ANTICANCER RESEARCH 37 : 6893-6898 (2017) doi:10.21873/anticanres.12152

The Genomic Alterations of 5 α- and Their Inhibitor ’s Effect in Bladder Cancer CHI-CHENG CHEN 1,2* , CHI-PING HUANG 1* , YI-TUNG TSAI 1, TENG-FU HSEIH 2 and CHIH-RONG SHYR 1

1Sex Hormone Research Center, Departments of Medical Laboratory Science and Biotechnology and Urology, Graduate Institute of Clinical Medical Science, China Medical University and Hospital, Taichung, Taiwan, R.O.C.; 2Department of Urology, Taichung Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taichung, Taiwan, R.O.C.

Abstract. Background/Aim: Since affect urothelial diagnosed with muscle-invasive disease, while only a small bladder cancer (UBC), we examined whether 5 α- reductases proportion of UBC patients have distant metastases detectable (5-AR) have genomic alterations in UBC and whether 5 α- at their initial diagnosis (2). Low-grade non-muscle-invasive inhibitors (5-ARI) affect UBC. Materials and tumors that recur frequently rarely progress to muscle Methods: The cBioPortal was used to analyze genomic invasion, but muscle-invasive tumors that are usually alternations of 5-ARs in UBC cancer genomic datasets. Next, diagnosed de novo frequently metastasize (3, 4). Almost half we used the Taiwan National Health Insurance Research the muscle-invasive UBC patients eventually develop obvious database to conduct a population-based case-control study to distant metastases (2), thus making UBC a deadly disease. investigate the effect of a 5-ARI, finasteride on UBC Comprehensive genomic characterization of UBC reveal a incidence. We also performed an XTT assay to examine the complex landscape of molecular alterations including direct effect of finasteride on UBC cells. Results: We found multiple involved in cell cycle regulation, chromatin that 5-AR genomic alternations were observed in 29% of UBC regulation, and kinase signaling pathways (3). These patients and patients with alternations had shorter disease- complicated molecular features cause various pathogenesis free survival. Also, the use of finasteride with >180 cDDDs and heterogeneity of UBC, resulting in the therapeutic reduced the risk of UBC. Finasteride could directly inhibit failures to obtain the effective molecularly targeted agents to UBC cell growth. Conclusion: Based on our findings, we treat this disease (3, 4). In the era of precision medicine, it is concluded that 5-AR could be explored as a therapeutic target proposed to match the most accurate and effective treatment for UBC with 5-ARIs. to each individual cancer patient based on their genomic alternations to find the therapeutic agents that target altered Urothelial Bladder cancer (UBC) is the most common cancer genes or pathways (5). Therefore, with this precision of the urinary tract with an estimated 79,030 new cases and medicine approach, it is possible to also find a potential 16,870 deaths projected to occur in the United States in 2017 therapeutic option in UBC based on its genomic alterations. (1). Most UBCs present as non-muscle invasive bladder An epidemiological study has shown that UBC occurs more cancer, but an approximate 25% of UBC patients are often in men than in women (male:female ratio is ~3:1) (1). In cell based experiments and animal models, male sex hormones, androgens and receptors have been demonstrated to be implicated in the development and *These Authors contributed equally to this study. progression of UBC (6-9). Using UPII-SV40T transgenic mice Correspondence to: Dr. T.F. Hsieh, Department of Urology, Taichung that spontaneously develop UBC, Johnson et al. reported that Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taichung 427, UBC progression was repressed by castration and restored by Taiwan, R.O.C. Tel: +886 436060666, e-mail: [email protected] and the administration of (DHT), a more Dr. C.R. Shyr, Research Center, Departments of Medical potent androgen than (10). Furthermore, Laboratory Science and Biotechnology and Urology, Graduate Institute Miyamoto showed that in ARKO mice models, DHT alone of Clinical Medical Science, China Medical University and Hospital, was able to promote bladder cancer development under Taichung 404, Taiwan, R.O.C. Tel: +886 422052121, e-mail: [email protected] chemical carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) induction (7), suggesting DHT is involved in UBC Key Words: Urothelial bladder cancer, 5 α- reductase, finasteride, development and progression. Furthermore, in a retrospective androgen, dihydrotestosterone. study, androgen deprivation therapy (ADT) using luteinizing

