Intratumoral Androgen Research Articles

Dehydroepiandrosterone (DHEA)-SO4 Depot and Castration-Resistant Prostate Cancer.

Dehydroepiandrosterone (DHEA)-SO4 of adrenal origin is the major C19 steroid in the serum. It is a precursor of intratumoral androgen biosynthesis in patients with advanced prostate cancer following chemical or surgical castration. DHEA is a product of the P450c17 (17α-hydroxylase-17,20-lyase) enzyme. Despite inhibition of P450c17 with new agents, e.g., Abiraterone acetate, Orterenol, and Galeterone, the level of enzyme inhibition rarely exceeds 90% leaving behind a significant depot for androgen biosynthesis within the tumor. For DHEA-SO4 to be utilized there is uptake by organic anion transporter polypeptides, deconjugation catalyzed by steroid sulfatase, and adaptive upregulation of prostate steroidogenic enzymes that will convert DHEA into either testosterone or dihydrotestosterone. The depot of DHEA-SO4 that remains after P450c17 inhibition and the adaptive responses that occur within the tumor to promote DHEA utilization contribute to mechanisms of drug resistance observed with P450c17 inhibitors. Knowledge of these mechanisms identify new targets for therapeutics that could be used to surmount drug resistance in prostate cancer.

HSD3B1 status as a biomarker of androgen deprivation resistance and implications for prostate cancer.

Patients with advanced prostate cancer who receive androgen deprivation therapy (ADT) almost invariably develop castration-resistant disease. The mechanism of resistance is largely based on synthesis of intratumoral androgens from adrenal precursors, requiring enzymatic action of 3β-hydroxysteroid dehydrogenase/Δ5→4 isomerase 1 (3β-HSD1), encoded by HSD3B1. A nucleotide polymorphism (1245A>C) in HSD3B1 results in a protein variant with increased steady-state levels and subsequently increased androgen synthesis from extragonadal precursors. Multiple clinical studies have shown that patients with the variant allele have significantly worse outcomes after ADT than those without, indicating that HSD3B1 variant status is a predictive biomarker of shortened ADT response. In addition, inheritance of the HSD3B1 variant is associated with extended responses to 17α-hydroxylase/17,20-lyase (CYP17A1) inhibition with a nonsteroidal agent, adding to evidence of increased tumour dependence on extragonadal androgens in patients who inherited the HSD3B1 variant. However, steroidal drugs with a 3β-hydroxyl, Δ5-structure, such as abiraterone, are also metabolized by 3β-HSD1, and 5α-abiraterone, a downstream metabolite, has been shown to activate the androgen receptor, potentially driving cancer progression. These data indicate a potential requirement to modify the treatment framework of patients harbouring variant HSD3B1.

Antiandrogens Reduce Intratumoral Androgen Concentrations and Induce Androgen Receptor Expression in Castration-Resistant Prostate Cancer Xenografts

The development of castration-resistant prostate cancer (CRPC) is associated with the activation of intratumoral androgen biosynthesis and an increase in androgen receptor (AR) expression. We recently demonstrated that, similarly to the clinical CRPC, orthotopically grown castration-resistant VCaP (CR-VCaP) xenografts express high levels of AR and retain intratumoral androgen concentrations similar to tumors grown in intact mice. Herein, we show that antiandrogen treatment (enzalutamide or ARN-509) significantly reduced (10-fold, P<0.01) intratumoral testosterone and dihydrotestosterone concentrations in the CR-VCaP tumors, indicating that the reduction in intratumoral androgens is a novel mechanism by which antiandrogens mediate their effects in CRPC. Antiandrogen treatment also altered the expression of multiple enzymes potentially involved in steroid metabolism. Identical to clinical CRPC, the expression levels of the full-length AR (twofold, P<0.05) and the AR splice variants 1 (threefold, P<0.05) and 7 (threefold, P<0.01) were further increased in the antiandrogen-treated tumors. Nonsignificant effects were observed in the expression of certain classic androgen-regulated genes, such as TMPRSS2 and KLK3, despite the low levels of testosterone and dihydrotestosterone. However, other genes recently identified to be highly sensitive to androgen-regulated AR action, such as NOV and ST6GalNAc1, were markedly altered, which indicated reduced androgen action. Taken together, the data indicate that, besides blocking AR, antiandrogens modify androgen signaling in CR-VCaP xenografts at multiple levels.

