 years 51 (1700) 79 (2634)

GenderMale 68 (2237) 99 (3257) 0978Female 56 (1842) 81 (2664)

Tumor sizele5 cm 78 (2600) 72 (2400)

lt0001gt5 cm 46 (1533) 104 (3467)

Tumor locationRectum 73 (2433) 103 (3433) 0873Colon 51 (1701) 73 (2433)

Serum CEAle5 ngmL 74 (2467) 79 (2633) 0012gt5 ngmL 50 (1667) 97 (3233)

Lymph node metastasisN0 53 (1767) 52 (1733) 0018N1-N2 71 (2367) 124 (4133)

Liver metastasisM0 109 (3633) 137 (4566) 0025M1 15 (500) 39 (1300)

Clinical stageI 17 (567) 12 (400)

0004II 23 (767) 16 (533)III 69 (2300) 109 (3633)IV 15 (500) 39 (1300)

group was distinctly better than that of the ATAD2 positiveone for samples separated according to lymphatic metastasisand liver metastasis (Figures 3(b)ndash3(e))

Furthermore univariate and multivariate analyses wereperformed to confirm the possibility of ATAD2 used as anindependent risk factor for poor prognosis in the 182 casesof CRC Univariate Cox regression analyses showed thatATAD2 expression tumor size serum CEA liver metastasisand clinical stage were significantly associated with overallsurvival (OS) (Table 2)Themultivariate Cox regression anal-ysis confirmed ATAD2 expression tumor size and clinicalstage as independent predictors of the OS in CRC patients(Table 2)

35 Effect of ATAD2 on CRC Cell Proliferation In Vitro Tobetter understand the biological function of ATAD2 wetransfected siRNAs-ATAD2 into SW480 and HCT116 cellsand the expression levels of endogenous ATAD2 proteinswere significantly suppressed by Western blot (Figure 4(a))Moreover we found that knockdown of ATAD2 resulted ina significant decrease in cell viability measured by CCK-8compared with control (Figures 4(b)-4(c))

si-co

ntro

l

si-AT

AD2

si-co

ntro

l

si-AT

AD2

ATAD2

GAPDH

SW480 HCT116

(a)

20

15

10

05

00

CCK8

-OD450

0 1 2 3 4 5

DayssiRNA-controlsiRNA-ATAD2

lowastlowast

lowastlowastlowast

lowastlowastlowast

SW480

(b)

lowastlowast

lowastlowastlowast

lowastlowastlowast

20

15

10

05

00

CCK8

-OD450

0 1 2 3 4 5


HCT116

(c)

Figure 4 Effect of siRNA-ATAD2 on the proliferation of SW480and HCT116 cells (a) ATAD2 knockdown efficiency was confirmedby qPCR in SW480 and HCT116 cells (b)-(c) siRNA-ATAD2decreased cell viability measured by CCK8 assays 119875 values werecalculated by Studentrsquos 119905-test (lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001) and theexperiment was repeated at least three times


Table 2 Univariate and multivariate analyses showing the overall survival in colorectal cancer

Prognostic parameter Univariate analysis Multivariate analysisHR 95 CI 119875 value HR 95 CI 119875 value

ATAD2 (high versus low) 2272 1479ndash3491 0000 1762 1113ndash2790 0016Age (gt65 versus le65) 1393 0911ndash2132 0127 mdash mdash mdashGender (male versus female) 1362 0905ndash2048 0139 mdash mdash mdashTumor size (gt5 cm versus le5 cm) 1842 1201ndash2825 0005 1698 1075ndash2681 0023Tumor location (colon versus rectum) 0770 0497ndash1194 0243 mdash mdash mdashSerum CEA (gt5 ngmL versus le5 ngmL) 1628 1063ndash2493 0025 1497 0979ndash2291 0063Lymph node metastasis (present versus absent) 1686 1099ndash2588 0017 1038 0644ndash1673 0879Liver metastasis (present versus absent) 3308 1777ndash6151 0000 1710 0777ndash3764 0183Clinical stage (I vs II vs III vs IV) 1983 1549ndash2539 0000 1611 1168ndash2221 0004HR hazard ratio CI confidence interval The bold number represents the 119875 values with significant differences

