Intratumoral Androgen Research Articles

Alternative Acts: Oncogenic Splicing of Steroidogenic Enzymes in Prostate Cancer.

Castration-resistant prostate cancer is characterized by loss of the androgen inactivation enzyme HSD17B2, emphasizing the importance of intratumoral androgens in tumor progression. Inactive isoforms generated by alternative splicing destabilize the wild-type enzyme, adding steroidogenesis to other prostate cancer drivers that undergo oncogenic splicing, highlighting aberrant splicing as a therapeutic target.See related article by Gao et al., p. 1291.

A 3-(4-nitronaphthen-1-yl) amino-benzoate analog as a bifunctional AKR1C3 inhibitor and AR antagonist: Head to head comparison with other advanced AKR1C3 targeted therapeutics

Drugs used for the treatment of castration resistant prostate cancer (CRPC) include Abiraterone acetate (Zytiga®) and Enzalutamide (XTANDI®). However, these drugs provide clinical benefit in metastatic disease for only a brief period before drug resistance emerges. One mechanism of drug resistance involves the overexpression of type 5 17-β-hydroxysteroid dehydrogenase (aldo-keto reductase 1C3 or AKR1C3), a major enzyme responsible for the formation of intratumoral androgens that activate the androgen receptor (AR). 3-((4-Nitronaphthalen-1-yl)amino)benzoic acid 1 is a “first-in-class” AKR1C3 competitive inhibitor and AR antagonist. Compound 1 was compared in a battery of in vitro studies with structurally related N-naphthyl-aminobenzoates, and AKR1C3 targeted therapeutics e.g. GTx-560 and ASP9521, as well as with R-bicalutamide, enzalutamide and abiraterone acetate. Compound 1 was the only naphthyl derivative that was a selective AKR1C3 inhibitor and AR antagonist in direct competitive binding assays and in AR driven reporter gene assays. GTx-560 displayed weak activity as a direct AR antagonist but had high potency in the AR reporter gene assay consistent with its ability to inhibit the co-activator function of AKR1C3. By contrast ASP9521 did not act as either an AR antagonist or block AR reporter gene activity. Compound 1 was the only compound that showed comparable potency to inhibit AKR1C3 and act as a direct AR antagonist. Compound 1 blocked the formation of testosterone in LNCaP-AKR1C3 cells, and the expression of PSA driven by the AKR1C3 substrate (4-androstene-3,17-dione) and by an AR agonist, 5α-dihydrotestosterone consistent with its bifunctional role. Compound 1 blocked the nuclear translocation of the AR at similar concentrations to enzalutamide and caused disappearance of the AR from cell lysates. R-biaclutamide and enzalutamide inhibited AKR1C3 at concentrations 200x greater than compound 1, suggesting that its bifunctionality can be explained by a shared pharmacophore that can be optimized.

Silencing of the transcriptional factor ZEB1 alters the steroidogenic pathway, and increases the concentration of testosterone and DHT in DU145 cells.

Prostate cancer (PCa) is the second most common type of male malignancy worldwide. The transcription factor zinc finger E‑box binding homeobox 1 (ZEB1) is associated with epithelial‑mesenchymal transition and is also involved in regulation of androgen receptor (AR) expression, the main ligands of which are testosterone and dihydrotestosterone (DHT). These androgens are synthesized through the steroidogenic pathway within the prostate, and their synthesis is altered in PCa. The present study aimed to determine the ZEB1‑induced alterations in androgen synthesis and AR expression in the DU145 PCa cell line. Reverse transcription‑quantitative polymerase chain reaction, western blotting and immunocytochemistry were used to determine the mRNA and protein expression levels, and cellular localization of steroidogenic pathway enzymes in the DU145 cell line in response to ZEB1 silencing. Furthermore, the concentrations of testosterone and DHT were detected in cell culture medium using ELISA. ZEB1‑silenced cells exhibited an increase in testosterone and DHT production, an increase in AR expression and an alteration in the steroidogenic pathway. In particular, steroidogenic acute regulatory protein and 5α‑reductase 2 expression levels were decreased, whereas cytochrome P450 family 17 subfamily A member 1, 5α‑reductase 1, aldo‑keto reductase family 1 member D1 and aldo‑keto reductase family 1 member C2 expression levels were increased. In conclusion, the present study provided novel information regarding the regulation of intratumoral androgen production in PCa, which is relevant for the progression of the disease to a castration‑resistant form.

