Oncogenic Propertyof Acrograninin Human Uterine ... · CathepsinK(pycnodysostosis) CTSK X82153 37.7 Hexabrachion(tenascinC,cytotactin) TNC X78565 15.87 Brain-specificpolypeptidePEP-19

Oncogenic Property of Acrogranin in Human UterineLeiomyosarcoma: Direct Evidence of GeneticContribution in In vivo TumorigenesisNoriomiMatsumura, Masaki Mandai, Masanori Miyanishi, Ken Fukuhara, Tsukasa Baba,Toshihiro Higuchi, Masatoshi Kariya, Kenji Takakura, and Shingo Fujii

Abstract To identify potential oncogenes that contribute to the development of uterine leiomyosarcoma,we conducted a cDNA microarray analysis between normal uterine smooth muscle and uterineleiomyosarcoma.We found that acrogranin (also named PCDGF or progranulin) is overex-pressed in uterine leiomyosarcoma.With immunohistochemical staining of 12 leiomyosarcomacases, we verified acrogranin expression in tumor cells. Furthermore, the intensity of acrograninexpression correlated with high histologic grade and poor prognosis. To directly analyze theoncogenic properties of acrogranin, we established an immortalized uterine smooth muscle cellline by transfection of human telomerase reverse transcriptase into primary culture. This cell lineretained the original characteristics of uterine smooth muscle cells, including spindle-shapedextension as well as expression of vimentin, estrogen receptor a, progesterone receptor, and a

smooth muscle actin. Transfection of acrogranin into the immortalized uterine smooth musclecells resulted in colony formation in soft agar, but the diameter of the colonies did not exceed100 Am.Transfection of both acrogranin and SV40 early region (SV40ER) into the immortalizeduterine smooth muscle cells resulted in an increased number of colonies and increased colonysize in soft agar versus transfection of SV40ER alone.We show that only immortalized uterinesmooth muscle cells expressing both acrogranin and SV40ER are capable of tumor formationin nude mice. Thus, acrogranin is overexpressed in uterine leiomyosarcoma cells, particularly inhigh-grade cases, and forced expression of acrogranin in immortalized uterine smooth musclecells contributes to malignant transformation, which suggest that acrogranin plays an importantrole in the pathogenesis of uterine leiomyosarcoma.

Leiomyosarcoma comprises only 1% of all gynecologicmalignancies and has an extremely poor prognosis (1).Although the majority of leiomyosarcomas are diagnosed atan early and resectable stage, 40% of these cases recur aftertherapy. Eighty-one percent of women with stage III tumors willhave tumor recurrence, and only 8% of women with stages II toIV tumors will survive 5 years (2, 3). As uterine leiomyosarcomais resistant to chemotherapy and radiotherapy (4, 5), develop-ing an efficient adjuvant therapy is expected to improve theprognosis of the disease.Because a significant effect was achieved for breast cancer

using antibody therapy directed against HER2, molecular

targeting is regarded as a promising strategy for treatment ofmalignant tumors (6). In this regard, it is becoming increas-ingly important to identify targets that play an important rolein the pathogenesis of specific tumors.Our objective was to identify molecules involved in the

pathogenesis of leiomyosarcoma that might serve as therapeutictargets. For this purpose, we conducted a cDNA microarrayanalysis and from these results we focused on acrogranin.Acrogranin (Genbank accession no. AF055008; NationalCancer Institute of Canada abbreviation, GRN), also calledPCDGF, progranulin, or proepithelin, is an 88 kDa glycopro-tein that was identified as a growth factor secreted from prostatecancer cell, a human teratoma cell (7, 8). Physiologically, acro-granin is known to play important roles in development andwound repair (9, 10). It has also been recently reported thatacrogranin is overexpressed in various malignant tumors, inclu-ding gliomas; breast, ovarian, and prostatic cancers; and hepa-tocellular carcinomas (11–16). In these tumors, the expressionlevel of acrogranin was shown to be a prognostic factor.Experimentally, overexpression of acrogranin in SW-13 adrenalcarcinoma cells and Madin-Darby canine kidney cells non-transformed renal epithelia results in the transfection-specificsecretion of acrogranin, acquisition of clonogenicity in semi-solid agar, and increased mitosis in monolayer culture (17).Acrogranin overproduction in SW-13 cells also increasedtumorigenicity in nude mice (17). Another study showed

Human Cancer Biology

Authors’ Affiliation: Department of Gynecology and Obstetrics, Faculty ofMedicine, Kyoto University, Kyoto, JapanReceived 9/13/05; revised11/27/05; accepted12/15/05.Grant support: Grants-in-aid for Scientific Research 15209057 (S. Fujii),17659514 (K. Takakura), and 17591729 (T. Higuchi) from Japan Society for thePromotion of Science.The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Requests for reprints: Masaki Mandai, Department of Gynecology andObstetrics, Faculty of Medicine, Kyoto University, 606-8507 Kyoto, Japan. Phone:81-75-751-3269; Fax: 81-75-761-3967; E-mail: [email protected].

