Intracrine Androgen Research Articles

Indomethacin to inhibit AKR1C3 intracrine androgen production and sensitizes prostate cancer (PCa) to enzalutamide.

161 Background: Enzalutamide (Enza) improves the treatment of metastatic castration-resistant PCa (mCRPC). Acquired resistance is the most common cause of treatment failure. Our objective was to determine mechanisms of resistance to Enza and develop novel therapy to overcome resistance. Methods: Enza-resistant PCa cells were established through continuous culture of C4-2B cells with Enza. Global gene expression array and western blots were used to assess gene expression in cell lines and validated in clinical specimens using Oncomine and GEO databases. The therapeutic effects of indomethacin were determined in cell culture and in mice carrying CWR22Rv1 xenografts. A Phase I/II clinical trial with Enza plus indomethacin has been approved by IRB. Results: Steroid biosynthesis pathways, especially AKR1C3, were significantly elevated in Enza resistant cells and was associated with high production of androgens. AKR1C3 was also highly expressed in metastatic and recurrent CaP and in Enza-resistant xenograft tumors. Overexpression of AKR1C3 conferred resistance to Enza. Inhibition of AKR1C3 expression by its specific shRNAs or AKR1C3 enzymatic activity by indomethacin re-sensitized Enza-resistant CaP cells to Enza treatment both in vitro and in vivo. Combination of indomethacin and Enza resulted in significant inhibition of Enza-resistant tumor growth. Indomethacin treatment in one patient with mCRPC induced tumor shrinkage and PSA decrease. In a Phase I/II trial funded by the Department of Defense, patients with mCRPC, good performance status (ECOG 0-2) and vital organ function, and no history of Enza or indomethacin treatment will be recruited. The co-primary objective is to assess the toxicity and prostate-specific antigen (PSA) response ( ≥ 50% reduction). We will also assess the overall response as determined by the Prostate Cancer Working Group 2 criteria, progression-free survival and molecular correlative studies. Conclusions: AKR1C3 activation is a critical resistance mechanism associated with Enza resistance. Indomethacin targets AKR1C3 and intratumor androgen synthesis, and can be potentially used as a therapeutic agent for mCRPC. Clinical trial results will be presented. Clinical trial information: to be registered.

Inhibition of AKR1C3 Activation Overcomes Resistance to Abiraterone in Advanced Prostate Cancer.

Abiraterone suppresses intracrine androgen synthesis via inhibition of CYP17A1. However, clinical evidence suggests that androgen synthesis is not fully inhibited by abiraterone and the sustained androgen production may lead to disease relapse. In the present study, we identified AKR1C3, an important enzyme in the steroidogenesis pathway, as a critical mechanism driving resistance to abiraterone through increasing intracrine androgen synthesis and enhancing androgen signaling. We found that overexpression of AKR1C3 confers resistance to abiraterone while downregulation of AKR1C3 resensitizes resistant cells to abiraterone treatment. In abiraterone-resistant prostate cancer cells, AKR1C3 is overexpressed and the levels of intracrine androgens are elevated. In addition, AKR1C3 activation increases intracrine androgen synthesis and enhances androgen receptor (AR) signaling via activating AR transcriptional activity. Treatment of abiraterone-resistant cells with indomethacin, an AKR1C3 inhibitor, overcomes resistance and enhances abiraterone therapy both in vitro and in vivo by reducing the levels of intracrine androgens and diminishing AR transcriptional activity. These results demonstrate that AKR1C3 activation is a critical mechanism of resistance to abiraterone through increasing intracrine androgen synthesis and enhancing androgen signaling. Furthermore, this study provides a preclinical proof-of-principle for clinical trials investigating the combination of targeting AKR1C3 using indomethacin with abiraterone for advanced prostate cancer. Mol Cancer Ther; 16(1); 35-44. ©2016 AACR.

Src promotes castration-recurrent prostate cancer through androgen receptor-dependent canonical and non-canonical transcriptional signatures.