6893 ANTICANCER RESEARCH 37 : 6893-6898 (2017) hormone-releasing hormone (LHRH), a standard treatment to missense mutations), CNA from GISTIC and RNA seq data with the treat advanced cancer (PC) patients has been shown default setting to perform query on all completed tumors in Bladder to significantly reduce the risk of UBC recurrence in PC Urothelial Carcinoma (TCGA) dataset. patients who had coincidental UBC (11). Based on these Cell culture and XTT assay. Human TCC-SUP bladder cancer cells evidence, we hypothesized that DHT could play a critical role were obtained from the American Type Culture Collection and in contributing to male dominance in UBC and AR-mediated maintained in DMEM supplemented with 10% fetal bovine serum. gender difference in UBC development and progression. To test the effects of finasteride on cell growth, the cells were 3 Therefore, the genomic alternations of 5 α- Reductases that seeded at 5 ×10 cells/well onto 96-well plates and incubated convert testosterone into the more potent DHT (12) could overnight. At time 0, media were replaced with fresh complete DMEM media supplemented with 10% fetal bovine serum (FBS) or influence UBC development and progression. Thus, it is 10% dextran-coated charcoal stripped FBS (DC-FBS) and then cells possible to use 5-ARI as a therapeutic agent for preventing were treated with finasteride (AdooQ BioScience, Irvine, CA, USA) and treating UBC development and progression. with concentrations as indicated. After 72 h, XTT assay kit (Sigma- There are three isoenzymes of 5-AR: 5 α- reductase Aldrich, St. Louis, MO, USA) was used to measure cell viability, 1, 2, and 3 ( SRD5A1, SRD5A2 and SRD5A3 ), which are which was expressed as a percentage of the absorbance measured expressed in many tissues: type 1 5 α- in the vehicle-treated cells. The mean of three parallel samples was (SRD5A1 ) is predominantly expressed in the skin, scalp, calculated. Experiments were performed in triplicate and standard deviations were calculated based on the mean of three experiments. sebaceous gland, , and brain; type 2 5 α- steroid reductase (SRD5A2 ) is found predominantly in androgen target organs Clinical observations. This study used the cancer dataset and LHID such as the prostate, genital skin, and seminal vesicles; 2010 of the NHIRD from 1 January, 2002 to 31 December, 2013. The whereas type 3 5 α- steroid reductase ( SRD5A3 ) is NHIRD is maintained by Taiwan’s National Health Research overexpressed in castration-resistant (13, 14). Institutes, and is made available to researchers (http:// nhird.nhri.org.tw/date_01_en.html). For cases, we first identified 5α- reductase inhibitor drugs are used to treat benign prostatic 16,784 patients who had received a first-time bladder cancer diagnosis hyperplasia and prostate cancer (15, 16). Finasteride, a 5-ARI, (ICD-9-CM code 188.X) between January 1, 2003 and December 31, mainly inhibits type II and III 5-AR, but is less effective on 2012 in cancer dataset. The date of receiving their first-time bladder type I 5-AR and was shown to decrease the mean serum level cancer diagnosis was defined as the index date. We then selected of DHT by 71% after 6 months of use (17, 18). controls from the remaining enrollees without bladder cancer diagnosis To test our hypothesis, we explored the UBC cancer genomics in LHID 2010. The controls included all patients without bladder dataset from The Cancer Genome Atlas (TCGA) projects to cancer. We attempted to reduce selection bias by bundling many confounding covariates that may be present in an observational study analyze the genomic alternations of 5-ARs in UBC. We also with this number of variables; we then implemented a systematic conducted a population-based survey to investigate whether the random sampling design to select a matching sample from the controls use of finasteride could affect the risk of UBC using Taiwan frequency matched by age and the year of the index date. The Insurance Research Database (NHIRD) from Taiwan National matching comparison to case ratio was 1 to 1. In this study, the year Health Insurance (NHI) program which is a mandatory general of the index date for cases was defined as the year in which the cases health insurance program, providing complete medical care received their first bladder cancer diagnosis. However, for controls, coverage to all Taiwanese residents. The cell-based experiments the index year was simply a matched year in which the controls had a healthcare utilization. Furthermore, we defined the first healthcare were also performed to directly determine the effect of utilization occurring in the index year as the index date for controls. finasteride on bladder cancer cells. The clinical observations and The defined daily dose (DDD) is a unit for measuring a in vitro experiments indicated that 5-AR genomic alternations prescribed amount of a drug; it is the assumed average daily were present and associated with a worse outcome in UBC maintenance dose of a drug consumed for its main indication in patients and there was a significantly decreased risk of UBC adults. The cumulative DDD (cDDD), which indicates the duration among patients who received finasteride compared with the of exposure, was estimated as the sum of dispensed DDD of 5- general population. Furthermore, finasteride could directly alpha-reductase inhibitor. To examine the dose-effect relationship, we categorized the use of 5ARI into three groups in each cohort by suppress the growth of bladder cancer cells. All suggest that 5- cDDD (0, 1 to 179, and more than 180). ARI could be beneficial to UBC patients as a therapeutic option The independent variables were co-morbidity (diabetes mellitus, to prevent and treat UBC patients who have genomic alternations cerebrovascular disease, chronic kidney disease, hypertension, and in 5-AR, which contribute to the progression of UBC. hyperlipidemia), geographical area of residence, and socioeconomic status (SES). Materials and Methods Statistical analysis. Disease-free survival curve was generated by The cBioPortal database analysis. We analyze the genomic the cBioPortal and displayed with values calculated using the log- alternations of three 5-AR genes using the cBioPortal tool, an open rank test and Kaplan-Meier plots. Statistical significance of the p platform that provides access to data of cancer genomic studies data ( p- values) was provided by the program. The conditional from the TCGA projects (19, 20). Our selected genomic profiles of logistic regression was used to measure the odds ratios (ORs) and all three 5-ARs included alterations (amplification, deep deletion, 95% confidence intervals (95% CIs) for the effect of finasteride on