F2006 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-05-2003

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diminution of acrogranin gene expression-impaired tumori-genicity of the breast cancer cell line MDA-MB-468 in immu-nodeficient mice (18). Thus far, it is unknown whetheroverexpression of acrogranin can transform human primary cells.Development of malignant tumor is a complicated process

involving multiple genetic and epigenetic alterations. Analyzingthe process of malignant transformation that is inducedexperimentally in cell culture is undoubtedly a powerfulapproach for uncovering the basic biological and biochemicalprinciples underlying development of malignant tumor. Be-cause the first report of in vitro malignant transformationof primary human cells by transfection of defined geneticelements (19), several groups, including us, reported in vitrogeneration of human cancer cells in various organs (20–25).However, there has been no report about in vitro malignanttransformation of uterine smooth muscle cells.Herein, we show that acrogranin is overexpressed in uterine

leiomyosarcoma compared with uterine smooth muscles and

leiomyomas and its expression is correlated with prognosis ofuterine leiomyosarcoma. We also show malignant transforma-tion of uterine smooth muscle cells by transfection of humantelomerase reverse transcriptase (hTERT), acrogranin , and SV40early region (SV40ER). These results suggest that acrograninmay play an important role in the pathogenesis of uterineleiomyosarcoma and further indicate that acrogranin may be auseful target of molecular therapy.

Materials andMethods

cDNA microarray analysis. cDNAmicroarray analysis was conductedusing tissues extracted from two cases of uterine leiomyosarcoma andthree cases of uterine smooth muscle as described previously (26).Briefly, from histologically diagnosed tissues, polyadenylate+ RNA wasextracted from tissues using an mRNA purification kit (AmershamBiosciences Corp., Piscataway, NJ). To analyze the average expressionvalues fromdifferent cases in one assay, the same amount of RNA extracted

Table1. cDNAmicroarray analysis showing stronger acrogranin expression inuterine leiomyosarcoma than in smoothmuscle

Gene name Symbol Genbank accession no. Ratio

Chitinase 3-like1 (cartilage glycoprotein-39) CHI3L1 Y08374 84.35Neuromedin B GPNMB X76534 55.36Cathepsin K (pycnodysostosis) CTSK X82153 37.7Hexabrachion (tenascin C, cytotactin) TNC X78565 15.87Brain-specific polypeptide PEP-19 PCP4 U52969 13.42Regulator of G-protein signaling 5 RGS5 AB008109 11.16Cadherin 3, P-cadherin (placental) CDH3 X63629 7.6Cerebellin1precursor CBLN1 M58583 7.41Thrombospondin 2 THBS2 L12350 7.13Ferritin, light polypeptide FTL M11147 6.9Human scaffold protein Pbp1 SYCL AF000652 6.29Cathepsin B CTSB L04288 6.24HumanmRNA for cysteine protease LGMN D55696 5.7Protein regulating cytokinesis1 PRC1 AF044588 5.5DNA-damage-inducible transcript1 GADD45A L24498 5.46Homo sapiens exportin t XPO1 AF039022 5.46Transforming growth factor, b3 TGFB3 X14885 5.06Integrin, a8 ITGA8 L36531 4.96HumanmRNA for pM5 protein NOMO X57398 4.84Tropomodulin TMOD1 M77016 4.64Microfibrillar-associated protein 4 MFAP4 L38486 4.62Acrogranin GRN AF055008 4.56Uridine phosphorylase UPP1 X90858 4.55Superoxide dismutase 2, mitochondrial SOD2 X07834 4.5MyosinVA (heavypolypeptide12, myoxin) MYO5A Y07759 4.48TYROprotein tyrosine kinase binding protein TYROBP AF019562 4.25Follistatin FST M19481 4.2Clathrin-associated/assembly/adaptor protein, large b1 AP2B1 M34175 4.06Ferritin, heavypolypeptide1 FTH1 L20941 4.05Ribonucleotide reductaseM1polypeptide RRM1 X59543 4.03Transketolase (Wernicke-Korsakoff syndrome) TKT L12711 3.96CD24 CD24 L33930 3.96Collagen, typeVI, a3 COL6A3 X52022 3.96Protease, serine,11 (IGF binding) PRSS11 D87258 3.92

(Continued on the following page)

Oncogenic Property of Acroganin

www.aacrjournals.org Clin Cancer Res 2006;12(5) March1, 20061403



from leiomyosarcoma cases and uterine smoothmuscle cases, respectively,was mixed individually and served for further analysis. Then, RNA waslabeled with Cy3-dCTP (leiomyosarcoma RNA) and Cy5-dCTP (uterinesmooth muscle RNA). Labeled probes were mixed with microarrayhybridization solution version 2 (Amersham Biosciences) and formamideto yield a final concentration of 50%. Each sample was hybridized ontotheUniGEMV cDNAmicroarray (IncyteGenomics, Inc., Saint Louis,MO).The UniGEM V array contains 7,800 unique and sequence-verified cDNAor expressed sequence tag elements. The Cy3/Cy5 signal ratio (leiomyo-sarcoma/uterine smooth muscle) was calculated and analyzed by GEMtools 2.4 (Incyte Genomics, Palo Alto, CA).

Construction of retroviral vectors and production of retroviruses.