4 Discussion

Studies focused on human clinical specimens and geneticallyengineered mouse models of CRC have led to a better under-standing of this genetic malignancy [22] ATAD2 throughdifferent mechanisms broadly participated in various tumortypes such as prostate cancer lung cancer breast cancer andovarian cancer [4 13 14 23] Previous studies indicated thatATAD2 directly interacted with the oncogene AIB1ACTRand played an important role in the recruitment of ER120572 topromote the expression of genes driving cancer cell prolifer-ation [24] Furthermore the ATPase domain of ATAD2 alsoenhanced the E2 induction of cyclin D1 and E2F1 expression[4] In addition ATAD2 was also directly associated withAR to activate AR-mediated transcription and was requiredto regulate the expression of androgen-induced genes thatcontrolled cancer cell proliferation and survival [4]

In the current studywe firstly assessedATAD2 expressionat mRNA level Both data from Oncomine database andour results showed that most primary CRC tissues exhibitedsignificantly higher mRNA expression of ATAD2 than theirmatched normal tissues This tendency was then confirmedby immunohistochemistry where 176 of 300 (5867) CRCtissues were found to have high protein expression ofATAD2 Previous studies had demonstrated that overex-pression of ATAD2 at the transcriptional level via miR-372regulation promotes tumor malignancy and consequentlyresults in poor outcome in liver adenocarcinoma patients[11] Our results revealed similar phenomenon that ATAD2has positive correlation with serum CEA level lymph nodemetastasis distant metastasis and clinical stage in CRCBesides patients with a high level of ATAD2 expression hadsignificantly shorter survival times compared to those witha low level of ATAD2 expression Furthermore univariateanalysis showed that elevated ATAD2 expression was signifi-cantly associated with the OS of CRC patients multivariateanalysis demonstrated that ATAD2 expression tumor sizeand clinical stage are independent risk factors for the prog-nosis of CRC patients Collectively these results suggest thatATAD2 may be involved in the initiation and progressionof CRC Finally we found that downexpression of ATAD2could dramatically suppress the proliferation of SW480 and

HCT116 cells which is in keeping with the previous studieson the other cancer cells However its underlying molecularmechanisms need to be further investigated

In conclusion our study demonstrated that ATAD2expression was increased in CRC tissues compared to adja-cent normal tissues and might be associated with patho-logical development of CRC In addition we found thathigh expression of ATAD2 could serve as an independentprognostic factor for CRC patients Therefore ATAD2 maybe an important clinical marker of therapy for CRC

Conflict of Interests

The authors declared that they have no competing interests

Authorsrsquo Contribution

Yang Luo Guang-Yao Ye and Shao-Lan Qin contributedequally to this work

References

[1] L-A Torre F Bray R-L Siegel et al ldquoGlobal cancer statistics2012rdquo Journal of Cancer Research and Clinical Oncology vol 65no 5 pp 87ndash108 2015

[2] Y-C Lee T-C Yin Y-T Chen et al ldquoHigh expression ofphospho-H2AX predicts a poor prognosis in colorectal cancerrdquoAnticancer Research vol 35 no 4 pp 2447ndash2453 2015

[3] M Ciro E Prosperini M Quarto et al ldquoATAD2 is a novelcofactor for MYC overexpressed and amplified in aggressivetumorsrdquo Cancer Research vol 69 no 21 pp 8491ndash8498 2009

[4] J X Zou L Guo A S Revenko et al ldquoAndrogen-induced coac-tivator ANCCA mediates specific androgen receptor signalingin prostate cancerrdquo Cancer Research vol 69 no 8 pp 3339ndash3346 2009

[5] R Beroukhim C H Mermel D Porter et al ldquoThe landscape ofsomatic copy-number alteration across human cancersrdquoNaturevol 463 no 7283 pp 899ndash905 2010

