Androgen Biosynthetic Enzymes Research Articles

Nuclear receptor ERRα contributes to castration-resistant growth of prostate cancer via its regulation of intratumoral androgen biosynthesis.

Enhanced intratumoral androgen biosynthesis and persistent androgen receptor (AR) signaling are key factors responsible for the relapse growth of castration-resistant prostate cancer (CRPC). Residual intraprostatic androgens can be produced by de novo synthesis of androgens from cholesterol or conversion from adrenal androgens by steroidogenic enzymes expressed in prostate cancer cells via different steroidogenic pathways. However, the dysregulation of androgen biosynthetic enzymes in CRPC still remains poorly understood. This study aims to elucidate the role of the nuclear receptor, estrogen-related receptor alpha (ERRα, ESRRA), in the promotion of androgen biosynthesis in CRPC growth.Methods: ERRα expression in CRPC patients was analyzed using Gene Expression Omnibus (GEO) datasets and validated in established CRPC xenograft model. The roles of ERRα in the promotion of castration-resistant growth were elucidated by overexpression and knockdown studies and the intratumoral androgen levels were measured by UPLC-MS/MS. The effect of suppression of ERRα activity in the potentiation of sensitivity to androgen-deprivation was determined using an ERRα inverse agonist.Results: ERRα exhibited an increased expression in metastatic CRPC and CRPC xenograft model, could act to promote castration-resistant growth via direct transactivation of two key androgen synthesis enzymes CYP11A1 and AKR1C3, and hence enhance intraprostatic production of dihydrotestosterone (DHT) and activation of AR signaling in prostate cancer cells. Notably, inhibition of ERRα activity by an inverse agonist XCT790 could reduce the DHT production and suppress AR signaling in prostate cancer cells.Conclusion: Our study reveals a new role of ERRα in the intratumoral androgen biosynthesis in CRPC via its transcriptional control of steroidogenic enzymes, and also provides a novel insight that targeting ERRα could be a potential androgen-deprivation strategy for the management of CRPC.

TMPRSS2-ERG fusions confer efficacy of enzalutamide in an in vivo bone tumor growth model

BackgroundCastrate Resistant Prostate Cancer (CRPC) is an advanced disease resistant to systemic traditional medical or surgical castration, and resistance is primarily attributed to reactivation of AR through multiple mechanisms. TMPRSS2-ERG fusions have been shown to regulate AR signaling, interfere with pro-differentiation functions, and mediate oncogenic signaling. We have recently shown that ERG regulates intra-tumoral androgen synthesis and thereby facilitates AR function in prostate cancer cells. We hypothesize that enzalutamide treatment will be more effective in cells/tumors with TMPRSS2-ERG translocations because these tumors have increased AR signaling.MethodsERG knockdown was performed with VCaP cells using lentiviral infections to generate VCaP ERGshRNA cells and control VCaP scr cells with scrambled shRNA. Cell-growth analysis was performed to determine the effect of enzalutamide. Reverse transcription, quantitative real-time PCR (RT-qPCR) was used to determine the expression of AR responsive genes. Luciferase tagged VCaP scr and shRNA infected cells were used in an intra-tibial animal model for bone tumor growth analysis and enzalutamide treatment used to inhibit AR signaling in bone tumors. Western blotting analyzed VCaP bone tumor samples for ERG, AR, AKR1C3 and HSD3B1 and HSD3B2 expression.ResultsEnzalutamide inhibited the growth of VCaP scr cells more effectively than shERG cells. Analysis of AR responsive genes shows that Enzalutamide treatment at 5 micromolar concentration inhibited by 85–90% in VCaP Scr cells whereas these genes were inhibited to a lesser extent in VCaP shERG cells. Enzalutamide treatment resulted in severe growth inhibition in VCaP scr shRNA cells compared to VCaP shERG cells. In bone tumor growth experiment, VCaP ERG shRNA cells grew at slower than VCaP scr shRNA cells. Androgen biosynthetic enzyme expression is lower VCaP shERG bone tumors compared to VCaP scr shRNA bone tumors and enzalutamide inhibited the enzyme expression in both types of tumors.ConclusionsThese data suggest that ERG transcription factor regulates androgen biosynthetic enzyme expression that enzalutamide treatment is more effective against VCaP bone tumors with an intact ERG expression, and that knocking down ERG in VCaP cells leads to a lesser response to enzalutamide therapy. Thus, ERG expression status in tumors could help stratify patients for enzalutamide therapy.