The plasmid containing hTERT cDNA (pcDNA3-hTERTn2; ref. 20) wascut to produce the hTERT cDNA fragment and this was inserted intothe retroviral vector pPGS-CITEneo. The plasmid containing acrogranincDNA (pCMV-SPORT6-acrogranin) was obtained from InvitrogenCorporation (Carlsbad, CA: clone ID 3457813). We converted pLHCXretroviral vector (Clontech, Franklin Lakes, NJ) to the Gatewaydestination vector by using the Gateway Vector Converting System

(Invitrogen). Then pLHCX-acrogranin plasmid was constructed usingGateway technology. The retroviral vector containing the SV40ER(pWB-SV40ER) was kindly provided by Dr. Jean J. Zhao (Departmentof Cancer Biology, Dana-Farber Cancer Institute, Harvard MedicalSchool, Boston, MA; ref. 22). The final construct was transduced bylipofection into the packaging cell line, Amphopack 293 (Clontech).Forty-eight hours later, the supernatants containing amphotropicviruses were collected. Virus was used to infect uterine smooth musclecells with 5 Ag/mL 1,5-dimethyl-1,5-diazaundecamethylene polyme-thobromide (Nacalai Tesque; Kyoto, Japan).

Generation of uterine smooth muscle cell lines. Uterine smoothmuscle tissue was obtained from a 48-year-old patient with regularmenstrual cycles who underwent surgery for uterine leiomyomas atKyoto University Hospital. Informed consent was obtained from thepatient before surgery. Uterine smooth muscle tissue was minced anddigested in DMEM (Nikken Biomedicals, Kyoto, Japan) containing0.02% collagenase (Wako Pure Chemical Industries Ltd., Osaka, Japan)for 4 hours at 37jC with agitation. The dispersed cells were plated inDMEM containing penicillin-streptomycin (2%, v/v; Nacalai Tesque)

Table1. cDNAmicroarray analysis showing stronger acrogranin expression inuterine leiomyosarcoma than in smoothmuscle (Cont’d)

Gene name Symbol Genbank accession no. Ratio

5-Hydroxytryptamine (serotonin) receptor 2B HTR2B X77307 3.92Ubiquitin-specific protease 9 USP9X X98296 3.9Chemokine (C-Cmotif) receptor1 CCR1 D10925 3.9Phosphoglucomutase1 PGM1 M83088 3.85Tubulin, c polypeptide TUBG1 M61764 3.78Serine/threonine kinase 2 STK2 L20321 3.78Hect domain and RLD 2 HERC2 AB002391 3.753-Phosphoglycerate dehydrogenase PHGDH AF006043 3.74BH-protocadherin (brain-heart) PCDH7 AB006757 3.64APK1antigen COVA1 S72904 3.61Matrix metalloproteinase 2 MMP2 M55593 3.52Ubiquitin-specific protease 9 USP9Y AF000986 3.5Integrin, a5 ITGA5 X06256 3.45Roundabout homologue1 ROBO1 AF040990 3.38p21 (CDKN1A)-activated kinase 2 PAK2 U24153 3.36Chloride channel 4 CLCN4 X77197 3.36Ribosomal protein S11 IDH2 X69433 3.3Inositol phosphate 5V-phosphatase 2 (synaptojanin 2) SYNJ2 AB002346 3.3ATP citrate lyase ACLY X64330 3.25IFN-c-inducible protein16 IFI16 M63838 3.23Insulin-like growth factor binding protein 3 precursor IGFBP3 M35878 3.2Enolase1 (a) ENO1 M55914 3.19Peptidyl-prolyl cis-trans isomeraseA PPIA X52851 3.15Equilibrative nucleoside transporter1 SLC29A1 U81375 3.12Protein S (a) PROS1 X12892 3.1Human leukemia virus receptor1 (GLVR1) SLC20A1 L20859 3.08Apolipoprotein C-I APOC1 X00570 3.08Calpastatin CAST D16217 3.08Ubiquitin-specific protease11 USP11 U44839 3.08Human arginine-rich protein (ARP) ARMET M83751 3.06TrkB NTRK2 U12140 3T-complex protein1, a subunit TCP1 X52882 3Thyroid hormone receptor, b THRB X04707 3

NOTE: cDNA microarray analysis was conducted between leiomyosarcoma and uterine smooth muscle tissues. The table lists genes for which expression ratios ofleiomyosarcoma/uterine smoothmuscle are >3. Expressed sequence tags are excluded.





and fetal bovine serum (10%, v/v, Asahi Technoglass, Funabashi,Japan) and maintained in a 37jC incubator ventilated with 5% CO2 in-room air. The following day, uterine smooth muscle cells were infectedfor 24 hours with the amphotropic hTERT retroviral vectors followed bysupplementation 48 hours later with G418 (500 Ag/mL). G418 wasused for 6 weeks to eliminate all noninfected cells and select for colonyformation of the hTERT-infected cells. Cell lines were generated byclonal selection and were maintained in DMEM with 10% fetal bovineserum and antibiotic. Among multiple colonies generated, one wasisolated and maintained. It was defined as ‘‘population = 1’’ when cellsbecome confluent in a 100 mm culture dish. When confluent, the cellswere trypsinized and seeded at a 1:4 split ratio. The pLHCX retroviralvector containing acrogranin cDNA or the pLHCX retroviral vector onlywere transfected into the immortalized uterine smooth muscle cell linein the same way and selected by hygromycin B (200 Ag/mL; Invitrogen).The pWB retroviral vector containing the SV40ER was similarlytransfected into these cells and transfectants were selected andmaintained using blastcidine (2 Ag/mL; InvivoGen, San Diego, CA).