6894 Chen et al : 5 α- Reductases in Bladder Cancer

UBC risk. In cell-based experiments, student’s t- test was performed which indicates the duration of exposure to estimate the sum of to determine whether there was a statistically significant difference dispensed DDD of 5-alpha-reductase inhibitor, finasteride. We between the control and treatment. All statistical analyses were then used conditional logistic regression measured odds ratio to performed using SPSS statistical software. Data are present as analyze the correlation between the cDDDs of finasteride and mean±standard deviation (SD). A p- value <0.05 was considered to be statistically significant. the risk of UBC. And we found that patients with high cDDDs (> 180) have a lower risk of UBC (Table II). Results To validate the clinical observations that 5-ARs are involved in the development and progression of UBC and To prove that the altered androgen pathway could be present 5-ARI could have an effect on the prevention and in UBC, we analyzed the mutations, copy number alterations intervention of UBC development and progression, we (CNA) as well as mRNA expression of 5-ARs that include examined whether finasteride could have an effect directly three isoenzymes encoded by 3 different genes: 3-oxo-5 α- on UBC cells. We then treated TCC-SUP cells, a bladder steroid 4-dehydrogenase 1 ( SRD5A1 ), SRD5A2 , and SRD5A3 cancer cell line derived from an undifferentiated, Grade IV by the cBioPortal tool on the Bladder Urothelial Carcinoma transitional cell carcinoma (21) with different concentrations (TCGA, Provisional) data on all 125 complete tumor samples of finasteride and then cells were incubated for 72 h. The that have mRNA, CNA and sequencing data (19, 20). The cells were harvested for XTT viability assay. The result TCGA network analyzed UBC samples consisted of showed that in the presence of 10% FBS which contains a chemotherapy naive, muscle-invasive, high-grade urothelial castrate level of testosterone (22.0±6.1 pg/mL), which still tumors (T2-T4a, Nx, Mx), as well as peripheral blood and/or could be converted to produce a physiologic ( i.e. 10 nM) tumor-adjacent, histologically normal-appearing bladder level of intracellular DHT by prostate cancer cells (22), all tissue to define the molecular landscape of UBC (3). The concentrations of finasteride suppressed cell growth (Figure results showed that there were total genomic alternations in 3A). We also examined the effect of finasteride in the 37 (29%) of 126 sequenced cases/patients (Figure 1A). For presence of 10% of dextran-coated charcoal stripped FBS the SRD5A1 , there were 15 cases (11.9%) of (DC-FBS), which removes most , and finasteride was amplification and 1 case of mutation in 126 cases. For the able to repress cell growth at higher does (Figure 3B). SRD5A2 gene, there were 2 cases (1.6%) of amplification and 1 case of deletion in 126 cases. And for the SRD5A3 gene, Discussion there was 1 case of amplification in 126 cases. Furthermore, mRNA upregulation was found to be present in 21 (17%) of UBC is three to four times more common in men than in 126 sequenced cases/patients with 11%, 0.8% and 5% on women, and represents the fourth most common cancer and SRD5A1, SRD5A2 , and SRD5A3 