Progression of prostate cancer (PC) to castration-recurrent growth (CRPC) remains dependent on sustained expression and transcriptional activity of the androgen receptor (AR). A major mechanism contributing to CRPC progression is through the direct phosphorylation and activation of AR by Src-family (SFK) and ACK1 tyrosine kinases. However, the AR-dependent transcriptional networks activated by Src during CRPC progression have not been elucidated. Here, we show that activated Src (Src527F) induces androgen-independent growth in human LNCaP cells, concomitant with its ability to induce proliferation/survival genes normally induced by dihydrotestosterone (DHT) in androgen-dependent LNCaP and VCaP cells. Src induces additional gene signatures unique to CRPC cell lines, LNCaP-C4-2 and CWR22Rv1, and to CRPC LuCaP35.1 xenografts. By comparing the Src-induced AR-cistrome and/or transcriptome in LNCaP to those in CRPC and LuCaP35.1 tumors, we identified an 11-gene Src-regulated CRPC signature consisting of AR-dependent, AR binding site (ARBS)-associated genes whose expression is altered by DHT in LNCaP[Src527F] but not in LNCaP cells. The differential expression of a subset (DPP4, BCAT1, CNTNAP4, CDH3) correlates with earlier PC metastasis onset and poorer survival, with the expression of BCAT1 required for Src-induced androgen-independent proliferation. Lastly, Src enhances AR binding to non-canonical ARBS enriched for FOXO1, TOP2B and ZNF217 binding motifs; cooperative AR/TOP2B binding to a non-canonical ARBS was both Src- and DHT-sensitive and correlated with increased levels of Src-induced phosphotyrosyl-TOP2B. These data suggest that CRPC progression is facilitated via Src-induced sensitization of AR to intracrine androgen levels, resulting in the engagement of canonical and non-canonical ARBS-dependent gene signatures.

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.

Regulators of Androgen Action Resource: a one-stop shop for the comprehensive study of androgen receptor action.

Androgen receptor (AR) is a ligand-activated transcription factor that is the main target for treatment of non-organ-confined prostate cancer (CaP). Failure of life-prolonging AR-targeting androgen deprivation therapy is due to flexibility in steroidogenic pathways that control intracrine androgen levels and variability in the AR transcriptional output. Androgen biosynthesis enzymes, androgen transporters and AR-associated coregulators are attractive novel CaP treatment targets. These proteins, however, are characterized by multiple transcript variants and isoforms, are subject to genomic alterations, and are differentially expressed among CaPs. Determining their therapeutic potential requires evaluation of extensive, diverse datasets that are dispersed over multiple databases, websites and literature reports. Mining and integrating these datasets are cumbersome, time-consuming tasks and provide only snapshots of relevant information. To overcome this impediment to effective, efficient study of AR and potential drug targets, we developed the Regulators of Androgen Action Resource (RAAR), a non-redundant, curated and user-friendly searchable web interface. RAAR centralizes information on gene function, clinical relevance, and resources for 55 genes that encode proteins involved in biosynthesis, metabolism and transport of androgens and for 274 AR-associated coregulator genes. Data in RAAR are organized in two levels: (i) Information pertaining to production of androgens is contained in a ‘pre-receptor level’ database, and coregulator gene information is provided in a ‘post-receptor level’ database, and (ii) an ‘other resources’ database contains links to additional databases that are complementary to and useful to pursue further the information provided in RAAR. For each of its 329 entries, RAAR provides access to more than 20 well-curated publicly available databases, and thus, access to thousands of data points. Hyperlinks provide direct access to gene-specific entries in the respective database(s). RAAR is a novel, freely available resource that provides fast, reliable and easy access to integrated information that is needed to develop alternative CaP therapies.Database URL: http://www.lerner.ccf.org/cancerbio/heemers/RAAR/search/

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.

Vitamin D in Prostate Cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is a progressive, noncurable disease induced by androgen receptor (AR) upon its activation by tumor tissue androgen, which is generated from adrenal steroid dehydroepiandrosterone (DHEA) through intracrine androgen biosynthesis. Inhibition of mCRPC and early-stage, androgen-dependent prostate cancer by calcitriol, the bioactive vitamin D3 metabolite, is amply documented in cell culture and animal studies. However, clinical trials of calcitriol or synthetic analogs are inconclusive, although encouraging results have recently emerged from pilot studies showing efficacy of a safe-dose vitamin D3 supplementation in reducing tumor tissue inflammation and progression of low-grade prostate cancer. Vitamin D-mediated inhibition of normal and malignant prostate cells is caused by diverse mechanisms including G1/S cell cycle arrest, apoptosis, prodifferentiation gene expression changes, and suppressed angiogenesis and cell migration. Biological effects of vitamin D are mediated by altered expression of a gene network regulated by the vitamin D receptor (VDR), which is a multidomain, ligand-inducible transcription factor similar to AR and other nuclear receptors. AR-VDR cross talk modulates androgen metabolism in prostate cancer cells. Androgen inhibits vitamin D-mediated induction of CYP24A1, the calcitriol-degrading enzyme, while vitamin D promotes androgen inactivation by inducing phase I monooxygenases (e.g., CYP3A4) and phase II transferases (e.g., SULT2B1b, a DHEA-sulfotransferase). CYP3A4 and SULT2B1b levels are markedly reduced and CYP24A1 is overexpressed in advanced prostate cancer. In future trials, combining low-calcemic, potent next-generation calcitriol analogs with CYP24A1 inhibition or androgen supplementation, or cancer stem cell suppression by a phytonutrient such as sulfarophane, may prove fruitful in prostate cancer prevention and treatment.