[6] A A Alizadeh M B Elsen R E Davis et al ldquoDistinct typesof diffuse large B-cell lymphoma identified by gene expressionprofilingrdquo Nature vol 403 no 6769 pp 503ndash511 2000


[7] X-J Ma R Salunga J T Tuggle et al ldquoGene expression profilesof human breast cancer progressionrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 100 no10 pp 5974ndash5979 2003

[8] C Caron C Lestrat S Marsal et al ldquoFunctional characteriza-tion of ATAD2 as a new cancertestis factor and a predictor ofpoor prognosis in breast and lung cancersrdquo Oncogene vol 29no 37 pp 5171ndash5181 2010

[9] S V Fernandez F M Robertson J Pei et al ldquoInflammatorybreast cancer (IBC) clues for targeted therapiesrdquo Breast CancerResearch and Treatment vol 140 no 1 pp 23ndash33 2013

[10] J-X Zou Z Duan J Wang et al ldquoKinesin family deregulationcoordinated by bromodomain protein ANCCA and histonemethyltransferase MLL for breast cancer cell growth survivaland tamoxifen resistancerdquo Molecular Cancer Research vol 12no 4 pp 539ndash549 2014

[11] G Wu H Liu H He et al ldquomiR-372 down-regulates theoncogene ATAD2 to influence hepatocellular carcinoma prolif-eration and metastasisrdquo BMC Cancer vol 14 article 107 2014

[12] G Wu X Lu Y Wang et al ldquoEpigenetic high regulation ofATAD2 regulates the Hh pathway in human hepatocellularcarcinomardquo International Journal of Oncology vol 45 no 1 pp351ndash361 2014

[13] E-V Kalashnikova A-S Revenko A-T Gemo et alldquoANCCAATAD2 overexpression identifies breast cancerpatients with poor prognosis acting to drive proliferation andsurvival of triple-negative cells through control of B-Myb andEZH2rdquo Cancer Research vol 70 no 22 pp 9402ndash9412 2010

[14] Y Zhang Y Sun Y Li et al ldquoANCCA protein expressionis a novel independent poor prognostic marker in surgicallyresected lung adenocarcinomardquo Annals of Surgical Oncologyvol 20 supplement 3 pp S577ndashS582 2013

[15] H Murakami S Ito H Tanaka E Kondo Y Kodera and HNakanishi ldquoEstablishment of new intraperitoneal paclitaxel-resistant gastric cancer cell lines and comprehensive geneexpression analysisrdquo Anticancer Research vol 33 no 10 pp4299ndash4307 2013

[16] D R Rhodes S Kalyana-Sundaram V Mahavisno et alldquoOncomine 30 genes pathways and networks in a collectionof 18000 cancer gene expression profilesrdquo Neoplasia vol 9 no2 pp 166ndash180 2007

[17] K Sugano K Maeda H Ohtani et al ldquoExpression of xCT asa predictor of disease recurrence in patients with colorectalcancerrdquo Anticancer Research vol 35 no 2 pp 677ndash682 2015

[18] S Kaiser Y-K Park J L Franklin et al ldquoTranscriptionalrecapitulation and subversion of embryonic colon developmentby mouse colon tumor models and human colon cancerrdquoGenome Biology vol 8 no 7 article R131 2007

[19] Y Hong T Downey K W Eu P K Koh and P Y CheahldquoA lsquometastasis-pronersquo signature for early-stage mismatch-repairproficient sporadic colorectal cancer patients and its impli-cations for possible therapeuticsrdquo Clinical and ExperimentalMetastasis vol 27 no 2 pp 83ndash90 2010

[20] M Skrzypczak K Goryca T Rubel et al ldquoModeling onco-genic signaling in colon tumors by multidirectional analysesof microarray data directed for maximization of analyticalreliabilityrdquo PLoS ONE vol 5 no 10 Article ID e13091 2010