Immunostaining of cultured cells. The hTERT-transfected uterinesmooth muscle cells were cultured for 3 days on multichambered cultureslides (BD Biosciences, San Jose, CA) in a 37jC incubator ventilated with

5% CO2 in room air. Cells were fixed and permeabilized by immersion in�20jC methanol for 15 minutes and �20jC acetone for 1 minute.Blocking was done with normal rabbit serum (Nichirei, Tokyo, Japan).Samples were incubated with primary antibodies at 4jC overnight andwashed. The cells were then incubated for 30 minutes with a 1:40dilution of FITC-conjugated rabbit anti-mouse IgG secondary antibody(DakoCytomation, Glostrup, Denmark) and washed. Propidium iodide(Nacalai Tesque) was used for nuclear staining. Samples were soaked withglycerol (DakoCytomation) and coverslipped. Photographs were takenwith a Zeiss microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY)equipped for epifluorescence. Primary antibodies used to stain culturedcells were all anti-human mouse monoclonal antibodies: anti–estrogenreceptor a (ERa; Nichirei), anti–progesterone receptor (PR; Nichirei),anti–a smooth muscle actin (a-SMA; DakoCytomation), anticytokeratin(Biomeda, Foster City, CA), and antivimentin (Santa Cruz Biotechnology,Santa Cruz, CA).

Immunostaining of tissue samples. Tissues were fixed in 10% buffered

formalin, embedded in paraffin, and sectioned. Sections were stained

with H&E. For the immunohistochemical staining, they were deparaffi-

nized in xylene and ethanol. Sections were blocked for endogenous

peroxidase with 0.3% H2O2 followed by sequential incubation

with normal goat serum, primary antibody, secondary antibody, and

peroxidase-conjugated streptavidin. Slides were then stained with 3,3V-diaminobenzidine and hematoxylin. We used the following antibodies as

primary antibodies: a goat polyclonal antiacrogranin antibody (Santa

Cruz Biotechnology), a rabbit polyclonal antivimentin antibody (Santa

Cruz Biotechnology), a mouse monoclonal anti-SV40 large T-antigen

antibody (Santa Cruz Biotechnology), and a mouse monoclonal

anticytokeratin antibody (Biomeda). For acrogranin and vimentin

staining, samples were retrieved with Target Retrieval Solution high pH

(DakoCytomation) for 15 minutes at 95jC. Then, SAB-PO kit (Nichirei),

which contains blocking serum, secondary antibodies, and peroxidase-

conjugated streptavidin, was used. For SV40 large T-antigen and

cytokeratin staining, samples were soaked in citrate buffer and retrieved

in a microwave for 15 minutes. To stain xenograft tumor tissues formed

in nude mice with murine monoclonal antibodies, Mouse Stain kit was

used according to the protocol of the manufacturer (Nichirei).Evaluation of acrogranin expression in human tissue samples. The

level of acrogranin protein expression was evaluated by immunohisto-chemical study and defined as follows: +, stronger than smooth musclebut weaker than endometrial glands; ++, equivalent to endometrialglands; +++, stronger than endometrial glands. Immunostaining of allhuman tissue samples was done simultaneously and under the sameconditions. Three persons (gynecologic pathologists) examined eachslide independently in a blind manner with final assignment ofexpression levels determined by consensus.

Reverse transcription-PCR. Total RNA was extracted from cultureduterine smooth muscle cell lines using Trizol reagent (Invitrogen).Reverse transcription-PCR (RT-PCR) was done with the One-StepRT-PCR kit (Qiagen, Hilden, Germany) according to the instructions ofthe manufacturer. ERa, PR, hTERT, acrogranin , and actin cDNAs wereamplified using specific primers for each transcript. PCR amplificationwas done as follows: denaturation at 94jC for 1 minute, annealing atprimer-specific temperatures (ERa, PR , 52jC; acrogranin, hTERT,actin , 55jC) for 1 minute, and extension at 72jC for 2 minutes. Theconstitutively expressed actin transcript was used as a reference toevaluate transcription of ER, hTERT , and PR . For evaluation of acrograninmRNA expression, actin was used as an internal control by multiplexingthe acrogranin- and actin-specific primers in each reaction. PCRs forERa and PR were terminated at 30 cycles, PCR for hTERT at 28 cycles, andPCR for acrogranin at 26 cycles. PCR for actin was terminated at 25 cyclesexcept for where actin was used as an internal control. Primers used wereas follows: ERa , forward 5V-ACAAGCGCCAGAGAGATGAT-3V, reverse 5V-CAGATTCATCATGCGGAACC-3V; PR , forward 5V-GTTGCTCTCCCA-CAGCCATT-3V, reverse 5V-TACAGCATCTGCCCACTGAC-3V; hTERT , for-ward 5V-CGGAAGAGTGTCTGGAGCAA-3V, reverse 5V-GGATGAAGCGG-AGTCTGGA-3V, acrogranin , forward 5V-TGGTTCACACCCGCTGCA-3V,

