Six1 Overexpression in Ovarian Carcinoma Causes Resistance to TRAIL-Mediated Apoptosis and Is Associated with Poor Survival Kian Behbakht, 1,2 Lubna Qamar, 1 Carrie S. Aldridge, 1 Ricardo D. Coletta, 1 Susan A. Davidson, 2 Andrew Thorburn, 3 and Heide L. Ford 1,2,4 Divisions of 1 Basic Reproductive Sciences and 2 Gynecologic Oncology in the Department of Obstetrics and Gynecology and Departments of 3 Pharmacology and 4 Biochemistry and Molecular Genetics, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado Abstract Tumorigenesis can arise from inappropriate activation of developmental genes in mature tissues. Here, we show that the developmental regulator Six1 is overexpressed in ovarian carcinoma cell lines (OCC) compared with normal ovarian surface epithelium. As observed in other cancers, Six1 overexpression in OCC leads to increased A-type cyclin expression and increased proliferation. In addition, Six1 overexpression renders OCC resistant to tumor necrosis factor–related apoptosis inducing ligand (TRAIL)–mediated apoptosis, and Six1 knockdown in the TRAIL-resistant SKOV3 ovarian carcinoma line dramatically sensitizes the cells to TRAIL. Because inactivation of the TRAIL response has been linked to metastasis, and because antibodies and recombinant ligand that activate the TRAIL pathway are currently in clinical trials against ovarian carcinoma, we screened normal ovarian and carcinoma specimens for Six1 mRNA. Six1 was overexpressed in 50% of the early-stage (stage I) and 63% of the late-stage (stages II, III, and IV) ovarian carcinomas examined, with late-stage carcinomas expressing f3-fold higher Six1 mRNA levels on average compared with early- stage tumors. Importantly, in patients with late-stage disease, high Six1 expression was associated with significantly shortened survival (P = 0.0015). These data suggest that Six1 may contribute to ovarian epithelial carcinogenesis by simultaneously increasing proliferation and decreasing TRAIL-mediated apoptosis and imply that Six1 may be an important determinant of TRAIL therapy response that should be considered in patient selection for TRAIL-related clinical trials. [Cancer Res 2007;67(7):3036–42] Introduction Ovarian carcinoma is the deadliest gynecologic malignancy and the fourth leading cause of death due to cancer in women (1). Because ovarian cancer is often diagnosed only after the disease has reached an advanced stage, the majority of patients require additionaltreatmentaftersurgicalremovalofthetumor.Although more than 70% of patients with advanced ovarian cancer respond to primary chemotherapy, most of them ultimately develop resistance, leading to an overall 5-year survival rate below 20% (2).Forthisreason,novelapproachesarebeingsoughttoovercome chemoresistance and to develop more effective therapies. Currently, biological therapies are being considered as the next approachinthefightagainstovariancancer.Thesetherapieshave thepotentialtoselectivelytargettumors,tominimizetoxicity,and to overcome the resistance often observed with conventional therapies (3). One such therapy involves activating the tumor necrosisfactor–relatedapoptosis-inducingligand(TRAIL)pathway that selectively induces apoptosis in tumor cells while sparing normalcells.Specifically,TRAILtriggersapoptosisrapidlythrough the extrinsic apoptotic pathway mediated by the death receptors DR4 and DR5 on the cell membrane (4–6). Upon TRAIL-mediated activation of the death receptor, its intracellular death domain attracts the Fas-associated death domain adaptor molecule (7, 8), whichfurtherrecruitstheinitiatorcaspase-8andcaspase-10tothe deathreceptortoformthedeath-inducingsignalingcomplex.This process ultimately results in the activation of the terminal executioner caspase-3, caspase-6, and caspase-7, thereby leading to cell death (9). Importantly, cell culture and mouse xenograft experiments have shown that TRAIL can exert selective cytotoxic activity against ovarian carcinoma cells with limited effects to normal cells (10). TRAIL has been implicated in several aspects of tumorigenesis, including innate immunosurveillance against tumors (11) and inhibitionoftumorinitiationandmetastases(11),andinresponse to conventional chemotherapy (12, 13). For example, TRAIL- deficient mice show increased tumor susceptibility in response to the chemical carcinogen methylchloanthrene and increased experimental and spontaneous metastasis (11). In addition, mutations in TRAIL receptors have been linked to metastatic breast cancer (14). Thus, activating the TRAIL pathway clinically could induce cell death and prevent metastatic disease, and those tumors that devise mechanisms to escape TRAIL-mediated apoptosis might be more metastatic. Inthisstudy,weshowthatthehomeoproteinSix1,animportant developmental regulator (15–20), is a modulator of the TRAIL pathway. Six1 is expressed during embryogenesis (21) but lost in most differentiated tissues (22) and has been implicated in the etiologyofnumerouscancers,includingbreastcancer(22–24)and rhabdomyosarcoma (25, 26). During normal development, Six1 stimulates the proliferation and survival of progenitor cells (17–20), and when expressed out of context, Six1 can aberrantly promote proliferation, contributing to tumorigenesis (23, 26). These observations have led us to postulate that inappropriate Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Present address for R.D. Coletta: Discipline of Pathology, University of Campinas Dental School, 13414-018 Piracicaba-SP, Brazil. Requests for reprints: KianBehbakhtorHeideL.Ford,DepartmentofObstetrics and Gynecology, Division of Basic Reproductive Sciences, Mail stop 8309, 12800 East 19thAvenue,P.O.Box6511,Aurora,CO80045.Phone:303-724-3522;Fax:303-724-3512; E-mail: kian.behbakht@uchsc.edu or heide.ford@uchsc.edu. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-3755 Cancer Res 2007; 67: (7). 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