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ERG/AKR1C3/AR Constitutes a Feed-Forward Loop for AR Signaling in Prostate Cancer Cells Katelyn Powell1, Louie Semaan1, M

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ERG/AKR1C3/AR Constitutes a Feed-Forward Loop for AR Signaling in Prostate Cancer Cells Katelyn Powell1, Louie Semaan1, M Published OnlineFirst March 9, 2015; DOI: 10.1158/1078-0432.CCR-14-2352 Biology of Human Tumors Clinical Cancer Research ERG/AKR1C3/AR Constitutes a Feed-Forward Loop for AR Signaling in Prostate Cancer Cells Katelyn Powell1, Louie Semaan1, M. Katie Conley-LaComb1, Irfan Asangani2, Yi-Mi Wu2, Kevin B. Ginsburg1, Julia Williams3, Jeremy A. Squire4, Krishna R. Maddipati5, Michael L. Cher1,5,6, and Sreenivasa R. Chinni1,5,6 Abstract Purpose: Intratumoral androgen synthesis in prostate cancer Results: We found that ERG regulated the expression of the ABE contributes to the development of castration-resistant prostate AKR1C3 in prostate cancer cells via direct binding to the AKR1C3 cancer (CRPC). Several enzymes responsible for androgen bio- gene. Knockdown of ERG resulted in reduced AKR1C3 expression, synthesis have been shown to be overexpressed in CRPC, thus which caused a reduction in both DHT synthesis and PSA expres- contributing to CRPC in a castrated environment. The TMPRSS2– sion in VCaP prostate cancer cells treated with 5a-androstane- ERG transcription factor has been shown to be present in primary dione (5a-Adione), a DHT precursor metabolite. Immunohisto- prostate cancer tumors as well as CRPC tumors. We hypothesize chemical staining revealed that ERG was coexpressed with that TMPRSS2–ERG fusions regulate androgen biosynthetic AKR1C3 in prostate cancer tissue samples. enzyme (ABE) gene expression and the production of androgens, Conclusions: These data suggest that AKR1C3 catalyzes the which contributes to the development of CRPC. biochemical reduction of 5a-Adione to DHT in prostate cancer Experimental design: We used a panel of assays, including cells, and that ERG regulates this step through upregulation of lentivirus transduction, gene expression, chromatin immunopre- AKR1C3 expression. Elucidation of ERG regulation of ABEs in CRPC cipitation and sequencing, liquid chromatography-mass spectro- may help to stratify TMPRSS2–ERG fusion-positive prostate cancer metric quantitation, immunocytochemistry, immunohistochem- patients in the clinic for anti–androgen receptor–driven therapies; istry, and bioinformatics analysis of gene microarray databases, to and AKR1C3 may serve as a valuable therapeutic target in the determine ERG regulation of androgen synthesis. treatment of CRPC. Clin Cancer Res; 21(11); 2569–79. Ó2015 AACR. Introduction ERG has been shown to have several biologic functions that facilitate its oncogenic properties in prostate cancer. It has been The TMPRSS2–ERG fusion gene has been shown to be present shown to regulate cellular migration and invasion, epithelial-to- in approximately 40% of prostate cancer tumors, including pri- mesenchymal transition, dedifferentiation, and AR signaling (16– mary and advanced prostate cancer (1–6). The TMPRSS2–ERG 19); however, the biologic functions of ERG contributing to the fusion gene results in androgen receptor (AR)–induced overex- development of castration-resistant prostate cancer (CRPC) have pression of the ERG transcription factor (7). The presence of not been fully elucidated. Interestingly, ERG fusion-positive pro- TMPRSS2–ERG in prostate cancer has been shown to be associ- state cancer patients responded better to abiraterone treatment, a ated with poor clinical prognosis (2, 8–10). ERG fusion-positive CYP17A1 enzyme inhibitor used to treat CRPC, compared with prostate cancer patients had significantly lower disease-free sur- ERG fusion-negative patients (5). These data are consistent with vival in watchful waiting cohorts (11, 12), and significantly greater ERG fusion-positive patients having a higher PSA recurrence, and risk of biochemical PSA recurrence compared with fusion-nega- suggests that ERG may regulate the synthesis of intratumoral tive prostate cancer patients (13–15). androgens, and AR activation in CRPC. Upregulation of ABEs and intratumoral androgen synthesis have been shown to facilitate the development of CRPC (20–22). 1Department of Urology, Wayne State University School of Medicine, This can be seen with the ability of abiraterone treatment to reduce Detroit, Michigan. 2Department of Pathology, University of Michigan, serum and intratumoral dihydrotestosterone (DHT) and testos- 3 Ann Arbor, Michigan. Department of Pathology and Molecular Med- terone levels in CRPC patients (23–25). One ABE that has recently icine, Queen's University, Kingston, Ontario, Canada. 4Department of Pathology and Forensic Medicine, University of Sao Paulo at Ribeirao~ become of particular interest is AKR1C3, because it has been Preto, Brazil. 