Fig. 1. Immunohistochemical study on acrogranin protein expression inuterine smooth muscle, leiomyoma, and various-grade leiomyosarcoma. Animmunohistochemical study was done among uterine leiomyosarcoma, leiomyoma,and smooth muscle tissues using an antiacrogranin antibody. A, leiomyosarcoma.B, leiomyosarcoma (high magnification). C, smoothmuscle (arrow, endometrialgland).D, leiomyoma. Strongexpressionwasobserved inendometrial glands,whichwas used as an internal control. Among leiomyosarcoma cases, the histologicallyhigh-grade group showed stronger expression of acrogranin than the low-gradegroup andmyxoid type. E, high grade. F, low grade.G, myxoid. Bar, 50 Am.





reverse 5V-GTTGGGCATTGGGCAGCA-3V, actin : forward 5V-CCGCAAA-GACCTGTACGCCA-3V, reverse 5V-TGGACTTGGGAGAGGACTGG-3V.

Assay for anchorage independence. A 2-fold concentration of DMEMsupplemented with fetal bovine serum and antibiotics was made frompowdered medium (Life Technologies), and 1% agarose was made withlow-melting-temperature agarose (Invitrogen). Then, 0.5% agarose and0.33% agarose in 1� DMEM was made by their mixture. For uterinesmooth muscle cells transfected with hTERT and acrogranin or emptyvector, colony formation in semisolid agar was assayed by suspending4� 104 cells in 800 AL of 0.33% agarose and placing this suspension onthe top of 800 AL solidified 0.5% agarose. Cultures were maintained at37jC in a 5% CO2 atmosphere. Colonies larger than 50 Am in diameterwere counted 3 weeks later. For cells further transfected with SV40ER ,colonies larger than 100 Am were counted 10 days later. Theexperiments were repeated four times individually.

Tumorigenicity assays. Six-week-old female BALB/C nude mice werepurchased from CLEA Japan (Tokyo, Japan). The animals receivedproper care according to the rules of the Kyoto University Committeeon Animal Care. Animal experiments were done in compliance with theUnited Kingdom Coordinating Committee on Cancer Research Guide-lines. In nude mice, 1 � 107 cells were injected s.c. per animal. If a s.c.mass was confirmed, the mice were sacrificed and the tumors wereresected. Cells were considered nontumorigenic if the mouse failed toform a tumor within 4 months of injection.

Statistical analysis. For analysis of between-group differences, Pvalues were determined using Mann-Whitney U test. P < 0.05 wasconsidered significant.

Results

cDNA microarray analysis. Table 1 lists highly expressedgenes in leiomyosarcoma from a cDNA microarray analysis of7,800 genes between leiomyosarcoma and uterine smoothmuscle. Acrogranin was the focus of the present study because of(a) the high ratio of acrogranin expression in leiomyosarcoma

compared with smooth muscle, (b) a previous report concerningpathogenesis of cancers (17, 18), and (c) the fact that acrograninmay be a useful molecular tumor marker and therapeutic targetin leiomyosarcoma, for which sensitive tumor markers andeffective cytotoxic agents do not currently exist.Immunohistochemical study for acrogranin expression. An

immunohistochemical study was done among uterine leiomyo-sarcoma, leiomyoma, and smooth muscle tissues usingan antiacrogranin antibody. Acrogranin protein expression wasobserved in uterine leiomyosarcoma, but expression wasweak or undetectable in smooth muscles and leiomyomas(Fig. 1A-D). Strong expression was observed in endometrialglands, which were used as an internal control. Uterineleiomyosarcoma was diagnosed using standard criteria (27).Furthermore, histologic grading was done based on previouslyestablished criteria (28). Among leiomyosarcoma cases, thehistologically high-grade group showed significantly strongerexpression of acrogranin than the low-grade group and myxoidtype (P < 0.05, m2 test; Fig. 1E-G; Table 2). Specifically, strongexpression was observed in five of six high-grade cases (case 11 isof high grade but with low acroganin expression). In contrast,strong expression was observed in only one (case 4) of six low-grade/myxoid cases. Prognoses of high-grade cases were ex-tremely poor (Table 2).Generation of a uterine smooth muscle cell line. To study the

effect of acrogranin overexpression in uterine smooth musclecells, we generated a uterine smooth muscle cell line by hTERTgene transfection into primary culture cells (Fig. 2). It waspreviously reported by several groups that uterine smoothmuscle cells could be immortalized by hTERT gene transfection(29–31). The cell line we generated maintained characteristicsof uterine smooth muscle cells (Fig. 2B and C). RT-PCR analysiswas done to confirm the expression of ERa, PR, and forced

Table 2. Summary of correlation amonghistologic grading, expression of acrogranin, and prognosis in uterineleiomyosarcoma cases

Case no. Age (y) Size (cm) Operation Adjuvant therapy Clinicalstage

Grade Follow-up Acrograninexpression

1 51 12 TAH ND I Low grade NED 60+mo +2 49 4 TAH+BSO CAP-F (CDDP-ADR-CPM-5FU)�2 I Low grade NED 60+mo +3 49 16 TAH+BSO ND I Low grade NED 60+mo +4 39 15 TAH+BSO ND I Low grade NED 60+mo ++5 65 10 TAH+BSO ND I Myxoid NED 51mo +6 22 20 TAH+BSO CDDP-THP-IFO�3 I Myxoid NED 50mo +7 52 12 TAH+ LSO ND I High grade NED 60+mo ++8 52 8.5 TAH +BSO CDDP-ADR�2,VP-16�2 I High grade DOD 8mo ++9 76 13 TAH+BSO ND II High grade DOD14mo +++10 44 9 TAH+BSO CDDP-THP-IFO�3 IV High grade DOD 24mo ++11 47 9 Tumor resection +