Androgen receptor and intracrine androgen signaling in endometrial carcinomas

The androgen receptor (AR) is known to play critical roles in the malignancy of prostate cancer as well as the management of male reproductive organs. Endometrial carcinoma, one of the major female cancers, is considered an androgen-related cancer. However, the importance of androgen signaling through its receptor in endometrial carcinomas has not yet been clarified. We recently demonstrated the significance of androgen signaling and intracrine dihydrotestosterone (DHT) in endometrial carcinomas as follows: 1) A positive status of androgen receptor (AR) was significantly associated with high rates of progression-free survival (PFS), but not with endometrial cancer-specific survival (ECSS) in endometrial carcinoma patients; 2) The potent androgen DHT was synthesized from testosterone by 5α-reductase in endometrial carcinoma tissues; and 3) endometrial carcinoma patients that were AR/5α-reductase type 1 double-negative had significantly worse PFS and ECSS. These findings suggest that androgen signaling exerts anti-cancer effects through the intratumoral DHT-AR pathway in endometrial carcinomas. In this highlight article, we describe androgen signaling in endometrial carcinomas, focusing mainly on our recent study entitled “The role of 5α-reductase type 1 associated with intratumoral dihydrotestosterone concentrations in human endometrial carcinoma” and discuss the findings of some previous related studies.

Intracrine androgens enhance decidualisation and modulate expression of human endometrial receptivity genes

Intracrine androgens enhance decidualisation and modulate expression of human endometrial receptivity genes

Intracrine Androgens and AKR1C3 Activation Confer Resistance to Enzalutamide in Prostate Cancer.

The introduction of enzalutamide and abiraterone has led to improvement in the treatment of metastatic castration-resistant prostate cancer. However, acquired resistance to enzalutamide and abiraterone therapies frequently develops within a short period in many patients. In the present study, we developed enzalutamide-resistant prostate cancer cells in an effort to understand the mechanisms of resistance. Global gene-expression analysis showed that the steroid biosynthesis pathway is activated in enzalutamide-resistant prostate cancer cells. One of the crucial steroidogenic enzymes, AKR1C3, was significantly elevated in enzalutamide-resistant cells. In addition, AKR1C3 is highly expressed in metastatic and recurrent prostate cancer and in enzalutamide-resistant prostate xenograft tumors. LC/MS analysis of the steroid metabolites revealed that androgen precursors such as cholesterol, DHEA and progesterone, as well as androgens are highly upregulated in enzalutamide-resistant prostate cancer cells compared to the parental cells. Knockdown of AKR1C3 expression by shRNA or inhibition of AKR1C3 enzymatic activity by indomethacin resensitized enzalutamide-resistant prostate cancer cells to enzalutamide treatment both in vitro and in vivo. In contrast, overexpression of AKR1C3 confers resistance to enzalutamide. Furthermore, the combination of indomethacin and enzalutamide resulted in significant inhibition of enzalutamide-resistant tumor growth. These results suggest that AKR1C3 activation is a critical resistance mechanism associated with enzalutamide resistance; targeting intracrine androgens and AKR1C3 will overcome enzalutamide resistance and improve survival of advanced prostate cancer patients.