Clinical relevance of androgen receptor alterations in prostate cancer.

Prostate cancer (PC) remains a leading cause of cancer-related deaths among men worldwide, despite continuously improved treatment strategies. Patients with metastatic disease are treated by androgen deprivation therapy (ADT) that with time results in the development of castration-resistant prostate cancer (CRPC) usually established as metastases within bone tissue. The androgen receptor (AR) transcription factor is the main driver of CRPC development and of acquired resistance to drugs given for treatment of CRPC, while a minority of patients have CRPC that is non-AR driven. Molecular mechanisms behind epithelial AR reactivation in CRPC include AR gene amplification and overexpression, AR mutations, expression of constitutively active AR variants, intra-tumoural and adrenal androgen synthesis and promiscuous AR activation by other factors. This review will summarize AR alterations of clinical relevance for patients with CRPC, with focus on constitutively active AR variants, their possible association with AR amplification and structural rearrangements as well as their ability to predict patient resistance to AR targeting drugs. The review will also discuss AR signalling in the tumour microenvironment and its possible relevance for metastatic growth and therapy.

Effects of cytokines derived from cancer-associated fibroblasts on androgen synthetic enzymes in estrogen receptor-negative breast carcinoma.

The tumor microenvironment plays pivotal roles in promotion of many malignancies. Cancer-associated fibroblasts (CAFs) have been well-known to promote proliferation, angiogenesis, and metastasis but mechanistic understanding of tumor-stroma interactions is not yet complete. Recently, estrogen synthetic enzymes were reported to be upregulated by co-culture with stromal cells in ER positive breast carcinoma (BC) but effects of co-culture on androgen metabolism have not been extensively examined. Therefore, we evaluated roles of CAFs on androgen metabolism in ER-negative AR-positive BC through co-culture with CAFs. Concentrations of steroid hormone in supernatant of co-culture of MDA-MB-453 and primary CAFs were measured using GC-MS. Cytokines derived from CAFs were determined using Cytokine Array. Expressions of androgen synthetic enzymes were confirmed using RT-PCR and Western blotting. Correlations between CAFs and androgen synthetic enzymes were analyzed using triple-negative BC (TNBC) patient tissues by immunohistochemistry. CAFs were demonstrated to increase expressions and activities of 17βHSD2, 17βHSD5, and 5α-Reductase1. IL-6 and HGF that were selected as potential paracrine mediators using cytokine array induced 17βHSD2, 17βHSD5, and 5α-Reductase1 expression. Underlying mechanisms of IL-6 paracrine regulation of 17βHSD2 and 17βHSD5 could be partially dependent on phosphorylated STAT3, while phosphorylated ERK could be involved in HGF-mediated 5α-Reductase1 induction. α-SMA status was also demonstrated to be significantly correlated with 17βHSD2 and 17βHSD5 status in TNBC tissues, especially AR-positive cases. Results of our present study suggest that both IL-6 and HGF derived from CAFs could contribute to the intratumoral androgen metabolism in ER-negative BC patients.

Commentary on: “Redirecting abiraterone metabolism to fine-tune prostate cancer anti-androgen therapy.” Li Z, Alyamani M, Li J, Rogacki K, Abazeed M, Upadhyay SK, Balk SP, Taplin ME, Auchus RJ, Sharifi N. Nature. 2016 May 25;533(7604):547–51.