Abstract 5422: TMPRSS2-ERG fusions confers efficacy of enzalutamide in an in vivo bone tumor growth model

Abstract Castrate Resistant Prostate Cancer (CRPC) is an advanced disease resistant to systemic anti-androgen therapies and resistance is primarily attributed to reactivation of AR through multiple mechanisms. TMPRSS2-ERG fusions have been shown to regulate AR signaling, interfere with pro-differentiation functions, and mediate oncogenic signaling. In fact, cooperation between AR and ERG drives invasive adenocarcinoma even in the castrate environment. We have recently shown that ERG regulates intra-tumoral androgen synthesis thereby facilitates AR function in CRPC disease. We hypothesize that enzalutamide treatment will be more effective in cells/tumors with TMPRSS2-ERG translocations because these tumors have increased AR signaling. In other words, AR blockade is likely to induce a greater treatment response in tumors with ERG gene fusions than in tumors without such fusions. ERG knockdown was performed with VCaP cells using lentiviral infections to generate VCaP ERGshRNA cells and control VCaP scr cells with scrambled shRNA. Cell-growth analysis was performed to determine the effect of enzalutamide. Luciferase tagged VCaP scr and shRNA infected cells were used in intra-tibial animal model for bone tumor growth analysis and enzalutamide treatment used to inhibit AR signaling in bone tumors. Reverse transcription, quantitative real-time PCR (RT-qPCR) was used to determine the expression of AR responsive genes and androgen biosynthetic enzyme genes; Western blotting (WB) samples, for ERG, AR and AKR1C3 in VCaP bone tumors. Enzalutamide inhibited VCaP scr more effectively than shERG cells. Analysis of AR responsive genes show that Enzalutamide treatment at 5 micro molar concentration inhibited 85-90% in VCaP Scr cells whereas these genes inhibited to lesser extent in VCaP shERG cells. In bone tumor growth experiment, VCaP ERG shRNA cells grown at slower than VCaP scr shRNA cells. Enzalutamide treatment resulted in a sever growth inhibition in VCaP scr shRNA cells compared to VCaP shERG cells. Androgen biosynthetic enzyme expression is lower VCaP shERG bone tumors compared to VCaP scr shRNA bone tumors and enzalutamide inhibited the enzyme expression in both types of tumors. These data suggest that ERG transcription factor regulates androgen biosynthetic enzyme expression and enzalutamide treatment is effective against VCaP bone tumors with an intact ERG expression, and knocking down ERG in VCaP cells leads lesser response for enzalutamide therapy. Citation Format: Sreenivasa R. Chinni, Louie Semaan, Michael Cher. TMPRSS2-ERG fusions confers efficacy of enzalutamide in an in vivo bone tumor growth model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5422.

Sprouty2 loss-induced IL6 drives castration-resistant prostate cancer through scavenger receptor B1.

Metastatic castration‐resistant prostate cancer (mCRPC) is a lethal form of treatment‐resistant prostate cancer and poses significant therapeutic challenges. Deregulated receptor tyrosine kinase (RTK) signalling mediated by loss of tumour suppressor Sprouty2 (SPRY2) is associated with treatment resistance. Using pre‐clinical human and murine mCRPC models, we show that SPRY2 deficiency leads to an androgen self‐sufficient form of CRPC. Mechanistically, HER2‐IL6 signalling axis enhances the expression of androgen biosynthetic enzyme HSD3B1 and increases SRB1‐mediated cholesterol uptake in SPRY2‐deficient tumours. Systemically, IL6 elevated the levels of circulating cholesterol by inducing host adipose lipolysis and hepatic cholesterol biosynthesis. SPRY2‐deficient CRPC is dependent on cholesterol bioavailability and SRB1‐mediated tumoral cholesterol uptake for androgen biosynthesis. Importantly, treatment with ITX5061, a clinically safe SRB1 antagonist, decreased treatment resistance. Our results indicate that cholesterol transport blockade may be effective against SPRY2‐deficient CRPC.

Randomized trial of enzalutamide versus bicalutamide in combination with androgen deprivation in metastatic hormone sensitive prostate cancer: A Prostate Cancer Clinical Trials Consortium trial.