[21] F Fan S Bellister J Lu et al ldquoThe requirement for freshlyisolated human colorectal cancer (CRC) cells in isolating CRCstem cellsrdquo British Journal of Cancer vol 112 no 3 pp 539ndash5462015

[22] W-N Wan Y-X Zhang X-M Wang et al ldquoATAD2 is highlyexpressed in ovarian carcinomas and indicates poor prognosisrdquoAsian Pacific Journal of Cancer Prevention vol 15 no 6 pp2777ndash2783 2014

[23] E-Y C Hsia E-V Kalashnikova A-S Revenko J X ZouA D Borowsky and H-W Chen ldquoDeregulated E2F and theAAA+ coregulator ANCCA drive proto-oncogene ACTRAIB1overexpression in breast cancerrdquo Molecular Cancer Researchvol 8 no 2 pp 183ndash193 2010

[24] A-S Revenko E-V Kalashnikova A-T Gemo J X Zouand H-W Chen ldquoChromatin loading of E2F-MLL complex bycancer-associated coregulator ANCCA via reading a specifichistone markrdquo Molecular and Cellular Biology vol 30 no 22pp 5260ndash5272 2010

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


10 (vv) fetal bovine serum (FBS) and 1 antibiotics at 37∘Cin a humidified incubator under 5 CO

2condition

The transfections were performed using Lipofectamine2000 (Invitrogen USA) Small interfering RNAs (siRNA)targeting ATAD2 and a negative control were obtained fromGenePharma Technology (Shanghai China) The transfec-tionwas performed according to themanufacturerrsquos protocol

23 Patients and Tissue Samples A total of 300 formalin-fixed paraffin-embedded CRC tissues were collected to per-form immunohistochemical staining from January 2005 toNovember 2014 at the Renji Hospital Shanghai Jiao TongUniversity School of Medicine China Moreover additional32 snap-frozen CRC tissues and corresponding adjacent non-cancerous tissues to isolate RNA which were also obtainedfrom Renji Hospital were enrolled in this study simultane-ously Inclusion criteria were histologically confirmed CRCand curative resection of tumor without preoperative orpostoperative adjuvant therapy Important clinical data suchas tumor location serum CEA level and clinical stage werecollected from each patientrsquos medical records The follow-uptime was calculated from the date of surgery to the date ofdeath or the last known follow-up All CRC tissue samplesin this study were obtained with patientsrsquo written informedconsent and all experiments have been approved by the ethicscommittee at local hospital

24 Real-Time Quantitative PCR Total RNA from pri-mary tumor and adjacent noncancerous tissue samples wasextracted using Trizol reagent (Takara Japan) and accord-ing to the manufacturerrsquos instructions reverse transcriptionwas performed by PrimeScript RT-PCR kit (Takara Japan)Real-time quantitative PCR (qPCR) was performed usinga 7500 real-time PCR system (Applied Biosystems IncUSA) The primers for ATAD2 were as follows forward51015840-GGAATCCCAAACCACTGGACA-31015840 reverse 51015840-GGT-AGCGTCGTCGTAAAGCACA-31015840 GAPDH mRNA wasused to standardize the relative expression of ATAD2 Theprimers for GAPDH were as follows forward 51015840-GCATTG-CCCTCAACGACCAC-31015840 reverse 51015840-CCACCACCCTGT-TGCTGTAG-31015840

25 Immunohistochemical Staining Four-micrometer-thicktissue sections were subjected to immunohistochemicalstaining with avidin-biotin-peroxidase complex systemwhich was performed as previously described [17] Tissuesections were incubated by anti-ATAD2 antibody (1 400Abcam Cambridge UK) at 4∘C overnight Immunohisto-chemical staining was scored by two independentpathologists according to intensity and percentage ofpositive cells simultaneously Staining intensity was scoredas follows 0 negative 1 weak staining 2 moderate staining3 strong staining and the percentage of positive cells wasscored on a scale of 0ndash4 (0 lt5 1 5ndash30 2 30ndash50 351ndash75 4 gt75) And the final score was designated aslow or high expression group using the percent of positivecell score times staining intensity score as follows low expressionwas defined as a total score lt 6 and high expression with atotal score ge 6