TAH +BSOCDDP-ADR-VCR�2, CAP-F

(CDDP-ADR-CPM-5FU)�2III High grade DOD 24mo +

12 60 8 Tumor resection+TAH +BSO

CAP-F (CDDP-ADR-CPM-5FU)�2 III High grade DOD 60mo +++

NOTE: The strength of acrogranin expression was evaluated as described in Materials and Methods. Twelve cases of uterine leiomyosarcoma were treated in KyotoUniversity Hospital during the last15 years.Abbreviations: TAH, total abdominal hysterectomy; BSO, bilateral salpingo-oophorectomy; LSO, left salpingo-oophorectomy; ND, not done; CDDP, Cisplatin; ADR,Adriamycin; CPM, Cyclophosphamide; 5FU, 5-Fluorouracil; THP, Therarubicin; IFO, Ifosfamide; VP-16, Etoposide; VCR,Vincristin; NED, no evidence of disease; DOD,died of disease.





expression of hTERT (data not shown). This cell line did notshow acrogranin gene expression as a primary culture (Fig. 3A).On the contrary, SKN (uterine leiomyosarcoma cell line)showed acrogranin gene expression (Fig. 3A).Impact of forced expression of acrogranin in uterine smooth

cells on anchorage-independent cell growth. Acrogranin wasretrovirally transfected into the immortalized smooth musclecell line and transfected cells were selected with hygromycin B.Forced expression of acrogranin in the immortalized uterinesmooth muscle cells was confirmed by RT-PCR (Fig. 3A). Theimmortalized uterine smooth muscle cell line (transfected onlywith hTERT gene) did not show increased cell proliferationcompared with early primary culture of uterine smooth musclecells as detected by WST-1 assay (data not shown). When this cellline was further transfected with acrogranin, it showed no changein cell proliferation, but showed anchorage-independent growth(Fig. 3B and C) to some extent. However, in this case, the colonysize did not exceed 100 Am. The immortalized uterine smoothmuscle cell line was also transfected with SV40ER (eventuallytransfected with hTERT + SV40ER). Transfection of SV40ERresulted in slight increase in cell proliferation and in more

apparent anchorage-independent growth (some of the coloniesexceeded 100 Am in size as shown in Fig. 3D). When the cell waseventually transfected with hTERT + SV40ER + acrogranin,anchorage-independent growth was markedly accelerated(Fig. 3D and E), but further stimulation of cell growth was notobserved. Cells (5 � 104/mL) were seeded in soft agar andcolonies larger than 100 Amwere counted 10 days later. The cellstransfected with hTERT + SV40ER + acrogranin formed signifi-cantly increased number of colonies in soft agar compared withcells transfected with hTERT + SV40ER (Fig. 3E).

Impact of forced expression of acrogranin in uterine smoothcells on tumor formation in nude mice. Cells used in Fig. 3 wereinoculated s.c. into nude mice at 6 to 8 weeks of age (1� 107 peranimal). Tumor formation and growth was assessed until atleast 4 months after the inoculation. Tumor formation in nudemice was observed when uterine smooth muscle cells trans-fected with hTERT, acrogranin , and SV40ER were inoculated(Fig. 4A; Table 3). In histologic analysis, tumor cells werearranged around tumor vessels and caused sudden massivehemorrhage necrosis, which are typical features of mesenchymaltumors (Fig. 4B). The mesenchymal character of the tumor was

Fig. 2. Generation of a uterine smoothmuscle cell line retaining the phenotype ofuterine smooth muscle cells by hTERTgenetransfection. Uterine smoothmuscle cellswere transfected with hTERTcDNA andselected with G418. A colony was isolatedand subcultured. A, proliferation of thehTERTgene ^ transfected uterine smoothmuscle cells continued over 300 days. Onehundred population doublings of these cellswere counted and, therefore, this cell lineis immortalized. B, these cells showedspindle-shaped extension under phase-contrast microscopy. C, immunostaining ofimmortalized smooth muscle cells observedin confocal microscopy [top left : propidiumiodide (PI) staining] showed expressionof vimentin, a-SMA, ERa, and PR, but didnot show expression of cytokeratin.





also confirmed by the immunohistochemical analysis: vimentin(+) and cytokeratin (�) (Fig. 4C). However, ERa, PR, and a-SMA were negative (data not shown). Acrogranin and SV40expression were confirmed in all tumor cells (Fig. 4D).