MP46-02 UP-REGULATION OF GLUCOSE METABOLISM BY NF-κB2/P52 MEDIATES ENZALUTAMIDE RESISTANCE IN PROSTATE CANCER CELLS

highly expressed in metastatic and recurrent prostate cancer and in enzalutamide resistant prostate xenograft tumors. Liquid Chromatography-Mass Spectrometry (LC-MS) analysis of the steroid metabolites revealed that androgen precursors such as cholesterol, DHEA and progesterone, as well as androgens are highly up regulated in enzalutamide resistant prostate cancer cells compared to the parental cells. Knock down of AKR1C3 expression by shRNA or inhibition of AKR1C3 enzymatic activity by indomethacin resensitized enzalutamide resistant prostate cancer cells to enzalutamide treatment both in vitro and in vivo. In contrast, overexpression of AKR1C3 confers resistance to enzalutamide. Furthermore, the combination of indomethacin and enzalutamide resulted in significant inhibition of enzalutamide-resistant tumor growth. These results suggest that AKR1C3 activation is a critical resistance mechanism associated with enzalutamide resistance. CONCLUSIONS: AKR1C3 activation and resulting in an increase in intracrine androgens are critical resistance mechanisms confer resistance to enzalutamide. Targeting AKR1C3 activation may provide a potential treatment strategy for metastatic prostate cancer patients who develop resistance to enzalutamide.

Abstract IA11: The role of bone microenvironment in the lethal progression of prostate cancer

Abstract Lethal prostate cancer (PCa) is clinically characterized by bone-homing and bone- forming metastases, and bone is frequently the initial organ involved in castrate-resistant progression. These clinical observations led investigators to develop bone-tropic radiopharmaceuticals to treat men with advanced PCa. The ability of the bone-homing alpha emitter (RAD-223) to prolong progression free and overall survival establishes the therapeutic relevance of targeting bone 1. The long accepted paradigm of PCa progression has been dominated by the model of evolution from androgen dependence to androgen independence under the selective pressure of castration. Thus, Src family kinases (SFKs) were investigated experimentally and clinically for the treatment of putative “androgen independent” PCa, as SFKs play a role in both tumor growth and bone turnover. The roles of SFKs in migration, invasion, survival of tumor cells, and osteoclast and osteoblast function in tumor microenvironment were supported by numerous clinical and laboratory observations 2, 3. Despite promising phase I /II reports of dasatinib, a multikinase and potent SFK inhibitor, in men with advanced PCa 4, 5, a phase III trial failed to yield the expected survival advantage 6. The result of this trial highlights the difficulties in translating data from cell line models to biologically heterogeneous clinical PCa and in transitioning from phase I/II to phase III clinical trials. The efficacy of dasatinib was not observed likely due to persistent AR signaling, which remains the primary driver of progression in bone by the transition from paracrine to intracrine androgen signaling 7. Based on these considerations, we are testing the efficacy of an androgen biosynthesis inhibitor in combination with dasatinib to better understand the relevance of mechanism(s). A co–clinical investigational strategy is being applied to better define biomarkers to predict clinical efficacy. These studies also highlight the importance of preclinical work that utilizes models that better reflect the heterogeneity of PCa. This problem is partly overcome by patient- derived xenografts (PDX) 8, 9 10. These models reflect many aspects of PCa in bone and can be used to identify therapy targets and better identify both mechanisms of drug efficacy and development of resistance. Studies in PDX have led to identification of FGF signaling as a therapy target 11. Additional studies in PDX implicated the tumor microenvironment in the striking bone responses observed with cabozantinib 13. These observations led us to propose a model of prostate cancer progression founded on the hypothesis that cancer progresses from being an endocrine-driven cancer (Dihydrotestosterone-dependent) to paracrine/intracrine driven (microenvironment-dependent), and then finally to a cell autochthonous phase 12. Continued studies should lead to decisions on therapy treatment that are based on an understanding of markers that predict disease progression. This new paradigm will also result in more effective therapy combinations.

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

Complexities of androgen receptor signalling in breast cancer.

While the clinical benefit of androgen-based therapeutics in breast cancer has been known since the 1940s, we have only recently begun to fully understand the mechanisms of androgen action in breast cancer. Androgen signalling pathways can have either beneficial or deleterious effects in breast cancer depending on the breast cancer subtype and intracellular context. This review discusses our current knowledge of androgen signalling in breast cancer, including the relationship between serum androgens and breast cancer risk, the prognostic significance of androgen receptor (AR) expression in different breast cancer subtypes and the downstream molecular pathways mediating androgen action in breast cancer cells. Intracrine androgen metabolism has also been discussed and proposed as a potential mechanism that may explain some of the reported differences regarding dichotomous androgen actions in breast cancers. A better understanding of AR signalling in this disease is critical given the current resurgence in interest in utilising contemporary AR-directed therapies for breast cancer and the need for biomarkers that will accurately predict clinical response.