AKR1C3 Inhibitor KV-37 Exhibits Antineoplastic Effects and Potentiates Enzalutamide in Combination Therapy in Prostate Adenocarcinoma Cells.

Aldo-keto reductase 1C3 (AKR1C3), also known as type 5 17 β-hydroxysteroid dehydrogenase, is responsible for intratumoral androgen biosynthesis, contributing to the development of castration-resistant prostate cancer (CRPC) and eventual chemotherapeutic failure. Significant upregulation of AKR1C3 is observed in CRPC patient samples and derived CRPC cell lines. As AKR1C3 is a downstream steroidogenic enzyme synthesizing intratumoral testosterone (T) and 5α-dihydrotestosterone (DHT), the enzyme represents a promising therapeutic target to manage CRPC and combat the emergence of resistance to clinically employed androgen deprivation therapy. Herein, we demonstrate the antineoplastic activity of a potent, isoform-selective and hydrolytically stable AKR1C3 inhibitor (E)-3-(4-(3-methylbut-2-en-1-yl)-3-(3-phenylpropanamido)phenyl)acrylic acid (KV-37), which reduces prostate cancer cell growth in vitro and in vivo and sensitizes CRPC cell lines (22Rv1 and LNCaP1C3) toward the antitumor effects of enzalutamide. Crucially, KV-37 does not induce toxicity in nonmalignant WPMY-1 prostate cells nor does it induce weight loss in mouse xenografts. Moreover, KV-37 reduces androgen receptor (AR) transactivation and prostate-specific antigen expression levels in CRPC cell lines indicative of a therapeutic effect in prostate cancer. Combination studies of KV-37 with enzalutamide reveal a very high degree of synergistic drug interaction that induces significant reduction in prostate cancer cell viability via apoptosis, resulting in >200-fold potentiation of enzalutamide action in drug-resistant 22Rv1 cells. These results demonstrate a promising therapeutic strategy for the treatment of drug-resistant CRPC that invariably develops in prostate cancer patients following initial treatment with AR antagonists such as enzalutamide. Mol Cancer Ther; 17(9); 1833-45. ©2018 AACR.

Androgen receptor: what we know and what we expect in castration-resistant prostate cancer

Androgen deprivation therapy is an important therapy for prostate cancer (PCa) in aging men. Under the background of castration, it is inevitable that prostate cancer will develop into castration-resistant prostate cancer (CRPC), which has a high mortality rate, after 2-3years. Androgen receptor (AR) plays a key role in PCa development and is essential to CRPC. More recent research studies have reported that the development of CRPC is largely due to altered mechanisms related to AR, so it is important for us to understand the roles of AR and detailed AR-related mechanisms in CRPC. The multiple AR-related mechanisms promoting the development of CRPC are as follows: (1) enhanced transformation and increased synthesis of intratumoral androgen; (2) AR overexpression, which enables CRPC to be hypersensitive to low levels of androgen; (3) AR cofactors, which enhanced AR transactivation; (4) AR-spliced variants, which mediated downstream gene expression without androgen; (5) the interaction between the AR pathway and classic tumor-related pathways; and» (6) AR mutations, which reduced AR specificity and enhanced AR transcription.