Abiraterone blocks androgen synthesis and prolongs survival in patients with castration-resistant prostate cancer, which is otherwise driven by intratumoral androgen synthesis. Abiraterone is metabolized in patients to Δ(4)-abiraterone (D4A), which has even greater antitumour activity and is structurally similar to endogenous steroidal 5α-reductase substrates, such as testosterone. Here, we show that D4A is converted to at least three 5α-reduced and three 5β-reduced metabolites in human serum. The initial 5α-reduced metabolite, 3-keto-5α-abiraterone, is present at higher concentrations than D4A in patients with prostate cancer taking abiraterone, and is an androgen receptor agonist, which promotes prostate cancer progression. In a clinical trial of abiraterone alone, followed by abiraterone plus dutasteride (a 5α-reductase inhibitor), 3-keto-5α-abiraterone and downstream metabolites were depleted by the addition of dutasteride, while D4A concentrations rose, showing that dutasteride effectively blocks production of a tumor-promoting metabolite and permits D4A accumulation. Furthermore, dutasteride did not deplete the three 5β-reduced metabolites, which were also clinically detectable, demonstrating the specific biochemical effects of pharmacological 5α-reductase inhibition on abiraterone metabolism. Our findings suggest a previously unappreciated and biochemically specific method of clinically fine-tuning abiraterone metabolism to optimize therapy.

Abstract 2170: Rb1 suppresses prostate cancer metastasis and lineage plasticity underlying castration resistance

Abstract Androgen deprivation therapy (ADT) is an effective treatment for metastatic prostate cancer (mPCa), but patients eventually relapse with ADT resistant disease. Well-characterized mechanisms of ADT resistance include AR amplification, intra-tumoral androgen synthesis, AR splice variants, and growth receptor bypass. All of these mechanisms function to maintain sufficient AR signaling for tumor growth and survival. Improved ADT like abiraterone acetate (AA) and enzalutamide (Enza) were developed to combat such resistance mechanisms associated with alterations in androgen receptor or androgen metabolism. While AA and Enza extend survival, clinical benefits are short-lived. A new form of resistance is increasingly appreciated in patients relapsing from AA or Enza, histologic transformation of prostate adenocarcinoma (PADC) to neuroendocrine prostate cancer (NEPC) variants. NEPC is lethal and the survival time is less than a year as effective targeted therapy is unavailable. NEPC typically exhibits reduced AR expression, increased expression of neuroendocrine markers, and visceral metastasis in the absence of rising PSA. Of note, NEPC possesses the similar genome rearrangements with adjacent PADC cells, indicating they share clonal origin. Thus, NEPC may arise by histologic transformation of PADC. Underlying mechanisms of histologic transformation are not understood and experimental models are limited, hindering development of effective remedies. RB1 loss is common in NEPC, but rare in PADC; genetic profiling shows human NEPC exhibit elevated levels of several epigenetic modifiers. We hypothesize that transdifferentiation from PADC to NEPC in the context of RB1 loss is due to epigenetic alterations and can be reversed or blocked by epigenetic targeted therapies. We established several genetically engineered mouse models (GEMMs) to test the role of Rb1, and we find Rb1 loss causes metastatic progression of PADC initiated by Pten deficiency. This Rb1/Pten deficient (DKO) PADC exhibits expression markers for both PADC and NEPC as seen in human patients. Yet, these tumors are sensitive to ADT but relapse with low AR expression and acquired Trp53 mutations. RNA profiling demonstrates the phenotype of DKO tumors is similar to human NEPC. Both human and mouse NEPC is accompanied by increased expression of epigenetic reprogramming factors like Sox2 and Ezh2. Clinically relevant Ezh2 inhibitors GSK126 and EPZ6438 can restore Enza sensitivity by reversing neuroendocrine transformation. This finding has been genetically validated using short-hairpin RNA(shRNA) in vitro. These results uncover genetic mutations driving prostate cancer lineage plasticity and suggest an epigenetic approach for extending the clinical benefits of ADT. Citation Format: Sheng-Yu Ku, Spencer Rosario, Yanqing Wang, Ping Mu, Mukund Seshadri, Zachary Goodrich, Maxwell Goodrich, David P. Labbé, Eduardo Cortez Gomez, Jianmin Wang, Henry W. Long, Bo Xu, Myles Brown, Massimo Loda, Charles L. Sawyers, Leigh Ellis, David G. Goodrich. Rb1 suppresses prostate cancer metastasis and lineage plasticity underlying castration resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2170. doi:10.1158/1538-7445.AM2017-2170

Abstract 478: Regulation of cross talk between AR and wnt/beta-catenin pathways in prostate cancer