respectively (Figure 1B). the eighth most common cause of cancer death in males in Disease-free survival Kaplan –Meier plot analysis showed the U.S.A. (1). This gender disparity is proposed to be that patients with genomic alterations on SRD5A1, SRD5A2, explained by the roles of androgens and androgen receptor and SRD5A3 genes had lower median survival (17.35 vs. (AR) in the development and progression of UBC, which 25.23 months) (Figure 2A). The network view of 5-AR have been examined in clinical observational and animal genes shows that their biologically interacted genes also have models (23). Furthermore, androgen deprivation therapy was genomic alterations in UBC and there are FDA approved shown to prevent bladder cancer recurrence (11), suggesting drugs (ex. finasteride) targeting genes in the network (Figure that androgen modulation could be a therapeutic option to 2B). These genes are involved in hormone regulation, have prevent or treat UBC. In the era of precision medicine, it is “catalysis-precedes” interactions with 5-ARs genes and their proposed to obtain molecular profiling of cancer samples and genomic alterations are mostly gene amplifications and find altered genes or pathways as targets for specific mRNA up-regulation as 5-AR genes do, suggesting that treatments. In the current study, by analyzing TCGA UBC hormone imbalance could play a critical role in UBC dataset, we found that 5-AR genes are found to be altered in development and progression. DNA and RNA levels. The main genomic alternations are Since finasteride is listed as a drug which could target 5-ARs gene amplification and mRNA up-regulations. We then in UBC, we wanted to know the relationship between further demonstrated that a 5-ARI, finasteride reduces the finasteride and UBC. We then performed a case-control study risk of UBC in our population-based study and in cell-based on the effect of finasteride on the risk of UBC in the Taiwan experiments, finasteride was also shown to suppress bladder National Health Insurance Research Database. We identified cancer cell growth. Therefore, our study provides a novel UBC patients as study cases and non-UBC patients, matched insight for a possible therapeutic intervention with 5-ARIs with gender and age as controls from 2002 to 2013. The for UBC patients whose genomic profiles of 5-AR genes are demographic data and comorbidities of the cohort are shown in altered since it is proposed to have clinical trials based on Table I. We used the cumulative defined daily doses (cDDDs), patients with relevant druggable genomic alterations (3).

6895 ANTICANCER RESEARCH 37 : 6893-6898 (2017)

Figure 1. The genomic alterations of 5-ARs (SRD5A1, SRD5A2 and SRD5A3) in UBC. (A) Oncoprint of mutations and CNAs in 5-ARs genes in UBC. (B) Heatmap of 5-AR gene mRNA expression in UBC.

Figure 2. (A) The disease-free survival analysis of patients with or without 5-AR genomic alterations using Kaplan –Meier plot. (B) The network view of biological interactions of 5-ARs to altered genes in UBC and FDA approved drugs targeting genes in the network. LHB: Luteinizing hormone beta polypeptide; CGA: glycoprotein hormones, alpha polypeptide; POMC: proopiomelanocortin; AKR1C3: aldo-keto reductase family 1 member C3.

