The Prostate 72:1542^1549 (2012) Role of Dutasteride in Pre-Clinical ETS Fusion-Positive Prostate Cancer Models Bushra Ateeq,1,2 Adaikkalam Vellaichamy,1,2 Scott A. Tomlins,1,2 Rui Wang,1,2 Qi Cao,1,2 Robert J. Lonigro,1,3 Kenneth J. Pienta,1,3,4 and Sooryanarayana Varambally1,2,3* 1Michigan Center forTranslational Pathology,University of Michigan Medical School, Ann Arbor, Michigan 2Department of Pathology,University of Michigan Medical School, Ann Arbor, Michigan 3Comprehensive Cancer Center,University of Michigan Medical School, Ann Arbor, Michigan 4Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan BACKGROUND. Androgens play a crucial role in prostate cancer, hence the androgenic pathway has become an important target of therapeutic intervention. Previously we discov- ered that gene fusions between the 50-untranslated region of androgen regulated gene TMPRSS2 and the ETS transcription factor family members were present in a majority of the prostate cancer cases. The resulting aberrant overexpression of ETS genes drives tumor progression. METHODS. Here, we evaluated the expression levels of 5a-reductase isoenzymes in pros- tate cancer cell lines and tissues. We tested the effect of dutasteride, a 5a-reductase inhibitor, in TMPRSS2–ERG fusion-positive VCaP cell proliferation and cell invasion. We also evaluat- ed the effect of dutasteride on the TMPRSS2–ERG fusion gene expression. Finally, we tested dutasteride alone or in combination with an anti-androgen in VCaP cell xenografts tumor model. RESULTS. Our data showed that 5a-reductase SRD5A1 and SRD5A3 isoenzymes that are responsible for the conversion of testosterone to DHT, are highly expressed in metastatic prostate cancer compared to benign and localized prostate cancer. Dutasteride treatment attenuated VCaP cell proliferation and invasion. VCaP cells pre-treated with dutasteride showed a reduction in ERG and PSA expression. In vivo studies demonstrated that dutaster- ide in combination with the anti-androgen bicalutamide significantly decreased tumor bur- den in VCaP cell xenograft model. CONCLUSIONS. Our findings suggest that dutasteride can inhibit ERG fusion-positive cell growth and in combination with anti-androgen, significantly reduce the tumor burden. Our study suggests that anti-androgens used in combination with dutasteride could syner- gistically augment the therapeutic efficacy in the treatment of ETS-positive prostate cancer. Prostate 72: 1542–1549, 2012. # 2012 Wiley Periodicals, Inc. KEY WORDS: prostate cancer; gene fusion; TMPRSS2–ERG; 5a-reductase; dutasteride INTRODUCTION Grant sponsor: GSK; Grant sponsor: Genentech Foundation Post- doctoral Fellowship; Grant sponsor: Expedition Inspiration. A majority of prostate cancer patients harbor gene Adaikkalam Vellaichamy present address is Department of Nano- fusions involving androgen regulated promoters and ETS medicine, The Methodist Hospital Research Institute, Houston, TX members of the family of transcription factors 77030. [1–3] that are known to play a critical role in prostate *Correspondence to: Dr. Sooryanarayana Varambally, PhD, Depart- tumorigenesis. Overexpression of ERG in primary or ment of Pathology, University of Michigan Medical School, Traver- immortalized benign prostate epithelial cells induces wood IV, Suite 100, 2900 Huron Parkway, Ann Arbor, MI 48109- @ an invasion-associated transcriptional program [4]. 0602. E-mail: soory med.umich.edu ERG Received 16 December 2011; Accepted 13 February 2012 Conversely, knockdown in VCaP cells, an andro- DOI 10.1002/pros.22509 gen-sensitive metastatic prostate cancer cell line har- Published online 13 March 2012 in Wiley Online Library boring the TMPRSS2–ERG gene fusion, decreases cell (wileyonlinelibrary.com). ß 2012 Wiley Periodicals,Inc. Role of Dutasteridein Regulating Gene Fusion 1543 proliferation and tumor growth in mice [5]. Other hyperplasia [16]. Recent report from a clinical trial studies demonstrated that VCaP cells and immortal- suggested that dutasteride could prove beneficial for ized benign prostate epithelial cells (RWPE-1) overex- low-risk prostate cancer patients [17]. Furthermore, pressing ERG directly engage components of the patients successfully treated with high doses of dutas- plasminogen activation pathway to mediate cellular teride were predominantly negative for TMPRSS2– invasion, potentially representing a downstream ERG ERG genetic rearrangement [18]. target susceptible to therapeutic intervention [4]. Fur- Since androgen induces the expression of thermore, transgenic mice expressing the ERG gene TMPRSS2–ERG, in the present study we investigated fusion product under androgen regulation develop the effect of dutasteride on the expression of ERG as mouse prostatic intraepithelial neoplasia (mPIN), a well as cellular phenotypes of the TMPRSS2–ERG precursor lesion of prostate cancer [4,6,7], and when rearrangement-positive prostate cancer cell line, VCaP. combined with other relevant genomic lesions such as Results from our study suggest that dual 5a-reductase loss of PTEN, result in the development of invasive inhibitor, dutasteride, inhibits the expression of carcinoma [7]. Recently, we showed that ERG binds TMPRSS2–ERG in vitro under androgen-deprived to androgen receptor (AR) and disrupts the regulation condition. We also demonstrate that dutasteride, in of a majority of its target genes. In addition, ERG acti- combination with bicalutamide, leads to greater re- vates the polycomb protein, EZH2, facilitating a stem- duction in VCaP tumor burden than either inhibitor cell-like dedifferentiation program, suggesting that alone. Thus, our study provides pre-clinical evidence TMPRSS2–ERG plays a key role in cancer by abrogat- for a potential therapeutic strategy for the treatment ing lineage-specific differentiation of the prostate [8]. of ETS fusion-positive prostate cancer. Androgen-AR axis plays a critical role in prostate tumorigenesis, thus inhibition of AR activity has been MATERIALS AND METHODS a major therapeutic strategy for prostate cancer treat- Cell Lines,Tissue Samples, ment. Anti-androgens that block AR function include and DutasterideTreatments bicalutamide (Casodex), flutamide, nilutamide, and the steroidal anti-androgen cyproterone acetate [9]. The VCaP prostate cancer cell line was derived Another potent AR inhibitor, apregnenolone-derived from a vertebral metastasis of a patient with castra- compound abiraterone, renders both potency and se- tion resistant metastatic prostate cancer and was lectivity in CYP17 inhibition and has demonstrated kindly provided by Kenneth Pienta (University of efficacy in reducing the weights of androgen-depen- Michigan). VCaP cells were cultured in DMEM Gluta- dent organs, such as prostate, seminal vesicles, and max medium supplemented with 10% fetal bovine se- testes [10]. Although responding initially to androgen rum (FBS; Invitrogen, Carlsbad, CA). LNCaP cells deprivation therapy by depletion of gonadal testoster- were purchased from the American Type Cell Culture one (T), metastatic tumors almost invariably develop (ATCC, used at passage number 30–40) and cultured into castration-resistant prostate cancer (CRPC). AR in RPMI-1640 medium supplemented with 10% FBS. amplification, gain-of-function mutations, and novel Both cell lines were maintained in a 5% CO2, 95% air- splice variant expression are thought to be responsi- humidified atmosphere at 378C. Prostate tissues were ble for this resistance [11]. The development of CRPC obtained from the radical prostatectomy series and is also dependent upon the intratumoral generation of the Rapid Autopsy Program, both of which are part the potent androgen, dihydrotestosterone (DHT). As of the University of Michigan Prostate Cancer Special- part of the androgen axis, the enzyme 5a-reductase is ized Program of Research Excellence (S.P.O.R.E.) Tis- responsible for the conversion of circulating T into sue Core. All samples were collected with informed DHT [12,13] and in CRPC, adrenal androgens are con- consent of the patients and prior institutional review verted to DHT by these enzymes. Three 5a-reductase board approval. For all tissue samples and cell lines, isoforms, SRD5A1, SRD5A2, and SRD5A3 have been total RNA was isolated with Trizol reagent (Invitro- characterized in the prostate [14]. In CRPC, SRD5A1 gen) according to the manufacturer’s instructions. is overexpressed and a recent study indicated that Various doses of dutasteride were used to test the SRD5A1 bypasses T and instead uses androstenedi- proliferative response. Cells were washed and sup- one as a substrate for 5a-reduction to produce 5a- plemented with phenol red-free medium containing androstanedione, which is then converted to DHT 5% charcoal dextran-treated FBS (Cambrex Corp., [15]. Inhibitors of 5a-reductases block the conversion East Rutherford, NJ) for 48 hr prior to androgen treat- of T to the more potent androgen, DHT resulting ment. For fusion gene expression studies, cells were in reduced androgenic activity in the prostate. pre-incubated with dutasteride for 2 hr and then 5a-Reductase inhibitors such as finasteride and dutas- treated with either T or DHT or dutasteride to test the teride are commonly used in cases of prostatic effect of dutasteride on the conversion of T to DHT. The Prostate 1544 Ateeq et al. Cell Proliferation Assay Forward primer: 50-TAGGCGCGAGCTAAGCAGG- AG-30 Equal numbers of VCaP cells were plated and 0 Reverse primer: 5 -GTAGGCACACTCAAACAACG- grown to 70% confluence before treatment with vary- 0 ACTGG-3