Abstract Prostate Cancer (PCa) is the second most commonly diagnosed cancer in the United States with 180,890 males diagnosed and is predicted to cause approximately 26,000 deaths in 2016. The current treatment of androgen deprivation therapy (ADT) initially depletes circulating androgens; however, intratumoral androgens rescue androgen receptor (AR) signaling and promotes the development of castration resistant prostate cancer (CRPC). The dysregulation of the Wnt/β-catenin signaling pathway has been implicated in the development of many cancers including PCa. The disruption of this signaling leads to the stabilization of β-catenin which upregulates many genes involved in tumorigenesis. Additionally, β-catenin acts as AR cofactor. Our previous studies show that AR is a direct target of miR-644a. We hypothesized that targeting the expression of both the β-catenin pathway and AR by conventional drugs and/or tumor suppressor miR-644a would have synergistic therapeutic benefits. In this study, we are investigating miR-644a mediated posttranscriptional downregulation of GSK3-β and β-catenin in the wnt/β-catenin signaling pathway. The effect of miR-644a in combination with inhibitors (β-catenin or GSK3-β) was also assessed using proliferation assays and a significant downregulation was observed. We will further study the posttranscriptional effects of tumor suppressor miR-644a on the wnt/β-catenin pathway and also the processing and regulation of miR-644a in PCa. Citation Format: Alexis Plaga, Girish C. Shukla. Regulation of cross talk between AR and wnt/beta-catenin pathways in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 478. doi:10.1158/1538-7445.AM2017-478

Abstract 4476: A novel nonsense mutation in androgen receptor confers resistance to CYP17 inhibitor treatment in prostate cancer

Abstract Background: The standard treatment for prostate cancer (PCa) is androgen deprivation therapy (ADT) that blocks AR transcriptional activity, which is responsible to the initiation and progression of PCa. However, ADT invariably leads to the castration-resistant PCa (CRPC) with restored activity of AR. CRPC can be further treated with more intensive ADTs, including CYP17-inhibitors to block intratumoral androgen synthesis and more potent AR antagonist. By analyzing the tumor mRNA from a CRPC patient biopsy who developed resistance to CYP17-inhibitor treatment, we identified a novel nonsense AR mutation on ligand binding domain (Q784*), which produces a C-terminal truncated form of AR protein containing only a fraction of ligand binding domain (LBD) and may mimic the function of some AR splice variants. We hypothesized that AR-Q784* may gain the androgen-independent activity, or may enhance the transcriptional activity of full-length AR (AR-FL) under low androgen environment through dimerization with AR-FL. Method: Luciferase reporter assays were applied to assess the transcriptional activity of AR-Q784* in absence or presence of androgens, and with or without AR-FL. Immunoblotting and immunofluorescence assays were used to examine the protein stability and cellular localization of AR-Q784*. Chromatin immunoprecipitation assays were also used to assess the chromatin binding ability of AR-Q784*. Moreover, the stable cell line that expresses endogenous AR-FL and tetracycline-inducible AR-Q784* was generated to determine the transcription activity on AR target genes and assess the effects on cell proliferation, particularly under low dose androgen condition. Result: Although it was constitutively expressed in nucleus, AR-Q784* does not elicit transcriptional activity despite the conditions of ligands. However, when it was co-expressed with AR-FL, AR-Q784* can enhance AR-mediated transcriptional activity and thus increased PCa cell proliferation particularly under low androgen conditions. Mechanistically, AR-Q784* can dimerize with AR-FL, enhance DNA binding ability of AR-FL, and strengthen AR recruitment of p300 coactivator. Conclusion: We show that CYP17 inhibitor treatment in CRPC may select for a distinct class of AR mutations/variations, including nonsense (AR-Q784*) or point-deletion mutations within LBD and certain splice variants that only lose a fraction of LBD domain. This class of AR mutants/variants is transcriptionally inactive but can enhance the activity of AR-FL due to their abilities of constitutive DNA binding and dimerization with AR-FL. However, this enhancing activity of those AR mutants/variants can be prevented by antagonist treatments such as enzalutamide. Therefore, this study provides an additional rationale for treating PCa patients with combination therapy of abiraterone and enzalutamide, which may prevent the selection for such AR mutations/variations in CRPC. Citation Format: Dong Han, Jude Owiredu, Kevin Valencia, Changmeng Cai. A novel nonsense mutation in androgen receptor confers resistance to CYP17 inhibitor treatment in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4476. doi:10.1158/1538-7445.AM2017-4476