Androgen Receptor Activation in Castration-Recurrent Prostate Cancer: The Role of Src-Family and Ack1 Tyrosine Kinases

There is growing appreciation that castration-recurrent prostate cancer (CR-CaP) is driven by the continued expression of androgen receptor (AR). AR activation in CR-CaP through various mechanisms, including AR overexpression, expression of AR splice variants or mutants, increased expression of co-regulator proteins, and by post-translational modification, allows for the induction of AR-regulated genes in response to very low levels of tissue-expressed, so-called intracrine androgens, resulting in pathways that mediate CaP proliferation, anti-apoptosis and oncogenic aggressiveness. The current review focuses on the role played by Src-family (SFK) and Ack1 non-receptor tyrosine kinases in activating AR through direct phosphorylation, respectively, on tyrosines 534 or 267, and how these modifications facilitate progression to CR-CaP. The fact that SFK and Ack1 are central mediators for multiple growth factor receptor signaling pathways that become activated in CR-CaP, especially in the context of metastatic growth in the bone, has contributed to recent therapeutic trials using SFK/Ack1 inhibitors in monotherapy or in combination with antagonists of the AR activation axis.

Vitamin D receptor agonist reduces intracrine androgens

Vitamin D receptor agonist reduces intracrine androgens

Steroid hormone synthetic pathways in prostate cancer.

While androgen deprivation therapy (ADT) remains the primary treatment for metastatic prostate cancer (PCa) since the seminal recognition of the disease as androgen-dependent by Huggins and Hodges in 1941, therapy is uniformly marked by progression to castration-resistant prostate cancer (CRPC) over a period of about 18 months, with an ensuing median survival of 1 to 2 years. Importantly, castration does not eliminate androgens from the prostate tumor microenvironment. Castration resistant tumors are characterized by elevated tumor androgens that are well within the range capable of activating the AR and AR-mediated gene expression, and by steroid enzyme alterations which may potentiate de novo androgen synthesis or utilization of circulating adrenal androgens. The dependence of CRPC on intratumoral androgen metabolism has been modeled in vitro and in vivo, and residual intratumoral androgens are implicated in nearly every mechanism by which AR-mediated signaling promotes castration-resistant disease.These observations suggest that tissue based alterations in steroid metabolism contribute to the development of CRPC and underscore these metabolic pathways as critical targets of therapy. Herein, we review the accumulated body of evidence which strongly supports intracrine (tumoral) androgen synthesis as an important mechanism underlying PCa progression. We first discuss the presence and significance of residual prostate tumor androgens in the progression of CRPC. We review the classical and non-classical pathways of androgen metabolism, and how dysregulated expression of these enzymes is likely to potentiate tumor androgen production in the progression to CRPC. Next we review the in vitro and in vivo data in human tumors, xenografts, and cell line models which demonstrate the capacity of prostate tumors to utilize cholesterol and adrenal androgens in the production of testosterone (T) and dihydrotestosterone (DHT), and briefly review the potential role of exogenous influences on this process. Finally, we discuss the emerging data regarding mechanisms of response and resistance to potent ligand synthesis inhibitors entering clinical practice, and conclude by discussing the implications of these findings for future therapy.

Abstract 3279: Enhanced in vivo inhibition of neuropeptide-mediated castration resistant prostate cancer progression by combination therapy with enzalutamide and saracatinib.