190 Background: Addition of abiraterone or docetaxel has overall survival (OS) benefit in metastatic hormone sensitive prostate cancer (mHSPC). The clinical outcomes with early enzalutamide in mHSPC are unknown. We compared the combinations of enzalutamide (Arm A) or bicalutamide (Arm B) each with LHRH analogue therapy in mHSPC. Methods: The primary endpoint was PSA nadir < 4 ng/ml after 7 months of therapy, as this has been recognized as a powerful surrogate for overall survival (OS) outcomes. A minimum of 29 African American (AA) patients were required to be enrolled to evaluate the effect of race on the primary endpoint. Secondary endpoints were toxicities, biochemical and radiologic progression free survival (PFS), and OS. Stratification was by presence of bone pain and race; AA or other. PSA was monitored monthly for first 7 months and then every 3 months. The target sample size was 82 evaluable patients but the study was stopped when early abiraterone showed OS benefit in mHSPC. Metastatic site biopsies were mandatory pretherapy and optional post therapy. Results: 71 men; 29 black, 41 white and 1 Asian were enrolled. The median age was 67 years (range 46-87 years) and median baseline PSA was 56.3 ng/ml in Arm A (4.2-10,431 ng/ml) and 60 (4.9-12,030 ng/ml) in Arm B. 27 pts (38.5%) had bone pain and 13 had visceral metastases. No seizures were noted in either arm. Grade 3+ AE’s on Arm A were: Hypertension (13%), infection (7%), and Snycope (7%) and on Arm B were: Hypertension (21%), Fatigue (7%), and Hematuria (7%). By intent to treat PSA nadir < 4ng/ml at month 7 was achieved in 96.3% pts in arm A and 66.7% pts in arm B and in 71.7% of AA men and 89.7% of Caucasians. The 6-month PSA remission duration rates after month 7 on Arms A and B were 86% and 79%, respectively. 53 (75%) biopsy samples had tumor tissue available. Tmprss2-Erg fusion gene expression and androgen biosynthetic enzyme levels were determined in metastatic biopsies and will be correlated with clinical endpoints. Conclusions: Early enzalutamide use in mHSPC has the potential to improve PSA remission rates and subsequently improve PFS and OS outcomes. Clinical trial information: NCT02058706.

Taxifolin suppresses rat and human testicular androgen biosynthetic enzymes

Taxifolin is a flavonoid. It has been used as a chemopreventive agent and supplement. It may have some beneficial effects to treat prostate cancer by suppressing androgen production in Leydig cells. The objective of the present study was to study the effects of taxifolin on androgen production of rat Leydig cells isolated from immature testis and some rat and human testosterone biosynthetic enzyme activities. Rat Leydig cells were incubated with 100μM taxifolin without (basal) or with 10ng/ml luteinizing hormone (LH), 10mM 8-bromoadenosine 3′,5′-cyclic monophosphate (8BR), and steroid enzyme substrates (20μM): 22R-hydroxychloesterol, pregnenolone, progesterone, and androstenedione. The medium concentrations of 5α-androstane-3α, 17β-diol (DIOL) and testosterone were measured. Taxifolin significantly suppressed basal, LH-stimulated, 8BR-stimulated, pregnenolone-mediated, and progesterone-mediated androgen production by Leydig cells. Further study demonstrated that taxifolin inhibited rat 3β-hydroxysteroid dehydrogenase and 17α-hydroxylase/17, 20-lyase with IC50 values of 14.55±0.013 and 16.75±0.011μM, respectively. Taxifolin also inhibited these two enzyme activities in human testis with IC50 value of about 100μM. Taxifolin was a competitive inhibitor for these two enzymes when steroid substrates were used. In conclusion, taxifolin may have benefits for the treatment of prostate cancer.

Current advances in intratumoral androgen metabolism in castration-resistant prostate cancer.

Androgen deprivation therapy is a cornerstone in the treatment of advanced prostate cancer and has extended the lives of countless patients. Unfortunately, many of these patients eventually succumb to metastatic castration-resistant prostate cancer (mCRPC). The efficacy of abiraterone acetate (AA, Zytiga) and enzalutamide (Enza, Xtandi) in the mCRPC setting prove that these tumors remain androgen-driven. We review recent studies that have shown that intratumoral androgen biosynthesis plays a significant role in the ever-evolving mCRPC tumor and we discuss the therapeutic implications of these findings. A novel abiraterone metabolite, 17-(pyridin-3-yl)androsta-4,16-dien-3-one (D4A), possesses robust antitumor activity in rodent models via the inhibition of androgen biosynthetic enzymes and antagonism of the androgen receptor. The TMPRSS2 : ERG fusion drives aldo-keto reductase 1C3 (AKR1C3) expression and activity to facilitate androgen biosynthesis and activate the androgen receptor in prostate cancer. Intracrine androgen formation and AKR1C3 expression and activity have been found to confer resistance to enzalutamide. These studies highlight the significant role that intratumoral androgen biosynthesis plays in the mCRPC tumor. The therapeutic implications include the inhibition of AKR1C3 in tumors that become resistant to current drugs such as abiraterone acetate or Enza and the potential administration of D4A as an mCRPC therapeutic.