Testicular vs adrenal sources of hydroxy-androgens in prostate cancer

Neoadjuvant androgen deprivation therapy (NADT) is one strategy for the treatment of early-stage prostate cancer; however, the long-term outcomes of NADT with radical prostatectomy including biochemical failure-free survival are not promising. One proposed mechanism is incomplete androgen ablation. In this study, we aimed to evaluate the efficiency of serum hydroxy-androgen suppression in patients with localized high-risk prostate cancer under NADT (leuprolide acetate plus abiraterone acetate and prednisone) and interrogate the primary sources of circulating hydroxy-androgens using our recently described stable isotope dilution liquid chromatography mass spectrometric method. For the first time, three androgen diols including 5-androstene-3β,17β-diol (5-adiol), 5α-androstane-3α,17β-diol (3α-adiol), 5α-androstane-3β,17β-diol (3β-adiol), the glucuronide or sulfate conjugate of 5-adiol and 3α-adiol were measured and observed to be dramatically reduced after NADT. By comparing patients that took leuprolide acetate alone vs leuprolide acetate plus abiraterone acetate and prednisone, we were able to distinguish the primary sources of these androgens and their conjugates as being of either testicular or adrenal in origin. We find that testosterone, 5α-dihydrotestosterone (DHT), 3α-adiol and 3β-adiol were predominately of testicular origin. By contrast, dehydroepiandrosterone (DHEA), epi-androsterone (epi-AST) and their conjugates, 5-adiol sulfate and glucuronide were predominately of adrenal origin. Our findings also show that NADT failed to completely suppress DHEA-sulfate levels and that two unappreciated sources of intratumoral androgens that were not suppressed by leuprolide acetate alone were 5-adiol-sulfate and epi-AST-sulfate of adrenal origin.

Androgen Receptor-Dependent and -Independent Mechanisms Involved in Prostate Cancer Therapy Resistance.

Despite the initial efficacy of androgen deprivation in prostate cancer, virtually all patients progress to castration-resistant prostate cancer (CRPC). Androgen receptor (AR) signaling is critically required for CRPC. A new generation of medications targeting AR, such as abiraterone and enzalutamide, has improved survival of metastatic CRPC (mCRPC) patients. However, a significant proportion of patients presents with primary resistance to these agents, and in the remainder, secondary resistance will invariably develop, which makes mCRPC the lethal form of the disease. Mechanisms underlying progression to mCRPC and treatment resistance are extremely complex. AR-dependent resistance mechanisms include AR amplification, AR point mutations, expression of constitutively active AR splice variants, and altered intratumoral androgen biosynthesis. AR-independent resistance mechanisms include glucocorticoid receptor activation, immune-mediated resistance, and neuroendocrine differentiation. The development of novel agents, such as seviteronel, apalutamide, and EPI-001/EPI-506, as well as the identification and validation of novel predictive biomarkers of resistance, may lead to improved therapeutics for mCRPC patients.

Androgen Signaling in Prostate Cancer.

The androgen-signaling axis plays a pivotal role in the pathogenesis of prostate cancer. Since the landmark discovery by Huggins and Hodges, gonadal depletion of androgens has remained a mainstay of therapy for advanced disease. However, progression to castration-resistant prostate cancer (CRPC) typically follows and is largely the result of restored androgen signaling. Efforts to understand the mechanisms behind CRPC have revealed new insights into dysregulated androgen signaling and intratumoral androgen synthesis, which has ultimately led to the development of several novel androgen receptor (AR)-directed therapies for CRPC. However, emergence of resistance to these newer agents has also galvanized new directions in investigations of prereceptor and postreceptor AR regulation. Here, we review our current understanding of AR signaling as it pertains to the biology and natural history of prostate cancer.