5Department of Pathology, Wayne State University shown to be highly upregulated in CRPC tumors (26). AKR1C3 6 School of Medicine, Detroit, Michigan. Department of Oncology, enzyme functions downstream of CYP17A1 enzyme in the DHT Wayne State University School of Medicine, Detroit, Michigan. synthesis pathway (22), and it may play a pivotal role in DHT Note: Supplementary data for this article are available at Clinical Cancer synthesis in CRPC. Intratumoral DHT synthesis bypasses the Research Online (http://clincancerres.aacrjournals.org/). synthesis of testosterone, and DHT is synthesized directly from Corresponding Author: Sreenivasa R. Chinni, Wayne State University School of androstanedione (5a-Adione) or androstanediol (22, 27, 28). fi Medicine, 540 East Can eld Avenue, Detroit, MI 48201. Phone: 313-577-1833; Inhibitors specific for AKR1C3 are currently being developed (29– Fax: 313-577-0057; E-mail: [email protected] 31), and may prove to be beneficial for treatment of CRPC. doi: 10.1158/1078-0432.CCR-14-2352 This study is focused on TMPRSS2–ERG transcription factor Ó2015 American Association for Cancer Research. upregulation of AKR1C3 expression and DHT synthesis in CRPC. www.aacrjournals.org 2569 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst March 9, 2015; DOI: 10.1158/1078-0432.CCR-14-2352 Powell et al. lentivector was used for ERG overexpression (Open BioSys- Translational Relevance tems).TruncatedERGandfull-lengthERGwereclonedintothe Intracrine androgen synthesis is a viable target for clinical pLOC lentivector. Cloning primers are listed in Supplementary management of patients undergoing traditional androgen Table S1. Lentiviral particles were generated in HEK293T cells deprivation therapies. Despite newer therapies such as abir- using a Trans-Lentiviral Packaging Kit (Thermo Fisher Scientif- aterone acetate, a drug that targets androgen biosynthetic ic). VCaP cells were infected with pGIPZ lentiviral particles and enzyme (ABE) Cyp17, the disease eventually relapses (abir- selected with (0.3 mg/mL) puromycin (Research Products Inter- aterone resistance). Elucidating molecular mechanisms con- national Corporation). LNCaP and BPH-1 cells were infected tributing to the expression of biosynthetic enzymes in andro- with pLOC lentiviral particles and selected with (2–4 mg/mL) gen production helps to understand the biologic mechanisms blasticidin (Research Products International Corporation). A contributing to the development of castration-resistant pros- pooled lentiviral cell population was used for all lentiviral tate cancer (CRPC). ERG factor regulates key enzymes that experiments. Transient transfections were performed using participate in DHT production in prostate cancer cells. ERG Lipofectamine2000 Reagent (Invitrogen) in OptiMEM media specifically binds to AKR1C3 gene, regulates its expression, (Invitrogen). promotes DHT production via 5a-androstanedione and results in androgen receptor (AR) activation. Further, both Western blotting and antibodies ERG factor and AKR1C3 are coexpressed in human prostate Western blotting was performed using SDS-PAGE with gel TMPRSS2– tumors and predict lower survival in patients. As transfer to a nitrocellulose membrane. Membranes were blocked ERG fusions are prevalent in prostate cancer, including CRPC, with 5% non-fat milk and probed with primary antibody in 5% and our data present a link between ERG expression, androgen milk. Membranes were probed with horseradish peroxidase– biosynthesis, and AR activation, this study suggests that linked secondary antibody in 5% milk. Protein bands were – TMPRSS ERG fusion-positive patients may have favorable detected using enhanced chemiluminescence substrate and auto- responses to anti-AR therapies. radiography film. Primary and secondary antibodies are listed in Supplementary Table S2. Densitometry was performed using ImageJ software (NIH). We have evidence, suggesting that AKR1C3 functions as a key ABE RT-PCR and RT-qPCR responsible for DHT synthesis via biochemical reduction of 5a- RNA was extracted from VCaP and LNCaP cells using the TRizol Adione into DHT (bypassing testosterone). Our data indicate that (Invitrogen) method. For RT-PCR experiments, cDNA was ampli- 5a-Adione is the primary DHT precursor metabolite, and that 5a- fied using an Eppendorf Mastercycler Realplex2 qPCR machine. Adione can induce high levels of AR activation in CRPC cells. PCR products were loaded onto a 0.8% agarose gel containing Elucidation of TMPRSS2–ERG regulation of AKR1C3 may help to ethidium bromide. For RT-qPCR experiments, cDNA was ampli- stratify ERG fusion-positive prostate cancer patients toward anti– fied with SYBR green from the SensiFAST SYBR Kit obtained from AR-targeted therapies. In addition, our data support the notion Bioline (BIO-98002). Gene expression was quantified with the that AKR1C3 is an attractive therapeutic target for CRPC previously
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