26 Western Blot Western blot was performed as previ-ously described [18] Cells were harvested after 48-hourtransfection and 40120583g of protein samples was resolvedby SDS-PAGE and transferred to PVDF membranes Themembranes were probed with the same primary antibodyused in immunostaining overnight at 4∘C and incubatedfor 1 hour with secondary antibody (Boston MA USA)The Western blotting analysis was repeated at least threetimes

27 Cell Proliferation Assay Cell viability was assessed bythe Cell Counting Kit 8 (CCK-8 Dojindo) Briefly controland treated SW480 and HCT116 cells were seeded into 96-well plates at an initial density of 3000 cellswell At eachtime point 10 120583L of CCK-8 solution was added to each welland incubated for 2 hours The absorbance was measured byscanning with amicroplate reader at 450 nmThe experimentwas repeated at least three times

28 Statistical Analysis Statistical analyses were performedby SPSS 190 (SPSS Inc Chicago USA) The expressionof ATAD2 mRNA in CRC tissues and corresponding non-cancerous tissues was analyzed with the Studentrsquos 119905-test Thedifferences of the relative absorbance value of CCK8 assaysalso were determined using Studentrsquos 119905-test The Chi-squaretest was used to analyze the relationship between ATAD2expression and clinicopathological features Survival rate wasevaluated by Kaplan-Meier method and differences betweensurvival curves were tested by the log-rank test 119875 values lessthan 005 were considered to be statistically significant

3 Results

31 ATAD2 Is Overexpressed in CRC at mRNA Level Toroundly investigate ATAD2 expression in CRC we ana-lyzed three independentmicroarray datasets fromOncominedatabase [19ndash21] The results showed that the mRNA expres-sion levels of ATAD2 were upregulated in most of CRCtissues compared with normal tissue (Figures 1(a)ndash1(c))Then 32 pairs of CRC and matched normal tissues weresubjected to qPCR Consistent with the data fromOncominedatabase ATAD2 was also overexpressed in 6875 (2232)of CRC patients at mRNA level (Figure 1(d))

32 ATAD2 Is Expressed Diversely in CRC at Protein Level Tofurther investigate the expression of ATAD2 at the proteinlevel we measured ATAD2 level in CRC cell lines andtissues The expression of ATAD2 protein was increasedin all five CRC cell lines compared with the nonmalig-nant HCoEpiC cells Moreover we found that the higherexpression of ATAD2 protein is in low differentiated CRCcell lines (SW480 HCT116 and LoVo) compared with well-differentiated cell lines (HT29 T84) (Figures 1(e)-1(f))Thenwe tested 300 CRC tissue samples by using the method ofimmunohistochemical staining and found that ATAD2 waslow expressed in the 124 (4133) of the total 300 CRCsamples while the remaining 176 (5867) samples remainedat a high expression level (Figures 2(a)ndash2(c))


10

05

00

minus05

minus10

minus15

minus20

minus25


1 2

log2

med

ian-

cent

ered

inte

nsity

(a)

55

50

45

40

35

30

25

20

15

10

05

00


0 1

log2

med

ian-

cent

ered

inte

nsity

(b)

1 2

45

40

35

30

25

20

15

10

05

00

minus05


log2

med

ian-

cent

ered

inte

nsity

(c)

80

60

40

20

0Rela

tive A

TAD2

mRN

A ex

pres

sion

leve

l

N T

P = 0001

(d)

Nonmalignantcells



ATAD2

GAPDH

HC

oEpi

C

SW480

HCT

116

LoVo

HT2

9

T84

(e)

HC

oEpi

C

SW480

HCT

116

LoVo

HT2

9

T84

Rela

tive A

TAD2

prot

ein

expr

essio

n le

vel 15

10

05

00

(f)