MP87-12 ADRENAL ANDROGENS FACILITATE PROSTATE CANCER CELL RESISTANCE TO ANDROGEN DEPRIVATION THERAPY

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2017MP87-12 ADRENAL ANDROGENS FACILITATE PROSTATE CANCER CELL RESISTANCE TO ANDROGEN DEPRIVATION THERAPY Michael Fiandalo, John Stocking, Elena Pop, Krystin Mantione, John Wilton, and James Mohler Michael FiandaloMichael Fiandalo More articles by this author , John StockingJohn Stocking More articles by this author , Elena PopElena Pop More articles by this author , Krystin MantioneKrystin Mantione More articles by this author , John WiltonJohn Wilton More articles by this author , and James MohlerJames Mohler More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2716AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate Cancer (CaP) growth and survival is dependent on the interaction of androgen receptor (AR) and testicular androgens, testosterone and dihydrotestosterone (DHT). Men that fail potential curative treatment are treated with androgen deprivation therapy (ADT). ADT is palliative and CaP recurs as the lethal phenotype. One mechanism that contributes to CaP recurrence is intratumoral intracrine androgen metabolism, which is the conversion of weak adrenal androgens (androstenedione [ASD] or dehydroepiandrosterone [DHEA]) to T or DHT. Mice lack adrenal androgens and therefore adrenal androgen contribute to CaP xenograft recurrence is overlooked. The hypothesis for these studies is adrenal androgen supplementation will facilitate androgen dependent human CaP CWR22 xenograft resistance to ADT. METHODS Mice were castrated, implanted with CWR22 and silastic tubes that contained T and empty silastic tubes that contained ASD or DHEA. T silastic tubes were removed after CWR22 tumor volumes reached 0.5 cc to simulate ADT. Digital calipers were used to measure changes in CWR22 tumor volume. Mouse serum, prostate and CWR22 were harvested at designated time points and liquid chromatography tandem-mass spectrometry (LC-MS/MS) was performed to measure androgen levels. RESULTS CWR22 xenografts treated with no adrenal androgen regressed and began to grow 120 days after T silastic tube removal. ASD treated CWR22 tumors did not regress; CWR22 tumor growth increased and mice were euthanized because tumors grew beyond veterinary limits. DHEA treated CWR22 did not regress and did not grow like ASD treated CWR22. However, CWR22 xenografts recurred faster than control CWR22 xenografts. Serum PSA levels correlated with tumor volume. CWR22 androgen levels decreased in all treatment groups, but T levels remained sufficient to activate AR in ASD treated CWR22, but not control or DHEA treated CWR22 xenografts. CONCLUSIONS DHEA and ASD supplementation facilitated CWR22 resistance to ADT. However, CWR22 xenografts responded differently to DHEA and ASD, which suggests that adrenal androgens may play different roles when CaP cells undergo ADT. These studies show that adrenal androgen contributes should be taken into account when in vivo studies are performed to test CaP responses to ADT or therapeutic agents. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1170 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Michael Fiandalo More articles by this author John Stocking More articles by this author Elena Pop More articles by this author Krystin Mantione More articles by this author John Wilton More articles by this author James Mohler More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

LuCaP Prostate Cancer Patient-Derived Xenografts Reflect the Molecular Heterogeneity of Advanced Disease an--d Serve as Models for Evaluating Cancer Therapeutics.

BACKGROUNDMetastatic prostate cancer is a common and lethal disease for which there are no therapies that produce cures or long‐term durable remissions. Clinically relevant preclinical models are needed to increase our understanding of biology of this malignancy and to evaluate new agents that might provide effective treatment. Our objective was to establish and characterize patient‐derived xenografts (PDXs) from advanced prostate cancer (PC) for investigation of biology and evaluation of new treatment modalities.METHODSSamples of advanced PC obtained from primary prostate cancer obtained at surgery or from metastases collected at time of death were implanted into immunocompromised mice to establish PDXs. Established PDXs were propagated in vivo. Genomic, transcriptomic, and STR profiles were generated. Responses to androgen deprivation and docetaxel in vivo were characterized.RESULTSWe established multiple PDXs (LuCaP series), which represent the major genomic and phenotypic features of the disease in humans, including amplification of androgen receptor, PTEN deletion, TP53 deletion and mutation, RB1 loss, TMPRSS2‐ERG rearrangements, SPOP mutation, hypermutation due to MSH2/MSH6 genomic aberrations, and BRCA2 loss. The PDX models also exhibit variation in intra‐tumoral androgen levels. Our in vivo results show heterogeneity of response to androgen deprivation and docetaxel, standard therapies for advanced PC, similar to the responses of patients to these treatments.CONCLUSIONSThe LuCaP PDX series reflects the diverse molecular composition of human castration‐resistant PC and allows for hypothesis‐driven cause‐and‐effect studies of mechanisms underlying treatment response and resistance. Prostate 77: 654–671, 2017. © 2017 The Authors. The Prostate Published by Wiley Periodicals, Inc.