AKR1C3, a crucial androgenic enzyme in prostate cancer, promotes epithelial-mesenchymal transition and metastasis through activating ERK signaling

BackgroundAKR1C3, as a crucial androgenic enzyme, facilitates intratumoral androgen biosynthesis and androgen receptor activation in castration-resistant prostate cancer (PCa). The data has shown that AKR1C3 expression is significantly elevated in clinical metastatic PCa specimens, indicating a potential role of AKR1C3 in PCa metastasis. MethodsC4-2, 22RV1-T, and PC-3 cells with higher AKR1C3 expression were selected and treated with several specific AKR1C3 shRNAs or small molecule inhibitor, and the cell migration and invasion abilities were detected by wound healing assay and Transwell assay. The expression of several epithelial-mesenchymal transition (EMT) markers (i.e., E-cadherin and vimentin) and the related transcription factors (i.e., ZEB1, TWIST1, and SLUG) was examined by Western blot or quantitative PCR assays, and the phosphorylation of AKT or ERK was detected by Western blot. Also, subcutaneous xenografts with 22RV1-T sublines were used to detect in vivo tumor growth, and the expression of E-cadherin, vimentin, and ZEB1 by immunohistochemical staining. The correlation between AKR1C3 and EMT marker expression in clinical specimens was analyzed. ResultsAKR1C3 was overexpressed in more aggressive PCa cell lines regardless of the androgen receptor status. Knockdown of AKR1C3 expression or inhibition of AKR1C3 activity could significantly suppress cell migration and invasion abilities in vitro, and increase E-cadherin expression but decrease vimentin expression, in which the phosphorylation of ERK and the EMT-associated transcription factor expression were specifically down-regulated. Also, knockdown of AKR1C3 could suppress PCa tumorigenesis and reverse EMT in vivo. Moreover, there was a significant correlation between AKR1C3 expression and EMT in human PCa specimens from public tissue microarray. ConclusionsAKR1C3 is a novel EMT driver in PCa metastasis through activating ERK signaling.

Abstract 480: Regulation of crosstalk between AR and Wnt/Beta-catenin pathways by miR-644a in prostate cancer

Abstract Prostate Cancer (PCa) is the most commonly diagnosed cancer in males with 161,360 people diagnosed in the United States and is predicted to cause 26,730 deaths in 2017. 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/B-catenin signaling pathway has been implicated in the development of many cancers including PCa. The disruption of this signaling leads to the stabilization of B-catenin which upregulates many genes involved in tumorigenesis. Additionally, B-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 B-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-B and B-catenin in the wnt/B-catenin signaling pathway. The effect of miR-644a in combination with inhibitors (B-catenin or GSK3-B) was also assessed using viability assays and a significant downregulation was observed. We will further study the posttranscriptional effects of tumor suppressor miR-644a on the wnt/B-catenin pathway as well as assess if miR-644a can inhibit the cross-talk of these pathways. Citation Format: Alexis R. Plaga, Girish C. Shukla. Regulation of crosstalk between AR and Wnt/Beta-catenin pathways by miR-644a in prostate cancer [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 480.

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.

Mathematical modeling of intracrine androgen metabolism in prostate cancer: Methodological aspects.

Progression of castration-recurrent/resistant prostate cancer (CRPC) relies in part on dihydrotestosterone derived from intratumoral androgen metabolism. Mathematical modeling provides a valuable tool for studies of androgen metabolism in CRPC. This modeling approach integrates existing knowledge about complex biologic systems and provides a means of interrogating the effects of various interventions. We sought to model a single reaction in the androgen biosynthesis network, namely the oxidation of androsterone (AND) to androstanedione (5α-dione) by four 3α-oxidoreductase enzymes, as an initial effort to establish the feasibility of our modeling approach. Models were constructed for two cell culture systems, a non-prostate cancer cell line (CV-1) and a prostate cancer cell line (LAPC-4), using the SimBiology app (version 5.3) in MATLAB (version 8.6). The models included components for substrate (AND), product (5α-dione), each of the four enzymes, and each of the four enzyme-substrate complexes. Each enzymatic reaction consisted of a reversible enzyme-substrate binding step and an irreversible catalysis step. Rates of change for each component were described using ordinary differential equations. Mathematical models were developed with model parameter values derived from literature sources or from existing experimental data, which included gene expression measurements and substrate and product concentrations determined using liquid chromatography-tandem mass spectrometry. The models for both cell lines adequately described substrate and product concentrations observed after 12 h treatment with AND. This modeling approach represents an adaptable, extensible and mechanistic framework that reflects androgen metabolism. The models can be expanded systematically to describe the complex androgen metabolic pathways important for study of novel therapies for CRPC.