200x 400x


(a)

200x400x


(b)


200x 400x

(c)






100

80

60

40

20

0

0 20 40 60 80 100 120 140

Ove

rall

surv

ival

()



P lt 0001P lt 0001


(a)

100

80

60

40

20

0

0 20 40 60 80 100 120 140

Ove

rall

surv

ival

()



P = 0002P = 00


(b)

100

80

60

40

20

0

0 20 40 60 80 100 120

Ove

rall

surv

ival

()



P = 0037


(c)

100

80

60

40

20

0

0 20 40 60 80 100 120 140

Ove

rall

surv

ival

()



P = 0001


(d)

100

80

60

40

20

0

0 20 40 60 80

Ove

rall

surv

ival

()



P = 0018


(e)





Low High119875

119899 = 124 119899 = 176



Tumor sizele5 cm 78 (2600) 72 (2400)

lt0001gt5 cm 46 (1533) 104 (3467)






0004II 23 (767) 16 (533)III 69 (2300) 109 (3633)IV 15 (500) 39 (1300)




si-co

ntro

l

si-AT

AD2

si-co

ntro

l

si-AT

AD2

ATAD2

GAPDH

SW480 HCT116

(a)

20

15

10

05

00

CCK8

-OD450

0 1 2 3 4 5


lowastlowast

lowastlowastlowast

lowastlowastlowast

SW480

(b)

lowastlowast

lowastlowastlowast

lowastlowastlowast

20

15

10

05

00

CCK8

-OD450

0 1 2 3 4 5


HCT116

(c)






4 Discussion









References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















10

05

00

minus05

minus10

minus15

minus20

minus25


1 2

log2

med

ian-

cent

ered

inte

nsity

(a)

55

50

45

40

35

30

25

20

15

10

05

00


0 1

log2

med

ian-

cent

ered

inte

nsity

(b)

1 2

45

40

35

30

25

20

15

10

05

00

minus05


log2

med

ian-

cent

ered

inte

nsity

(c)

80

60

40

20

0Rela

tive A

TAD2

mRN

A ex

pres

sion

leve

l

N T

P = 0001

(d)

Nonmalignantcells



ATAD2

GAPDH

HC

oEpi

C

SW480

HCT

116

LoVo

HT2

9

T84

(e)

HC

oEpi

C

SW480

HCT

116

LoVo

HT2

9

T84

Rela

tive A

TAD2

prot

ein

expr

essio

n le

vel 15

10

05

00

(f)



200x 400x


(a)

200x400x


(b)


200x 400x

(c)






100

80

60

40

20

0

0 20 40 60 80 100 120 140

Ove

rall

surv

ival

()



P lt 0001P lt 0001


(a)

100

80

60

40

20

0

0 20 40 60 80 100 120 140

Ove

rall

surv

ival

()



P = 0002P = 00


(b)

100

80

60

40

20

0

0 20 40 60 80 100 120

Ove

rall

surv

ival

()



P = 0037


(c)

100

80

60

40

20

0

0 20 40 60 80 100 120 140

Ove

rall

surv

ival

()



P = 0001


(d)

100

80

60

40

20

0

0 20 40 60 80

Ove

rall

surv

ival

()



P = 0018


(e)





Low High119875

119899 = 124 119899 = 176



Tumor sizele5 cm 78 (2600) 72 (2400)

lt0001gt5 cm 46 (1533) 104 (3467)






0004II 23 (767) 16 (533)III 69 (2300) 109 (3633)IV 15 (500) 39 (1300)




si-co

ntro

l

si-AT

AD2

si-co

ntro

l

si-AT

AD2

ATAD2

GAPDH

SW480 HCT116

(a)

20

15

10

05

00

CCK8

-OD450

0 1 2 3 4 5


lowastlowast

lowastlowastlowast

lowastlowastlowast

SW480

(b)

lowastlowast

lowastlowastlowast

lowastlowastlowast

20

15

10

05

00

CCK8

-OD450

0 1 2 3 4 5


HCT116

(c)