Suppression of rat and human androgen biosynthetic enzymes by apigenin: Possible use for the treatment of prostate cancer

Apigenin is a natural flavone. It has recently been used as a chemopreventive agent. It may also have some beneficial effects to treat prostate cancer by inhibiting androgen production. The objective of the present study was to investigate the effects of apigenin on the steroidogenesis of rat immature Leydig cells and some human testosterone biosynthetic enzyme activities. Rat immature Leydig cells were incubated for 3h with 100μM apigenin without (basal) or with 1ng/ml luteinizing hormone (LH), 10mM 8-bromoadenosine 3′,5′-cyclic monophosphate (8BR), and 20μM of the following steroid substrates: 22R-hydroxychloesterol (22R), pregnenolone (P5), progesterone (P4), and androstenedione (D4). The medium levels of 5α-androstane-3α, 17β-diol (DIOL), the primary androgen produced by rat immature Leydig cells, were measured. Apigenin significantly inhibited basal, 8BR, 22R, PREG, P4, and D4 stimulated DIOL production in rat immature Leydig cells. Further study showed that apigenin inhibited rat 3β-hydroxysteroid dehydrogenase, 17α-hydroxylase/17, 20-lyase, and 17β-hydroxysteroid dehydrogenase 3 with IC50 values of 11.41±0.7, 8.98±0.10, and 9.37±0.07μM, respectively. Apigenin inhibited human 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 with IC50 values of 2.17±0.04 and 1.31±0.09μM, respectively. Apigenin is a potent inhibitor of rat and human steroidogenic enzymes, being possible use for the treatment of prostate cancer.

Regulation of androgen action during establishment of pregnancy.

During the establishment of pregnancy, the ovarian-derived hormones progesterone and oestradiol regulate remodelling of the endometrium to promote an environment that is able to support and maintain a successful pregnancy. Decidualisation is characterised by differentiation of endometrial stromal cells that secrete growth factors and cytokines that regulate vascular remodelling and immune cell influx. This differentiation process is critical for reproduction, and inadequate decidualisation is implicated in the aetiology of pregnancy disorders such as foetal growth restriction and preeclampsia. In contrast to progesterone and oestradiol, the role of androgens in regulating endometrial function is poorly understood. Androgen receptors are expressed in the endometrium, and androgens are reported to regulate both the transcriptome and the secretome of endometrial stromal cells. In androgen-target tissues, circulating precursors are activated to mediate local effects, and recent studies report that steroid concentrations detected in endometrial tissue are distinct to those detected in the peripheral circulation. New evidence suggests that decidualisation results in dynamic changes in the expression of androgen biosynthetic enzymes, highlighting a role for pre-receptor regulation of androgen action during the establishment of pregnancy. These results suggest that such enzymes could be future therapeutic targets for the treatment of infertility associated with endometrial dysfunction. In conclusion, these data support the hypothesis that androgens play a beneficial role in regulating the establishment and maintenance of pregnancy. Future studies should be focussed on investigating the safety and efficacy of androgen supplementation with the potential for utilisation of novel therapeutics, such as selective androgen receptor modulators, to improve reproductive outcomes in women.

Intracrine Androgens Enhance Decidualization and Modulate Expression of Human Endometrial Receptivity Genes.

The endometrium is a complex, steroid-dependent tissue that undergoes dynamic cyclical remodelling. Transformation of stromal fibroblasts (ESC) into specialised secretory cells (decidualization) is fundamental to the establishment of a receptive endometrial microenvironment which can support and maintain pregnancy. Androgen receptors (AR) are present in ESC; in other tissues local metabolism of ovarian and adrenal-derived androgens regulate AR-dependent gene expression. We hypothesised that altered expression/activity of androgen biosynthetic enzymes would regulate tissue availability of bioactive androgens and the process of decidualization. Primary human ESC were treated in vitro for 1–8 days with progesterone and cAMP (decidualized) in the presence or absence of the AR antagonist flutamide. Time and treatment-dependent changes in genes essential for a) intra-tissue biosynthesis of androgens (5α-reductase/SRD5A1, aldo-keto reductase family 1 member C3/AKR1C3), b) establishment of endometrial decidualization (IGFBP1, prolactin) and c) endometrial receptivity (SPP1, MAOA, EDNRB) were measured. Decidualization of ESC resulted in significant time-dependent changes in expression of AKR1C3 and SRD5A1 and secretion of T/DHT. Addition of flutamide significantly reduced secretion of IGFBP1 and prolactin and altered the expression of endometrial receptivity markers. Intracrine biosynthesis of endometrial androgens during decidualization may play a key role in endometrial receptivity and offer a novel target for fertility treatment.

Comparison of the Effects of Dibutyl and Monobutyl Phthalates on the Steroidogenesis of Rat Immature Leydig Cells.