11-Oxygenated androgen precursors are the preferred substrates for aldo-keto reductase 1C3 (AKR1C3): Implications for castration resistant prostate cancer

The progression of castration resistant prostate cancer (CRPC) is driven by the intratumoral conversion of adrenal androgen precursors to potent androgens. The expression of aldo-keto reductase 1C3 (AKR1C3), which catalyses the reduction of weak androgens to more potent androgens, is significantly increased in CRPC tumours. The oxidation of androgens to their inactive form is catalysed by 17β-hydroxysteroid dehydrogenase type 2 (17βHSD2), but little attention is given to the expression levels of this enzyme. In this study, we show that the 11-oxygenated androgen precursors of adrenal origin are the preferred substrate for AKR1C3. In particular we show that the enzymatic efficiency of AKR1C3 is 8- and 24-fold greater for 11-ketoandrostenedione than for the classic substrates androstenedione and 5α-androstanedione, respectively. Using three independent experimental systems and a computational model we subsequently show that increased ratios of AKR1C3:17βHSD2 significantly favours the flux through the 11-oxygenated androgen pathway as compared to the classical or 5α-androstanedione pathways. Our findings reveal that the flux through the classical and 5α-androstanedione pathways are limited by the low catalytic efficiently of AKR1C3 towards classical androgens combined with the high catalytic efficiency of 17βHSD2, and that the expression of the oxidative enzyme therefore plays a vital role in determining the steady state concentration of active androgens. Using microarray data from prostate tissue we confirm that the AKR1C3:17βHSD2 ratio is significantly increased in patients undergoing androgen deprivation therapy as compared to benign tissue, and further increased in patients with CRPC. Taken together this study therefore demonstrates that the ratio of AKR1C3:17βHSD2 is more important than AKR1C3 expression alone in determining intratumoral androgen levels and that 11-oxygenated androgens may play a bigger role in CRPC than previously anticipated.

AR Signaling in Prostate Cancer Regulates a Feed-Forward Mechanism of Androgen Synthesis by Way of HSD3B1 Upregulation.

3βHSD1 enzymatic activity is essential for synthesis of potent androgens from adrenal precursor steroids in prostate cancer. A germline variant in HSD3B1, the gene that encodes 3βHSD1, encodes for a stable enzyme, regulates adrenal androgen dependence, and is a predictive biomarker of poor clinical outcomes after gonadal testosterone deprivation therapy. However, little is known about HSD3B1 transcriptional regulation. Generally, it is thought that intratumoral androgen synthesis is upregulated after gonadal testosterone deprivation, enabling development of castration-resistant prostate cancer. Given its critical role in extragonadal androgen synthesis, we sought to directly interrogate the transcriptional regulation of HSD3B1 in multiple metastatic prostate cancer cell models. Surprisingly, we found that VCaP, CWR22Rv1, LNCaP, and LAPC4 models demonstrate induction of HSD3B1 upon androgen stimulation for approximately 72 hours, followed by attenuation around 120 hours. 3βHSD1 protein levels mirrored transcriptional changes in models harboring variant (LNCaP) and wild-type (LAPC4) HSD3B1, and in these models androgen induction of HSD3B1 is abrogated via enzalutamide treatment. Androgen treatment increased flux from [3H]-dehydroepiandrosterone to androstenedione and other downstream metabolites. HSD3B1 expression was reduced 72 hours after castration in the VCaP xenograft mouse model, suggesting androgen receptor (AR) regulation of HSD3B1 also occurs in vivo. Overall, these data suggest that HSD3B1 is unexpectedly positively regulated by androgens and ARs. These data may have implications for the development of treatment strategies tailored to HSD3B1 genotype status.