Discussion

Uterine leiomyosarcoma is a disease with extremely poorprognosis, is highly aggressive, and is resistant to chemo-therapies. At present, surgical intervention is virtually the onlymeans of treatment for uterine leiomyosarcoma (4, 5). However,molecular-targeting therapies against malignant tumors haverecently shown remarkable achievements (6). To improve theprognosis of uterine leiomyosarcoma, we aimed to search for keyoncogenes that play an important role in their pathogenesis andthat could serve as a molecular target for treatment. For thispurpose, we conducted a cDNA microarray analysis between

uterine leiomyosarcoma and normal uterine smoothmuscle andshowed that several factors, such as brain-specific polypeptidePEP-19, may be associated with the pathogenesis of leimyosar-coma (26). However, in terms of tumorigenesis of leiomyosar-coma, merely comparing the expression of potential oncogenesbetween normal and malignant tissues is not sufficient becausethe results obtained may be the consequence of malignanttransformation and, therefore, not necessarily the cause. Toobtain more direct evidence for a genetic contribution to tumoroccurrence, we established a method in which a candidateoncogene was transfected into phenotypically normal smoothmuscle cells and oncogenicity was determined using soft agarassays or tumor formation in nude mice.Among the various genes that exhibited differential expres-

sion between normal and malignant uterine smooth muscles,acrogranin, which is an autocrine/paracrine growth factor, waschosen as our research target. This was largely because, thus far,

Fig. 3. Effect of transfection of with acrogranin and SV40ER genes on anchorage-independent growth of the immortalized uterine smoothmuscle cells. A, acrograninmRNA expression in SKN, uterine leiomyosarcoma cells, and in the immortalized uterine smooth muscle cells. I-SM, immortalized uterine smooth muscle cell line by hTERTtransfection. Acrogranin mRNA expressionwas analyzed by RT-PCR (26 cycles). As an internal control, actin primers were also included in the reactions. SKN (uterineleiomyosarcoma cell line) showed acrogranin gene expression. Acrogranin was not detected in the immortalized uterine smoothmuscle cell line and in the uterine smoothmuscle cells in primary culture.The acrogranin gene was retrovirally transfected into the immortalized smoothmuscle cell line and transfected cells were selected withhygromycin B. Forced expression of acrogranin was confirmed in the immortalized uterine smoothmuscle cells by RT-PCR. B, anchorage-independent growth ofacrogranin-transfected immortalized uterine smoothmuscle cell line. Immortalized symmetrical cells (5� 104/mL) were seeded in soft agar and colonies larger than 50 Amwere counted 20 days later. C, number of colonies in soft agar in (B); the data are shown as the sum of colony numbers in 800 AL (eight wells of a 96-well plate).Thisexperiment was done four times independently. Column, mean; bar, SE.D, accelerated anchorage-independent growth of the immortalized uterine smoothmuscle cellstransfected with acrogranin and SV40ER genes.The uterine smoothmuscle cells used in (B) were further transfected with SV40ER using a retroviral vector and transfectedcells were selected by blastcidine. Cells (5� 104/mL) were seeded in soft agar and colonies larger than100 Amwere counted10 days later. A, phase-contrast microscopyof colonies in soft agar. E, number of formed colonies in soft agar in (D). Data are colony numbers in100 AL (one well of a 96-well plate).This experiment was done fourtimes independently. Columns, mean; bars, SE. *, P < 0.001.





clinically successful molecular targets include growth factors ortheir receptors, such as HER2 or vascular endothelial growthfactor. Acrogranin is a pluripotent growth factor that mediatescell cycle progression and cell motility (32, 33). Structurally, itbears no homology to the well-established growth factorfamilies (34). In vivo , acrogranin is expressed at a high levelin epithelial cells that undergo rapid turnover, notably in theintestinal deep crypt and epidermal keratinocytes, suggesting arole of acrogranin in the regulation of epithelial proliferation(35). It is scarce in connective tissue, endothelia, and muscle inhealthy adult tissues.

First, we examined the expression of acrogranin in uterinesmooth muscle tumors and showed that it is highly expressedin uterine leiomyosarcoma and in the leiomyosarcoma cell lineSKN (Figs. 1 and 3A; Table 2) In immunohistochemical studies,the intensity of its expression correlated with the histologicgrade and prognosis although the number of samples analyzedare relatively few (Table 2). In other types of malignancies,acrogranin is highly expressed in aggressive cancer cell lines andclinical specimens, including breast, hepatocellular, ovarian,and prostatic cancers, as well as gliomas (11–16, 18).Accordingly, we hypothesized that acrogranin may play acritical role in tumorigenesis in uterine leiomyosarcoma.

We attempted to evaluate the oncogenic property ofacrogranin in a more direct fashion, namely by transfectingits gene into phenotypically normal uterine smooth musclecells. It has been reported that uterine smooth muscle cellsare immortalized by transfection of hTERT cDNA and theseimmortalized cells retain their original characteristics (29–31).However, to our knowledge, there have been no attempts totransform immortalized uterine smooth muscle cells by trans-fecting potential oncogenes. We first transfected hTERT cDNAinto uterine smooth muscle cells and one of the selectedcolonies was maintained. These cells expressed ERa, PR, and a-SMA (Fig. 2C), showing the original character of primaryuterine smooth muscle cells. This cell line did not showanchorage-independent growth or tumorigenicity in nude miceand, therefore, was suitable to examine the oncogenic functionof acrogranin.

Although it was reported that overexpression of acrogranin inleiomyosarcoma cell line promote proliferation (35), it did notpromote cell proliferation in immortalized uterine smoothmuscle cells as detected by WST-1 assay. However, it resulted incolony formation in soft agar, which was only microscopicallydetected; three to four colonies of 4 � 104 cells were obtainedthat were only f50 Am in diameter and never exceeded 100Am (Fig. 3B). These cells did not form tumors in nude mice(Table 3). These results may show a potential oncogenicfunction of acrogranin, but also suggest that coexpression ofhTERT and acrogranin is not sufficient to transform uterinesmooth muscle cells.