Androgen synthesis in prostate cancer: do all roads lead to Rome?

The accumulation of high concentrations of signalling androgens within prostate tumours that progress despite use of androgen-deprivation therapy is a clinically important mechanism of the development of castration-resistant prostate cancer. In the past 5 years, data from a number of studies have increased our understanding of the enzymes and substrates involved in intratumoural androgen biosynthesis, and have implicated three competing pathways, which are likely to account for these observations. These pathways ('canonical', 'backdoor' and '5α-dione'), which can all ultimately generate the potent signalling androgen, dihydrotestosterone, involve many of the same enzymes, but differ in terms of substrate preference, reaction sequence and the organs and tissues in which they occur. For this reason, the relative importance of each pathway to the development and progression of prostate cancer remains controversial. In this Review, we describe the current understanding of androgen synthesis and the evidence for its role in castration resistance, and examine the evidence supporting and or rebutting the relevance of each pathway to patients with prostate cancer.

Targeting of CYP17A1 Lyase by VT-464 Inhibits Adrenal and Intratumoral Androgen Biosynthesis and Tumor Growth of Castration Resistant Prostate Cancer.

Cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1) is a validated treatment target for the treatment of metastatic castration-resistant prostate cancer (CRPC). Abiraterone acetate (AA) inhibits both 17α-hydroxylase (hydroxylase) and 17,20-lyase (lyase) reactions catalyzed by CYP17A1 and thus depletes androgen biosynthesis. However, coadministration of prednisone is required to suppress the mineralocorticoid excess and cortisol depletion that result from hydroxylase inhibition. VT-464, a nonsteroidal small molecule, selectively inhibits CYP17A1 lyase and therefore does not require prednisone supplementation. Administration of VT-464 in a metastatic CRPC patient presenting with high tumoral expression of both androgen receptor (AR) and CYP17A1, showed significant reduction in the level of both dehydroepiandrosterone (DHEA) and serum PSA. Treatment of a CRPC patient-derived xenograft, MDA-PCa-133 expressing H874Y AR mutant with VT-464, reduced the increase in tumor volume in castrate male mice more than twice as much as the vehicle (P < 0.05). Mass spectrometry analysis of post-treatment xenograft tumor tissues showed that VT-464 significantly decreased intratumoral androgens but not cortisol. VT-464 also reduced AR signaling more effectively than abiraterone in cultured PCa cells expressing T877A AR mutant. Collectively, this study suggests that VT-464 therapy can effectively treat CRPC and be used in precision medicine based on androgen receptor mutation status.

Antiandrogens reduce intratumoral androgen concentrations and induce androgen receptor expression in castration-resistant VCaP xenografts

Antiandrogens reduce intratumoral androgen concentrations and induce androgen receptor expression in castration-resistant VCaP xenografts

Dihydrotestosterone synthesis pathways from inactive androgen 5α-androstane-3β,17β-diol in prostate cancer cells: Inhibition of intratumoural 3β-hydroxysteroid dehydrogenase activities by abiraterone.

Intratumoural dihydrotestosterone (DHT) synthesis could be an explanation for castration resistance in prostate cancer (PC). By using liquid chromatography-mass spectrometry, we evaluated the intratumoral DHT synthesis from 5α-androstane-3β,17β-diol (3β-diol), which is inactive androgen metabolized from DHT. 3β-diol had biochemical potential to be converted to DHT via three metabolic pathways and could stimulate PC cell growth. Especially, 3β-diol was not only converted back to upstream androgens such as dehydroepiandrosterone (DHEA) or Δ5-androstenediol but also converted directly to DHT which is the main pathway from 3β-diol to DHT. Abiraterone had a significant influence on the metabolism of DHEA, epiandrosterone and 3β-diol, by the inhibition of the intratumoural 3β-hydroxysteroid dehydrogenase (3β-HSD) activities which is one of key catalysts in androgen metabolic pathway. The direct-conversion of 3β-diol to DHT was catalysed by 3β-HSD and abiraterone could inhibit this activity of 3β-HSD. These results suggest that PC had a mechanism of intratumoural androgen metabolism to return inactive androgen to active androgen and intratumoural DHT synthesis from 3β-diol is important as one of the mechanisms of castration resistance in PC. Additionally, the inhibition of intratumoural 3β-HSD activity could be a new approach to castration-resistant prostate cancer treatment.