Corroboratively, Morales et al. reported that finasteride was with AST (with, 12.5% vs. without, 30.1%) and 3.1% patients associated with a reduced incidence of bladder cancer (hazard without AST, but no patients with AST progressed to muscle ratio: 0.634; 95% confidence interval=0.493-0.816; p= 0.0004) invasive bladder cancer (25). In consistent with our results, as observed by self-report in the Prostate, Lung, Colorectal, these studies also suggest the potential therapeutic option and Ovarian cancer screening trial, large prospective screening using 5-ARI to prevent and treat UBC. study (24). A recent study examined the intravesical The majority of patients with UBC are treated with surgery recurrence rate among men with non-muscle invasive bladder and chemotherapy and treatment for muscle-invasive bladder cancer, who have or have not received androgen suppression cancer has not advanced beyond cisplatin based combination therapy (AST) by androgen deprivation therapy for prostate chemotherapy and surgery in the past 30 years with no FDA cancer or another 5-ARI, , for benign prostatic approved targeted agents (3). Tumor heterogeneity and hyperplasia and found lower intravesical recurrence in patients acquired resistance because of somatically acquired genetic,

6896 Chen et al : 5 α- Reductases in Bladder Cancer

Figure 3. The effect of finasteride on TCC-SUP cell growth by XTT assay. Cells were plated in cell culture plates for 24 h. Then cells were incubated in MEM containing 10% FBS (A) or 10% DC-FBS (B) and treated with 0, 0.1, 1 and 10 ( μM) finasteride respectively for 72 h. The cell viability was determined with the XTT assay. Data are presented as mean±SD *p<0.05, **p<0.01.

Table I. Baseline characteristics of the patients from 2002 to 2013 in Table II. Conditional logistic regression measured odds ratio and 95% Taiwan (n=21,948). confidence intervals.

Variables Control, N ( %) Case s, N ( %) p-Value Characteristics Crude Adjusted

Total 16784 (50.0) 16784 (50.0) OR 95%CI OR 95%CI Mean age, years(±SD) 68.6±13.0 68.6±13.0 - CCI score <0.001 5ARI 0-1 12927 (77.0) 12033 (71.7) 0 cDDD 1.00 Ref. 1.00 Ref. 2-3 3005 (17.9) 3797 (22.6) 1-179 cDDD 0.90 0.78-1.03 0.93 0.79-1.09 >3 852 (5.1) 954 (5.7) ≥180 cDDD 0.82 0.71-0.95** 0.84 0.70-0.99* Comorbidities Diabetes mellitus 3029 (18.0) 3307 (19.7) <0.001 cDDD: Cumulative defined daily dose; OR: odds ratio; CI: confidence Cerebrovascular Disease 1981 (11.8) 1696 (10.1) <0.001 interval. Adjusted OR: Adjusted for comorbidities, socioeconomic Chronic kidney Disease 950 (5.7) 1888 (11.2) <0.001 status, geographic region. * p<0.05, ** p<0.01. Hypertension 5827 (34.7) 6526 (38.9) <0.001 Hyperlipidemia 2670 (15.9) 2062 (12.3) <0.001 Socioeconomic status <0.001 Low 6008 (35.8) 12122 (72.2) metastatic bladder cancer who achieved a durable (>2 years) Moderate 5881 (35.0) 3962 (23.6) High 4895 (29.2) 700 (4.2) and ongoing complete response to everolimus, a drug targeting Geographic region <0.001 the mTORC1 (mammalian target of rapamycin) was obtained Northern 8058 (48.0) 7275 (43.3) and revealed a two-base-pair deletion in the TSC1 (tuberous Central 2978 (17.7) 2821 (16.8) sclerosis complex 1) gene and a nonsense mutation in the NF2 Southern 5155 (30.7) 6237 (37.2) (neurofibromatosis type 2), both associating with mTORC1 Eastern 593 (3.5) 451 (2.7) dependence, which may explain the patient’s drug response (26). This finding demonstrates the feasibility of precision medicine that the genomic alternations of patients may aid in the identification of targeted anticancer drugs that UBC epigenetic, transcriptomic, and proteomic alterations in cancer patients most likely would respond to. 5-ARIs, investigated in cell cones may contribute to chemotherapy’s failure and our present study, may hold the promise in the treatment of resistance to targeted therapy in UBC. However, the molecular UBC with this precision medicine approach. profiling of cancers would provide valuable information about In conclusion, our study found that there are genomic genomic alterations in cancer, which may grant cancer cells alternations of 5-AR in UBC patients and finasteride could growth and survival advantages, but become Achilles’ heel of reduce the risk of UBC and inhibit cancer cell growth. These the malignancy, becoming possible actionable targets (3). A results suggest that the efficacy and feasibility of 5-AR single-patient case reported that with whole-genome inhibitors, such as finasteride, to prevent or treat UBC sequencing, the tumor genetic profile of a patient with patients with altered 5-AR genes.