Mechanisms and Approaches for Overcoming Enzalutamide Resistance in Prostate Cancer.

Enzalutamide, a second-generation small-molecule inhibitor of the androgen receptor (AR), has been approved for patients who failed with androgen deprivation therapy and have developed castration-resistant prostate cancer. More than 80% of these patients develop bone metastases. The binding of enzalutamide to the AR prevents the nuclear translocation of the receptor, thus inactivating androgen signaling. However, prostate cancer cells eventually develop resistance to enzalutamide treatment. Studies have found resistance both in patients and in laboratory models. The mechanisms of and approaches to overcoming such resistance are significant issues that need to be addressed. In this review, we focus on the major mechanisms of acquired enzalutamide resistance, including genetic mutations and splice variants of the AR, signaling pathways that bypass androgen signaling, intratumoral androgen biosynthesis by prostate tumor cells, lineage plasticity, and contributions from the tumor microenvironment. Approaches for overcoming these mechanisms to enzalutamide resistance along with the associated problems and solutions are discussed. Emerging questions, concerns, and new opportunities in studying enzalutamide resistance will be addressed as well.

Intratumoral androgen levels are linked to TMPRSS2-ERG fusion in prostate cancer.

Intratumoral androgen biosynthesis is one of the mechanisms involved in the progression of prostate cancer, and an important target for novel prostate cancer therapies. Using gas chromatography-tandem mass spectrometry and genome-wide RNA sequencing, we have analyzed androgen concentrations and androgen-regulated gene expression in cancerous and morphologically benign prostate tissue specimens and serum samples obtained from 48 primary prostate cancer patients. Intratumoral dihydrotestosterone (DHT) concentrations were significantly higher in the cancerous tissues compared to benign prostate (P < 0.001). The tissue/serum ratios of androgens were highly variable between the patients, indicating individual patterns of androgen metabolism and/or uptake of androgens within the prostate tissue. An unsupervised hierarchical clustering analysis of intratissue androgen concentrations indicated that transmembrane protease, serine 2/ETS-related gene (TMPRSS2-ERG)-positive patients have different androgen profiles compared to TMPRSS2-ERG-negative patients. TMPRSS2-ERG gene fusion status was also associated with an enhanced androgen-regulated gene expression, along with altered intratumoral androgen metabolism, demonstrated by reduced testosterone concentrations and increased DHT/testosterone ratios in TMPRSS2-ERG-positive tumors. TMPRSS2-ERG-positive and -negative prostate cancer specimens have distinct intratumoral androgen profiles, possibly due to activation of testosterone-independent DHT biosynthesis via the alternative pathway in TMPRSS2-ERG-positive tumors. Thus, patients with TMPRSS2-ERG-positive prostate cancer may benefit from novel inhibitors targeting the alternative DHT biosynthesis.

Nuclear Receptor LRH-1 Functions to Promote Castration-Resistant Growth of Prostate Cancer via Its Promotion of Intratumoral Androgen Biosynthesis.