Dibutyl phthalate (DBP) is a widely used synthetic phthalic diester and monobutyl phthalate (MBP) is its main metabolite. DBP can be released into the environment and potentially disrupting mammalian male reproductive endocrine system. However, the potencies of DBP and MBP to inhibit Leydig cell steroidogenesis and their possible mechanisms are not clear. Immature Leydig cells isolated from rats were cultured with 0.05–50 μM DBP or MBP for 3 h in combination with testosterone synthesis regulator or intermediate. The concentrations of 5α-androstanediol and testosterone in the media were measured, and the mRNA levels of the androgen biosynthetic genes were detected by qPCR. The direct actions of DBP or MBP on CYP11A1, CYP17A1, SRD5A1, and AKR1C14 activities were measured. MBP inhibited androgen production by the immature Leydig cell at as low as 50 nM, while 50 μM was required for DBP to suppress its androgen production. MBP mainly downregulated Cyp11a1 and Hsd3b1 expression levels at 50 nM. However, 50 μM DBP downregulated Star, Hsd3b1, and Hsd17b3 expression levels and directly inhibited CYP11A1 and CYP17A1 activities. In conclusion, DBP is metabolized to more potent inhibitor MBP that downregulated the expression levels of some androgen biosynthetic enzymes.

Androgen receptor targeting drugs in castration-resistant prostate cancer and mechanisms of resistance.

Reactivated androgen receptor (AR) signaling drives castration-resistant prostate cancer (CRPC). The novel AR targeting drugs abiraterone and enzalutamide have improved survival of CRPC patients. However, resistance to these agents develops and patients ultimately succumb to CRPC. Potential mechanisms of resistance include the following: 1) Expression of AR splice variants, such as the AR-V7 isoform, which lacks the ligand-binding domain; 2) AR missense mutations in the ligand-binding domain, such as F876L and T877A; and 3) Mutation or overexpression of androgen biosynthetic enzymes or glucocorticoid receptor. Several novel agents may overcome resistance mechanisms. Galeterone acts through multiple mechanisms that include degradation of AR protein and is being evaluated in CRPC patients positive for AR-V7. EPI-001 and related compounds inhibit AR splice variants by targeting the N-terminal transactivation domain of AR. Promising therapies and novel biomarkers, such as AR-V7, may lead to improved outcomes for CRPC patients.

The Steroidogenic Enzyme AKR1C3 Regulates Stability of the Ubiquitin Ligase Siah2 in Prostate Cancer Cells

Re-activation of androgen receptor (AR) activity is the main driver for development of castration-resistant prostate cancer. We previously reported that the ubiquitin ligase Siah2 enhanced AR transcriptional activity and prostate cancer cell growth. Among the genes we found to be regulated by Siah2 was AKR1C3, which encodes a key androgen biosynthetic enzyme implicated in castration-resistant prostate cancer development. Here, we found that Siah2 inhibition in CWR22Rv1 prostate cancer cells decreased AKR1C3 expression as well as intracellular androgen levels, concomitant with inhibition of cell growth in vitro and in orthotopic prostate tumors. Re-expression of either wild-type or catalytically inactive forms of AKR1C3 partially rescued AR activity and growth defects in Siah2 knockdown cells, suggesting a nonenzymatic role for AKR1C3 in these outcomes. Unexpectedly, AKR1C3 re-expression in Siah2 knockdown cells elevated Siah2 protein levels, whereas AKR1C3 knockdown had the opposite effect. We further found that AKR1C3 can bind Siah2 and inhibit its self-ubiquitination and degradation, thereby increasing Siah2 protein levels. We observed parallel expression of Siah2 and AKR1C3 in human prostate cancer tissues. Collectively, our findings identify a new role for AKR1C3 in regulating Siah2 stability and thus enhancing Siah2-dependent regulation of AR activity in prostate cancer cells.

ERG/AKR1C3/AR Constitutes a Feed-Forward Loop for AR Signaling in Prostate Cancer Cells.

Intratumoral androgen synthesis in prostate cancer contributes to the development of castration-resistant prostate cancer (CRPC). Several enzymes responsible for androgen biosynthesis have been shown to be overexpressed in CRPC, thus contributing to CRPC in a castrated environment. The TMPRSS2-ERG transcription factor has been shown to be present in primary prostate cancer tumors as well as CRPC tumors. We hypothesize that TMPRSS2-ERG fusions regulate androgen biosynthetic enzyme (ABE) gene expression and the production of androgens, which contributes to the development of CRPC. We used a panel of assays, including lentivirus transduction, gene expression, chromatin immunoprecipitation and sequencing, liquid chromatography-mass spectrometric quantitation, immunocytochemistry, immunohistochemistry, and bioinformatics analysis of gene microarray databases, to determine ERG regulation of androgen synthesis. We found that ERG regulated the expression of the ABE AKR1C3 in prostate cancer cells via direct binding to the AKR1C3 gene. Knockdown of ERG resulted in reduced AKR1C3 expression, which caused a reduction in both DHT synthesis and PSA expression in VCaP prostate cancer cells treated with 5α-androstanedione (5α-Adione), a DHT precursor metabolite. Immunohistochemical staining revealed that ERG was coexpressed with AKR1C3 in prostate cancer tissue samples. These data suggest that AKR1C3 catalyzes the biochemical reduction of 5α-Adione to DHT in prostate cancer cells, and that ERG regulates this step through upregulation of AKR1C3 expression. Elucidation of ERG regulation of ABEs in CRPC may help to stratify TMPRSS2-ERG fusion-positive prostate cancer patients in the clinic for anti-androgen receptor-driven therapies; and AKR1C3 may serve as a valuable therapeutic target in the treatment of CRPC.