6897 ANTICANCER RESEARCH 37 : 6893-6898 (2017)

Conflicts of Interest 13 Uemura M, Tamura K, Chung S, Honma S, Okuyama A, Nakamura Y and Nakagawa H: Novel 5 alpha-steroid reductase The Authors disclosed no conflicts of interest. (SRD5A3 , type-3) is overexpressed in hormone-refractory prostate cancer. Cancer Sci 99 : 81-86, 2008. Acknowledgements 14 Steers WD: 5alpha-reductase activity in the prostate. Urology 58 : 17-24, 2001. This work was supported by the MOST grants (MOST 103-2633- 15 Tarter TH and Vaughan ED Jr.: Inhibitors of 5alpha-reductase in B-039-003- and MOST 106-2314-B-303-017-), CMU grant the treatment of benign prostatic hyperplasia. Curr Pharm Des (CMU105-S-51), CMUH grant (DMR-CELL-17020), Buddhist Tzu 12 : 775-783, 2006. Chi General Hospital grant (TTCRD 104-12) and in part by Taiwan 16 Hudak SJ, Hernandez J and Thompson IM: Role of 5 alpha- Ministry of Health and Welfare Clinical Trial Center (MOHW105- reductase inhibitors in the management of prostate cancer. Clin TDU-B-212-133019). Interv Aging 1: 425-431, 2006. 17 McConnell JD, Wilson JD, George FW, Geller J, Pappas F and References Stoner E: Finasteride, an inhibitor of 5 alpha-reductase, suppresses prostatic dihydrotestosterone in men with benign 1 Siegel RL, Miller KD and Jemal A: Cancer Statistics, 2017. CA prostatic hyperplasia. J Clin Endocrinol Metab 74 : 505-508, Cancer J Clin 67 : 7-30, 2017. 1992. 2 Dinney CP, McConkey DJ, Millikan RE, Wu X, Bar-Eli M, 18 Azzouni F, Godoy A, Li Y and Mohler J: The 5 alpha-reductase Adam L, Kamat AM, Siefker-Radtke AO, Tuziak T, Sabichi AL, family: a review of basic biology and their role in Grossman HB, Benedict WF and Czerniak B: Focus on bladder human diseases. Adv Urol 2012 : 530121, 2012. cancer. Cancer Cell 6: 111-116, 2004. 19 Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer 3 The Cancer Genome Atlas Research N: Comprehensive SO, Sun Y, Jacobsen A, Sinha R, Larsson E, Cerami E, Sander molecular characterization of urothelial bladder carcinoma. C and Schultz N: Integrative analysis of complex cancer Nature 507 : 315-322, 2014. genomics and clinical profiles using the cBioPortal. Science 4 Knowles MA and Hurst CD: Molecular biology of bladder Signaling 6: pl1-pl1, 2013. cancer: new insights into pathogenesis and clinical diversity. Nat 20 Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, Rev Cancer 15 : 25-41, 2015. Jacobsen A, Byrne CJ, Heuer ML, Larsson E, Antipin Y, Reva B, 5 Friedman AA, Letai A, Fisher DE and Flaherty KT: Precision Goldberg AP, Sander C and Schultz N: The cBio cancer genomics medicine for cancer with next-generation functional diagnostics. portal: an open platform for exploring multidimensional cancer Nat Rev Cancer 15 : 747-756, 2015. genomics data. Cancer Discov 2: 401-404, 2012. 