Targeting of steroidogenic enzymes (e.g., abiraterone acetate targeting CYP17A1) has been developed as a novel therapeutic strategy against metastatic castration-resistant prostate cancer (CRPC). However, resistance to steroidal inhibitors inevitably develops in patients, the mechanisms of which remain largely unknown. Liver receptor homolog-1 (LRH-1, NR5A2) is a nuclear receptor, originally characterized as an important regulator of some liver-specific metabolic genes. Here, we report that LRH-1, which exhibited an increased expression pattern in high-grade prostate cancer and CRPC xenograft models, functions to promote de novo androgen biosynthesis via its direct transactivation of several key steroidogenic enzyme genes, elevating intratumoral androgen levels and reactivating AR signaling in CRPC xenografts as well as abiraterone-treated CRPC tumors. Pharmacologic inhibition of LRH-1 activity attenuated LRH-1-mediated androgen deprivation and anti-androgen resistance of prostate cancer cells. Our findings not only demonstrate the significant role of LRH-1 in the promotion of intratumoral androgen biosynthesis in CRPC via its direct transcriptional control of steroidogenesis, but also suggest targeting LRH-1 could be a potential therapeutic strategy for CRPC management.Significance: These findings not only demonstrate the significant role of the nuclear receptor LRH-1 in the promotion of intratumoral androgen biosynthesis in CRPC via its direct transcriptional control of steroidogenesis, but also suggest targeting LRH-1 could be a potential therapeutic strategy for CRPC management. Cancer Res; 78(9); 2205-18. ©2018 AACR.

Abiraterone survival benefit is independent of pain, prostate-specific antigen and Gleason sum in men with chemo-naïve metastatic castration-resistant prostate cancer (analysis of COU-AA-302)

Advanced prostate cancer (PC), including metastatic castration-resistant prostate cancer (mCRPC) represents the lethal form of PC, yet remains largely dependent on androgen signaling (1). Abiraterone acetate (AA) is a steroid 17-hydroxylase/C17,20-lyase (CYP17) inhibitor targeting testicular, adrenal and intratumoral androgen production.

A Novel Mechanism to Drive Castration-Resistant Prostate Cancer

Androgen receptor signaling is critical for prostate adenocarcinoma, even after androgen deprivation therapy. Persistence of intratumoral androgens has been found in castration-resistant prostate cancer and attributed to increased in situ synthesis. Recently, Sharifi and colleagues reported an additional mechanism that can enhance local androgenic exposure: downregulation of an androgen-inactivating enzyme.

A phase Ib/II trial of indomethacin and enzalutamide to treat castration-resistant prostate cancer (CRPC).

TPS394 Background: Enzalutamide (Enza) and abiraterone (Abi) are commonly used to treat CRPC. Resistance is the most common cause of treatment failure. We discovered that a critical steroidogenic enzyme AKR1C3 was significantly elevated and contributed to intratumoral androgen synthesis in Enza-resistant prostate cancer cells and tumors. Overexpression of AKR1C3 induced androgen receptor variant 7 (AR-V7) expression, while inhibition of AKR1C3 downregulated AR-V7. We then discovered that indomethacin (Indo) inhibited AKR1C3 activation and sensitized resistant CRPC cells to Enza and Abi. One patient accidentally took Indo and achieved biochemical as well as radiological response of his prostate cancer. These findings prompted us to design a clinical trial to test the combination of Indo with Enza for the treatment of CRPC and to study the underlying mechanisms of action and resistance. Methods: This investigator-initiated single-arm Phase Ib/II trial enrolls patients with progressive CRPC after Abi, adequate vital organ function, ECOG performance status 0-2, and serum testosterone &lt; 50 ng/dl. Major exclusion criteria include prior Enza treatment, brain metastasis and history of seizure. In the Phase Ib cohort, patients receive Enza 160 mg po qd and Indo 50 mg po tid to determine toxicity. The Phase II expansion will enroll 26 patients with 21 evaluable patients. This sample size provides 90% power to detect, at the 0.05 level (1-sided), the difference between a PSA response rate of 50% expected with the study treatment and a historical control of 20% with Enza alone. Co-primary endpoints are safety and PSA response of ≥50% decrease. Secondary endpoints include overall response rate as determined by the Prostate Cancer Working Group 2 criteria (PCWG2), progression-free survival and overall survival. Molecular correlative studies are exploratory endpoints. Serum and intratumoral androgen levels, full-length AR, AR-V7 and AKR1C3 will be measured to assess the effect of the combination therapy. To date, 4 patients have been enrolled to the trial (clinicaltrials.gov Identifier No: NCT02935205; this trial is funded by DoD Prostate Cancer Research Program IMPACT award). Clinical trial information: NCT02935205.