Antiandrogenic mechanisms of pesticides in human LNCaP prostate and H295R adrenocortical carcinoma cells.

Several pesticides suspected or known to have endocrine disrupting effects were screened for pro- or antiandrogenic properties by determining their effects on proliferation, prostatic-specific antigen (PSA) secretion and androgen receptor (AR) expression, and AR phosphorylation in androgen-dependent LNCaP human prostate cancer cells, as well as on the expression and catalytic activity of the enzyme CYP17 in H295R human adrenocortical carcinoma cells, an in vitro model of steroidogenesis. Effects on SRD5A gene expression were determined in both cell lines. Benomyl, vinclozolin, and prochloraz, but not atrazine, concentration dependently (1-30 μM) decreased dihydrotestosterone (DHT)-stimulated proliferation of LNCaP cells. All pesticides except atrazine decreased DHT-stimulated PSA secretion, AR nuclear accumulation, and AR phosphorylation on serines 81 and 213 in LNCaP cells. Benomyl and prochloraz, but not vinclozolin or atrazine, decreased levels of CYP17 gene and protein expression, as well as catalytic activity in H295R cells. In the case of prochloraz, some of these effects corresponded with cytotoxicity. H295R cells expressed AR protein and SRD5A1, but not SRD5A2 transcripts. SRD5A1 gene expression in H295R cells was increased by 10 nM DHT, whereas in LNCaP cells significant induction was observed by 0.1 nM DHT. AR protein expression in H295R cells was not increased by DHT. Vinclozolin decreased DHT-induced SRD5A1 gene expression in LNCaP, but not H295R cells, indicating a functional difference of AR between the cell lines. In conclusion, pesticides may exert antiandrogenic effects through several mechanisms that are cell type-specific, including AR antagonism and down-regulation or catalytic inhibition of androgen biosynthetic enzymes, such as CYP17 and SRD5A1.

Abstract 456: ERG regulation of intracrine androgen production and castration-resistant prostate cancer progression

Abstract Introduction and Objective: Intratumoral androgen synthesis in prostate cancer (PC) contributes to the development of castration-resistant prostate cancer (CRPC). Several enzymes responsible for androgen biosynthesis have been shown to be overexpressed in CRPC, thus, contributing to CRPC in a castrated environment. However, little is known regarding their mode of regulation and expression in this context. The TMPRSS2-ERG transcription factor has been shown to be present in primary PC tumors as well as CRPC tumors; however, its biological function contributing to the development CRPC in the context of intratumoral androgen synthesis is not known. We hypothesize that TMPRSS2-ERG fusions regulate androgen biosynthetic enzyme (ABE) gene expression and the production of androgens, which contributes to the development of CRPC. Methods: We used a variety of methods including lentiviral knockdown and overexpression of ERG, qPCR, Western blot, ChIP-PCR, and Mass Spectrometry. Results: We found that (a) shRNA knockdown of ERG in VCaP PC cells significantly reduces the expression of ABE's AKR1C3, HSD17B6, and HSD17B4; (b) overexpression of truncated ERG or wild-type ERG in LNCaP PC cells significantly increases the expression of AKR1C3 and HSD17B6; (c) ERG binds to the AKR1C3 gene in VCaP PC cells; and (d) DHT synthesis is reduced in VCaP shERG cells treated with 5α-Androstanedione, a DHT precursor metabolite, compared to VCaP shScrambled controls. Conclusions: These data suggest that AKR1C3 catalyzes the biochemical reduction of 5α-Androstanedione to DHT in PC cells, and that ERG may regulate this step through upregulation of AKR1C3 expression. Elucidation of ERG regulation of ABEs in CRPC may help to stratify TMPRSS2-ERG fusion-positive PC patients in the clinic for anti-hormone driven therapies. Additionally, not all PC tumors express CYP17A1 enzyme, therefore this study emphasizes the importance of investigating ABEs other than CYP17A1, such as AKR1C3 and HSD17B6. In summary, this study addresses alternative ABE drug targets as well as possible drug resistance modes in PC treatment. Citation Format: Katelyn A. Powell, Louie Semaan, Krishna R. Maddipati, Katie (Mary) Conley-Lacomb, Yanfeng Li, Michael Cher, Sreenivasa R. Chinni. ERG regulation of intracrine androgen production and castration-resistant prostate cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 456. doi:10.1158/1538-7445.AM2014-456