6 Hsieh TF, Chen CC, Ma WL, Chuang WM, Hung XF, Tsai YR, 21 Nayak SK, O’Toole C and Price ZH: A cell line from an Lin MH, Zhang Q, Zhang C, Chang C and Shyr CR: Epidermal anaplastic transitional cell carcinoma of human urinary bladder. growth factor enhances androgen receptormediated bladder Br J Cancer 35 : 142-151, 1977. cancer progression and invasion via potentiation of AR 22 Sedelaar JP and Isaacs JT: Tissue culture media supplemented transactivation. Oncol Rep 30 : 2917-2922, 2013. with 10% fetal calf serum contains a castrate level of 7 Miyamoto H, Yang Z, Chen YT, Ishiguro H, Uemura H, Kubota testosterone. Prostate 69 : 1724-1729, 2009. Y, Nagashima Y, Chang YJ, Hu YC, Tsai MY, Yeh S, Messing 23 Dobruch J, Daneshmand S, Fisch M, Lotan Y, Noon AP, Resnick EM and Chang C: Promotion of bladder cancer development and MJ, Shariat SF, Zlotta AR and Boorjian SA: Gender and bladder progression by androgen receptor signals. J Natl Cancer Inst 99 : cancer: a collaborative review of etiology, biology, and 558-568, 2007. outcomes. Eur Urol 69 : 300-310, 2016. 8 Tanahashi NK, Suzawa N and Azuma C: Effects of sex 24 Morales EE and Grill S: Finasteride reduces risk of bladder hormones on oncogenesis in rat urinary bladder by N-butyl-N- cancer in a large prospective screening study. Eur Urol 69 : 407- (4-hydroxybutyl)-nitrosamine. Int J Clin Pharmacol Biopharm 410, 2016. 15 : 101-105, 1977. 25 Shiota M, Kiyoshima K, Yokomizo A, Takeuchi A, Kashiwagi 9 Imada S, Akaza H, Ami Y, Koiso K, Ideyama Y and Takenaka E, Dejima T, Takahashi R, Inokuchi J, Tatsugami K, and Eto M: T: Promoting effects and mechanisms of action of androgen in Suppressed recurrent bladder cancer after androgen suppression bladder carcinogenesis in male rats. Eur Urol 31 : 360-364, 1997. with androgen deprivation therapy or 5alpha-reductase inhibitor. 10 Johnson AM, O’Connell MJ, Miyamoto H, Huang J, Yao JL, J Urol 197 : 308-313, 2017. Messing EM and Reeder JE: Androgenic dependence of 26 Iyer G, Hanrahan AJ, Milowsky MI, Al-Ahmadie H, Scott SN, exophytic tumor growth in a transgenic mouse model of bladder Janakiraman M, Pirun M, Sander C, Socci ND, Ostrovnaya I, cancer: a role for thrombospondin-1. BMC Urol 8: 7, 2008. Viale A, Heguy A, Peng L, Chan TA, Bochner B, Bajorin DF, 11 Izumi K, Taguri M, Miyamoto H, Hara Y, Kishida T, Chiba K, Berger MF, Taylor BS and Solit DB: Genome sequencing Murai T, Hirai K, Suzuki K, Fujinami K, Ueki T, Udagawa K, identifies a basis for everolimus sensitivity. Science 338 : 221, Kitami K, Moriyama M, Miyoshi Y, Tsuchiya F, Ikeda I, 2012. Kobayashi K, Sato M, Morita S, Noguchi K and Uemura H: Androgen deprivation therapy prevents bladder cancer recurrence. Oncotarget 5: 12665-12674, 2014. 12 Tindall DJ and Rittmaster RS: The rationale for inhibiting Received August 31, 2017 5alpha-reductase isoenzymes in the prevention and treatment of Revised September 25, 2017 prostate cancer. J Urol 179 : 1235-1242, 2008. Accepted September 26, 2017

6898