Modulation of AKR1C2 by curcumin decreases testosterone production in prostate cancer.

Intratumoral androgen biosynthesis has been recognized as an essential factor of castration‐resistant prostate cancer. The present study investigated the effects of curcumin on the inhibition of intracrine androgen synthesis in prostate cancer. Human prostate cancer cell lines, LNCaP and 22Rv1 cells were incubated with or without curcumin after which cell proliferation was measured at 0, 24, 48 and 72 hours, respectively. Prostate tissues from the transgenic adenocarcinoma of the mouse prostate (TRAMP) model were obtained after 1‐month oral administration of 200 mg/kg/d curcumin. Testosterone and dihydrotestosterone concentrations in LNCaP prostate cancer cells were determined through LC‐MS/MS assay. Curcumin inhibited cell proliferation and induced apoptosis of prostate cancer cells in a dose‐dependent manner. Curcumin decreased the expression of steroidogenic acute regulatory proteins, CYP11A1 and HSD3B2 in prostate cancer cell lines, supporting the decrease of testosterone production. After 1‐month oral administration of curcumin, Aldo‐Keto reductase 1C2 (AKR1C2) expression was elevated. Simultaneously, decreased testosterone levels in the prostate tissues were observed in the TRAMP mice. Meanwhile, curcumin treatments considerably increased the expression of AKR1C2 in prostate cancer cell lines, supporting the decrease of dihydrotestosterone. Taken together, these results suggest that curcumin's natural bioactive compounds could have potent anticancer properties due to suppression of androgen production, and this could have therapeutic effects on prostate cancer.

PTEN Loss Promotes Intratumoral Androgen Synthesis and Tumor Microenvironment Remodeling via Aberrant Activation of RUNX2 in Castration-Resistant Prostate Cancer.

Purpose: Intratumoral androgen synthesis (IAS) is a key mechanism promoting androgen receptor (AR) reactivation and antiandrogen resistance in castration-resistant prostate cancer (CRPC). However, signaling pathways driving aberrant IAS remain poorly understood.Experimental Design: The effect of components of the AKT-RUNX2-osteocalcin (OCN)-GPRC6A-CREB signaling axis on expression of steroidogenesis genes CYP11A1 and CYP17A1 and testosterone level were examined in PTEN-null human prostate cancer cell lines. Pten knockout mice were used to examine the effect of Runx2 heterozygous deletion or abiraterone acetate (ABA), a prodrug of the CYP17A1 inhibitor abiraterone on Cyp11a1 and Cyp17a1 expression, testosterone level and tumor microenvironment (TME) remodeling in vivoResults: We uncovered that activation of the AKT-RUNX2-OCN-GPRC6A-CREB signaling axis induced expression of CYP11A1 and CYP17A1 and testosterone production in PTEN-null prostate cancer cell lines in culture. Deletion of Runx2 in Pten homozygous knockout prostate tumors decreased Cyp11a1 and Cyp17a1 expression, testosterone level, and tumor growth in castrated mice. ABA treatment also inhibited testosterone synthesis and alleviated Pten loss-induced tumorigenesis in vivoPten deletion induced TME remodeling, but Runx2 heterozygous deletion or ABA treatment reversed the effect of Pten loss by decreasing expression of the collagenase Mmp9.Conclusions: Abnormal RUNX2 activation plays a pivotal role in PTEN loss-induced IAS and TME remodeling, suggesting that the identified signaling cascade represents a viable target for effective treatment of PTEN-null prostate cancer, including CRPC. Clin Cancer Res; 24(4); 834-46. ©2017 AACR.

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.