Importance of 5α-Reductase Gene Polymorphisms on Circulating and Intraprostatic Androgens in Prostate Cancer

Polymorphisms in the genes SRD5A1 and SRD5A2 encoding androgen biosynthetic 5α-reductase enzymes have been associated with an altered risk of biochemical recurrence after radical prostatectomy in localized prostate cancer. To gain potential insights into SRD5A biologic effects, we examined the relationship between SRD5A prognostic markers and endogenous sex-steroid levels measured by mass spectrometry in plasma samples and corresponding prostatic tissues of patients with prostate cancer. We report that five of the seven SRD5A markers differentially affect sex-steroid profiles of dihydrotestosterone and its metabolites in both the circulation and prostatic tissues of patients with prostate cancer. Remarkably, a 32% increase in intraprostatic testosterone levels was observed in the presence of the high-risk SRD5A rs2208532 polymorphism. Moreover, SRD5A2 markers were associated predominantly with circulating levels of inactive glucuronides. Indeed, the rs12470143 SRD5A2 protective allele was associated with high circulating androstane-3α, 17β-diol-17-glucuronide (3α-diol-17G) levels as opposed to lower levels of both 3α-diol-17G and androsterone-glucuronide observed with the rs2208532 SRD5A2 risk allele. Moreover, SRD5A2 rs676033 and rs523349 (V89L) risk variants, in strong linkage disequilibrium, were associated with higher circulating levels of 3α-diol-3G. The SRD5A2 rs676033 variant further correlated with enhanced intraprostatic exposure to 5α-reduced steroids (dihydrotestosterone and its metabolite 3β-diol). Similarly, the SRD5A1 rs166050C risk variant was associated with greater prostatic exposure to androsterone, whereas no association was noted with circulating steroids. Our data support the association of 5α-reductase germline polymorphisms with the hormonal milieu in patients with prostate cancer. Further studies are needed to evaluate if these variants influence 5α-reductase inhibitor efficacy.

Abstract 359: Neuropeptides promote castration-resistant prostate cancer through intracrine androgen biosynthesis

Abstract Introduction and Objectives: Neuroendocrine differentiation is associated with castration resistant prostate cancer (CRPC) with detectable increased serum levels of chromogranin A in patients. Neuropeptides secreted from the neuroendocrine cells are believed to activate androgen receptor (AR) in low or no androgen environments. We hypothesize that neuropeptides sustain CRPC by promoting intracrine androgen synthesis. Methods: We engineered LNCaP cells overexpressing gastrin-releasing peptide (GRP) and examined the level of androgen biosynthetic enzymes by RT-PCR and Western blots. Inhibition of androgen-free growth of LNCaP-GRP cells was performed with siRNA to AKR1C3 or CYP17A1 with or without Src inhibitor saracatinib. PSA and total testosterone levels were assessed in LNCaP-GRP cells, serum from tumor-bearing animals and xenografts. Results: The androgen biosynthetic enzymes were up-regulated in LNCaP-GRP cells, with AKR1C3, CYP17A1 and HSD17B2 displaying over 40 fold increase compared to the control line by real-time RT-PCR analysis. Transfection of siRNA against AKR1C3 and CYP17A1 inhibited LNCaP-GRP cell growth in androgen-free medium. Specific Src family kinase inhibitor saracatinib inhibits androgen-independent LNCaP-GRP cell growth in vitro and tumor metastasis in vivo. Saracatinib also reduced the expression of AKR1C3, AKR1C1/2 and HSD3B1/2 in GRP cells at both RNA and protein levels. When combined with siRNA for AKR1C3 and CYP17A1, saracatinib further inhibited androgen-independent LNCaP-GRP cell growth. Testosterone was detectable by EIA assay in LNCaP-GRP cell lysates (109.1 and 44.5 pg/mg protein vs. 18.3 in control), serum from castrated mice bearing GRP tumors (2235.5 pg/ml serum) and xenograft tissue (59.2 pg/mg tumor). These enhanced T levels were greatly reduced when treated with saracatinib, to 594 pg/ml serum and 12 pg/mg tumor, respectively. Conclusions: Secretion of neuropeptides promotes CRPC through up-regulation of intracrine androgen biosynthesis. Saracatinib not only targets neuropeptide-mediated AR activation but also blocks steroidogenesis in CRPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 359. doi:10.1158/1538-7445.AM2011-359