4 Discussion









References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















200x 400x


(a)

200x400x


(b)


200x 400x

(c)






100

80

60

40

20

0

0 20 40 60 80 100 120 140

Ove

rall

surv

ival

()



P lt 0001P lt 0001


(a)

100

80

60

40

20

0

0 20 40 60 80 100 120 140

Ove

rall

surv

ival

()



P = 0002P = 00


(b)

100

80

60

40

20

0

0 20 40 60 80 100 120

Ove

rall

surv

ival

()



P = 0037


(c)

100

80

60

40

20

0

0 20 40 60 80 100 120 140

Ove

rall

surv

ival

()



P = 0001


(d)

100

80

60

40

20

0

0 20 40 60 80

Ove

rall

surv

ival

()



P = 0018


(e)





Low High119875

119899 = 124 119899 = 176



Tumor sizele5 cm 78 (2600) 72 (2400)

lt0001gt5 cm 46 (1533) 104 (3467)






0004II 23 (767) 16 (533)III 69 (2300) 109 (3633)IV 15 (500) 39 (1300)




si-co

ntro

l

si-AT

AD2

si-co

ntro

l

si-AT

AD2

ATAD2

GAPDH

SW480 HCT116

(a)

20

15

10

05

00

CCK8

-OD450

0 1 2 3 4 5


lowastlowast

lowastlowastlowast

lowastlowastlowast

SW480

(b)

lowastlowast

lowastlowastlowast

lowastlowastlowast

20

15

10

05

00

CCK8

-OD450

0 1 2 3 4 5


HCT116

(c)






4 Discussion









References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

80

60

40

20

0

0 20 40 60 80 100 120 140

Ove

rall

surv

ival

()



P lt 0001P lt 0001


(a)

100

80

60

40

20

0

0 20 40 60 80 100 120 140

Ove

rall

surv

ival

()



P = 0002P = 00


(b)

100

80

60

40

20

0

0 20 40 60 80 100 120

Ove

rall

surv

ival

()



P = 0037


(c)

100

80

60

40

20

0

0 20 40 60 80 100 120 140

Ove

rall

surv

ival

()



P = 0001


(d)

100

80

60

40

20

0

0 20 40 60 80

Ove

rall

surv

ival

()



P = 0018


(e)





Low High119875

119899 = 124 119899 = 176



Tumor sizele5 cm 78 (2600) 72 (2400)

lt0001gt5 cm 46 (1533) 104 (3467)






0004II 23 (767) 16 (533)III 69 (2300) 109 (3633)IV 15 (500) 39 (1300)




si-co

ntro

l

si-AT

AD2

si-co

ntro

l

si-AT

AD2

ATAD2

GAPDH

SW480 HCT116

(a)

20

15

10

05

00

CCK8

-OD450

0 1 2 3 4 5


lowastlowast

lowastlowastlowast

lowastlowastlowast

SW480

(b)

lowastlowast

lowastlowastlowast

lowastlowastlowast

20

15

10

05

00

CCK8

-OD450

0 1 2 3 4 5


HCT116

(c)






4 Discussion









References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Low High119875

119899 = 124 119899 = 176



Tumor sizele5 cm 78 (2600) 72 (2400)

lt0001gt5 cm 46 (1533) 104 (3467)






0004II 23 (767) 16 (533)III 69 (2300) 109 (3633)IV 15 (500) 39 (1300)




si-co

ntro

l

si-AT

AD2

si-co

ntro

l

si-AT

AD2

ATAD2

GAPDH

SW480 HCT116

(a)

20

15

10

05

00

CCK8

-OD450

0 1 2 3 4 5


lowastlowast

lowastlowastlowast

lowastlowastlowast

SW480

(b)

lowastlowast

lowastlowastlowast

lowastlowastlowast

20

15

10

05

00

CCK8

-OD450

0 1 2 3 4 5


HCT116

(c)






4 Discussion









References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















4 Discussion









References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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