We then transfected the SV40ER into these uterine smoothmuscle cells. When transfected with the SV40ER, hTERT-immortalized uterine smooth muscle cells showed obviousanchorage-independent growth but did not form tumors innude mice (Fig. 3D; Table 3). On the other hand, coexpressionof hTERT, acrogranin , and SV40ER resulted in more extensiveanchorage-independent growth and tumor formation in nudemice (Figs. 3D and 4A; Table 3). This result indicates that

Fig. 4. Tumor formation in nude mice by uterine smoothmuscle cells transfectedwith hTERT, acrogranin, and SV40ER. A, a representative photograph oftumor-bearing mice. Arrow, a representative tumor. B, histologic analysis of thetumor formed inmice. In H&E staining, histologically massive hemorrhage andcoagulative necrosis existed in tumor tissues (left). In highmagnification (right),tumor cells were arranged around tumor vessels. C, immunohistochemical study oftumors.Tumorswere positive for vimentin andnegative for cytokeratin, showing thatthe tumor originated frommesenchymal cells.D, forced expression of acrograninand SV40 in tumor cells was confirmed by the immunohistochemical study.Bar, 50 Am.

Table 3. Summary of tumor formation in nudemice

Smoothmuscle cells + hTERT + empty vector 0/3Smoothmuscle cells + hTERT + acrogranin 0/4Smoothmuscle cells + hTERT + empty vector + SV40ER 0/7Smoothmuscle cells + hTERT + acrogranin + SV40ER 6/8

NOTE: 1�107 cells were inoculated s.c. in nude mice at 6 to 8 weeks of age.Tumor formation and growth was not assessed until at least 4 months post-inoculation.





coexpression of these three genes are necessary and sufficientto transform uterine smooth muscle cells. A number ofnonmitogenic actions of acrogranin relevant to cancer areknown, e.g., decreased susceptibility to anoikis, increasedangiogenesis, invasion, and motility (10, 11, 18). As theexpression of acrogranin did not accelerate cell proliferation inmonolayer (data not shown), our results suggest oncogenic, butnonproliferative, actions of acrogranin.Studies on in vitro malignant transformation have revealed

that to transform primary human cells, the following foursignals are thought to be necessary: (a) elongation of telomerelength by hTERT expression, (b) perturbation of p53 and reti-noblastoma by SV40LT expression or other methods, (c) per-turbation of PP2A by SV40 small tumor antigen expression orother methods, and (d) constitutive RAS signaling by trans-fection of the oncogenic allele of RAS (36–39). In general,signals from growth factors are transmitted through tyrosinekinase type receptors to RAS (40). As such, constitutively acti-vated RAS signaling can be used to substitute for persistentgrowth factor signaling. Actually, as we previously reported,ovarian surface epithelial cells immortalized by coexpression ofhTERT and SV40 are transformed by c-ERBB2 instead ofoncogenic RAS (20). However, there has been no report thatoverexpression of growth factors can substitute for transfectionof oncogenic RAS.Signals generated by acrogranin are thought to be strong.

Embryonic fibroblasts from mice in which the insulin-likegrowth factor-I receptor was deleted (i.e., R� cells) fail to dividewhen stimulated by well-established growth factors. They do,

however, proliferate when exposed to acrogranin (41). Acro-granin activates the mitogen-activated protein kinase andphosphatidylinositol-3 kinase signaling cascades (42). Activatedphosphatidylinositol-3 kinase can substitute, at least in part, foroncogenic RAS signal in de novo transformation (22). Hence, it isone of the possible mechanisms that acrogranin exert oncoge-nicity via phosphatidylinositol-3 kinase activation by substitut-ing RAS signaling. However, we do not have direct evidence forthat yet and other possibilities cannot be excluded.In summary, we showed that acrogranin was overexpressed in

uterine leiomyosarcoma and their higher histologic grade wascorrelated with higher expression of acrogranin. Transfection ofhTERT, SV40ER , and acrogranin in uterine smooth muscle cellsresulted in transformation of primary cultured cells. These dataindicate that acrogranin may be a promising target fordeveloping effective molecular targeting therapy for treatmentof uterine leiomyosarcoma. In addition, acrogranin may alsoserve as a specific diagnostic marker to identify leiomyosarcomasof the uterus, the diagnosis of which is clinically difficult.Moreover, our study established a new method to evaluate theoncogenic property of specific genes in the process of malignanttransformation of hTERT-immortalized and SV40-activatedhuman smooth muscle cells.

Acknowledgments

We thank Dr. SusanMurphy at Duke UniversityMedical Center for her useful ad-vice in the preparation of the manuscript and Michiko Muraoka for her great contri-bution as a laboratory technician.

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2006;12:1402-1411. Clin Cancer Res Noriomi Matsumura, Masaki Mandai, Masanori Miyanishi, et al.

TumorigenesisIn vivoinLeiomyosarcoma: Direct Evidence of Genetic Contribution

Oncogenic Property of Acrogranin in Human Uterine

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