Abstract 4554: Regulation of intracrine androgen synthesis by NF-kappaB2/p52

Abstract Introduction: Benign prostatic hyperplasia and the initial stages of prostate cancer (CaP) exhibit androgen dependence, but androgen ablation results only in temporary regression of CaP, with progression to castration-resistant prostate cancer (CRPC). Androgen receptor (AR) signaling remains active in CRPC due to aberrant activation of the AR. Synthesis of intracrine androgens has emerged as one of the mechanisms by which AR is activated in CRPC after androgen ablation. Even though levels of androgen biosynthetic enzymes have been shown to be elevated in CRPC, their regulation is not completely understood. In this study, we examined the role of NF-kappaB2/p52 in intracrine androgen synthesis and castration-resistant progression of CaP. Methods: Expression levels of androgen biosynthetic enzymes were measured in CaP cells with or without expression of NF-kappaB2/p52 using real-time RT-PCR and western blotting. Regulation by NF-kappaB2/p52 was examined using luciferase reporter assays and plasmids containing regulatory elements of androgen biosynthetic enzymes. Intracrine levels of androgens were measured using EIA in tumors obtained from castrated mice. Results: Expression levels of androgen biosynthetic enzymes including AKR1C3, CYP17A1, HSD3B2, and SRD5A1 were found to be elevated in CaP cells expressing NF-kappaB2/p52. Luciferase assays showed that NF-kappaB2/p52 regulates their expression directly by binding to their promoters and inducing transcription. The levels of total testosterone in CaP cells expressing NF-kappaB2/p52 were approximately 3-fold higher than control cells as measured using EIA. Intraprostatic androgen levels were found to be at 1002 ± 232 pg/g tissue, compared to 377.8 ± 105 pg/g tissue in control tumors obtained by orthotopic implantation of CaP cells expressing NF-kappaB2/p52. These data suggest that CaP cells synthesize detectable levels of testosterone in the absence of exogenous steroid precursors and overexpression of NF-kappaB2/p52 can increase this process, possibly by enhancing the expression of genes encoding steroidogenic enzymes. Conclusions: Intraprostatic androgen synthesis in recurrent prostate tumors contributes significantly to resistance to androgen ablation and development of CRPC. NF-kappaB2/p52 regulates the expression levels of steroidogenic enzymes and thereby enhances synthesis of intracrine androgens and aberrant activation of the AR. Coupled with our previous studies, these data suggest that antagonizing NF-kappaB2/p52 signaling may prove beneficial in CRPC therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4554. doi:10.1158/1538-7445.AM2011-4554

1454 TETRANDRINE BLOCKS EMERGENCE OF CASTRATE RESISTANT PHENOTYPE BY PROMOTING APOPTOSIS AND SENSITIZING PROSTATE CANCER CELLS TO ANDROGEN DEPRIVATION

zymes were measured in CaP cells with or without expression of NF-kappaB2/p52 using real-time RT-PCR and western blotting. Regulation by NF-kappaB2/p52 was examined using luciferase reporter assays and plasmids containing regulatory elements of androgen biosynthetic enzymes. Intracrine levels of androgens were measured using EIA in tumors obtained from castrated mice. RESULTS: Expression levels of androgen biosynthetic enzymes including AKR1C3, CYP17A1, HSD3B2, and SRD5A1 were found to be elevated in CaP cells expressing NF-kappaB2/p52. Luciferase assays showed that NF-kappaB2/p52 regulates their expression directly by binding to their promoters and inducing transcription. The levels of total testosterone in CaP cells expressing NF-kappaB2/ p52 were approximately 3-fold higher than control cells as measured using EIA. Intraprostatic androgen levels were found to be at 1002 232 pg/g tissue, compared to 377.8 105 pg/g tissue in control tumors obtained by orthotopic implantation of CaP cells expressing NF-kappaB2/p52. These data suggest that CaP cells synthesize detectable levels of testosterone in the absence of exogenous steroid precursors and overexpression of NF-kappaB2/p52 can increase this process, possibly by enhancing the expression of genes encoding steroidogenic enzymes. CONCLUSIONS: Intraprostatic androgen synthesis in recurrent prostate tumors contributes significantly to resistance to androgen ablation and development of CRPC. NF-kappaB2/p52 regulates the expression levels of steroidogenic enzymes and thereby enhances synthesis of intracrine androgens and aberrant activation of the AR. Coupled with our previous studies, these data suggest that antagonizing NF-kappaB2/p52 signaling may prove beneficial in CRPC therapy.