Discovery of (R)-2-(6-Methoxynaphthalen-2-yl)butanoic Acid as a Potent and Selective Aldo-keto Reductase 1C3 Inhibitor.

Type 5 17β-hydroxysteroid dehydrogenase, aldo-keto reductase 1C3 (AKR1C3) converts Δ(4)-androstene-3,17-dione and 5α-androstane-3,17-dione to testosterone (T) and 5α-dihydrotestosterone, respectively, in castration resistant prostate cancer (CRPC). In CRPC, AKR1C3 is implicated in drug resistance, and enzalutamide drug resistance can be surmounted by indomethacin a potent inhibitor of AKR1C3. We examined a series of naproxen analogues and find that (R)-2-(6-methoxynaphthalen-2-yl)butanoic acid (in which the methyl group of R-naproxen was replaced by an ethyl group) acts as a potent AKR1C3 inhibitor that displays selectivity for AKR1C3 over other AKR1C enzymes. This compound was devoid of inhibitory activity on COX isozymes and blocked AKR1C3 mediated production of T and induction of PSA in LNCaP-AKR1C3 cells as a model of a CRPC cell line. R-Profens are substrate selective COX-2 inhibitors and block the oxygenation of endocannabinoids and in the context of advanced prostate cancer R-profens could inhibit intratumoral androgen synthesis and act as analgesics for metastatic disease.

Sulforaphane increases the efficacy of anti-androgens by rapidly decreasing androgen receptor levels in prostate cancer cells.

Prostate cancer (PCa) cells utilize androgen for their growth. Hence, androgen deprivation therapy (ADT) using anti-androgens, e.g. bicalutamide (BIC) and enzalutamide (ENZ), is a mainstay of treatment. However, the outgrowth of castration resistant PCa (CRPC) cells remains a significant problem. These CRPC cells express androgen receptor (AR) and utilize the intratumoral androgen towards their continued growth and invasion. Sulforaphane (SFN), a naturally occurring isothiocyanate found in cruciferous vegetables, can decrease AR protein levels. In the present study, we tested the combined efficacy of anti-androgens and SFN in suppressing PCa cell growth, motility and clonogenic ability. Both androgen-dependent (LNCaP) and androgen-independent (C4-2B) cells were used to monitor the effects of BIC and ENZ, alone and in combination with SFN. Co-exposure to SFN significantly (p<0.005) enhanced the anti-proliferative effects of anti-androgens and downregulated expression of the AR-responsive gene, prostate specific antigen (PSA) (p<0.05). Exposure to SFN decreased AR protein levels in a time- and dose-dependent manner with almost no AR detected at 24h with 15µM SFN (p<0.005). This rapid and potent AR suppression by SFN occurred by both AR protein degradation, as suggested by cycloheximide (CHX) co-exposure studies, and by suppression of AR gene expression, as evident from quantitative RT-PCR experiments. Pre-exposure to SFN also reduced R1881-stimulated nuclear localization of AR, and combined treatment with SFN and anti-androgens abrogated the mitogenic effects of this AR-agonist (p<0.005). Wound-healing assays revealed that co-exposure to SFN and anti-androgens can significantly (p<0.005) reduce PCa cell migration. In addition, long-term exposures (14 days) to much lower concentrations of these agents, SFN (0.2µM), BIC (1µM) and/or ENZ (0.4µM) significantly (p<0.005) decreased the number of colony forming units (CFUs). These findings clearly suggest that SFN may be used as a promising adjunct agent to augment the efficacy of anti-androgens against aggressive PCa cells.