Abstract Introduction: It has been shown that overexpression of the neuropeptides gastrin-releasing peptide (GRP) in LNCaP cells renders the cells castration-resistant through both androgen receptor (AR) activation and intracrine androgen biosynthesis. Aberrant activation of AR by GRP is mediated through Src kinase activation, which may be inhibited by the specific SFK inhibitor saracatinib. Inhibition of cognate AR activation requires anti-androgen. We hypothesize that treatment of this kind of CRPC demands the combination of saracatinib and new generation anti-androgen enzalutamide (MDV-3100). Materials and methods: LNCaP GRP-Pro cells grown in CS medium were treated with control, saracatinib (2 μM), enzalutamide (10 μM) or the combination. Cell proliferation over 7 days was monitored by MTT assays. Cell cycle distributions over 72 hours of treatment were analyzed by flow cytometry. Cells were collected to prepare nuclear and cytosolic lysates respectively and examined by Western Blotting analysis for AR, pSrc, pAkt, LC-3 and β-actin. Expression of full-length AR and variants were compared by RT-qPCR. Orthotopic injection of GRP-Pro cells into castrated SCID male mice was used as the animal model for drug treatment studies. Results: Enzalutamide or saracatinib alone only slightly inhibited CR cell growth but the combination brought it down to 27% of the control. Apoptosis was also enhanced with the combination. Enzalutamide only inhibited AR nuclear translocation in regular serum-kept GRP-Pro cells, while saracatinib inhibited the translocation in CS conditions. In the orthotopic model, neither treatments with saracatinib (25 mg/kg/daily) or enzalutamide (10 mg/kg/daily) alone showed any effect on tumor take, tumor weight or serum PSA levels. However, the combination treatment drastically reduced the tumor weights (0.66±0.13 g for controls vs. 0.24±0.07 g for the combo) and serum PSA levels (120.79±27.87 ng/ml vs. 19.19±7.45 ng/ml). Conclusions: For GRP mediated CRPC growth, neither saracatinib or enzalutamide alone is sufficient to inhibit AR translocation and tumor progression. Combination of the two significantly improved the treatment for the CRPC orthotopic tumor model. Citation Format: Joy C. Yang, Hao G. Ngyuen, Allen C. Gao, Christopher P. Evans. Enhanced in vivo inhibition of neuropeptide-mediated castration resistant prostate cancer progression by combination therapy with enzalutamide and saracatinib. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3279. doi:10.1158/1538-7445.AM2013-3279

The role of intracrine androgen metabolism, androgen receptor and apoptosis in the survival and recurrence of prostate cancer during androgen deprivation therapy.

Prostate cancer (CaP) is the most frequently diagnosed cancer and leading cause of cancer death in American men. Almost all men present with advanced CaP and some men who fail potentially curative therapy are treated with androgen deprivation therapy (ADT). ADT is not curative and CaP recurs as the lethal phenotype. The goal of this review is to apply our current understanding of CaP and castration-recurrent CaP (CR-CaP) to earlier studies that characterized ADT and the molecular mechanisms that facilitate the transition from androgen-stimulated CaP to CR-CaP. Reexamination of earlier studies also may provide a better understanding of how more newly recognized mechanisms, such as intracrine metabolism, may be involved with the early events that allow CaP survival after initiation of ADT and subsequent development of CR-CaP.

Effect of abiraterone acetate and prednisone compared with placebo and prednisone on pain control and skeletal-related events in patients with metastatic castration-resistant prostate cancer: exploratory analysis of data from the COU-AA-301 randomised trial

Bone metastases are a major cause of morbidity in metastatic castration-resistant prostate cancer. Abiraterone acetate potently disrupts intracrine androgen receptor signalling pathways implicated in the progression of the disease, including bone metastases. We assessed data for pain control and skeletal-related events prospectively collected as part of the randomised, phase 3 COU-AA-301 trial of abiraterone acetate plus prednisone versus placebo plus prednisone in patients with metastatic castration-resistant prostate cancer after docetaxel chemotherapy. The COU-AA-301 trial enrolled patients with metastatic castration-resistant prostate cancer in whom one or two lines of chemotherapy (one docetaxel based) had been unsuccessful and who had Eastern Cooperative Oncology Group performance statuses of 2 or less. Pain intensity and interference of pain with daily activities were assessed with the Brief Pain Inventory-Short Form questionnaire at baseline, day 15 of cycle 1, and day 1 of each treatment cycle thereafter until discontinuation. We assessed, with prospectively defined response criteria that incorporated analgesic use, clinically meaningful changes in pain intensity and interference with daily living. We measured time to first occurrence of skeletal-related events, which we defined as pathological fracture, spinal cord compression, palliative radiation to bone, or bone surgery, and regularly assessed them throughout the study. Pain palliation was assessed in patients who had clinically significant baseline pain, whereas all other analyses were done in the overall intention-to-treat population. COU-AA-301 is registered with ClinicalTrials.gov, number NCT00638690. Median follow-up was 20·2 months (IQR 18·4-22·1). In patients with clinically significant pain at baseline, abiraterone acetate and prednisone resulted in significantly more palliation (157 of 349 [45·0%] patients vs 47 of 163 [28·8%]; p=0·0005) and faster palliation (median time to palliation 5·6 months [95% CI 3·7-9·2] vs 13·7 months [5·4-not estimable]; p=0·0018) of pain intensity than did prednisone only. Palliation of pain interference (134 of 223 [60·1%] vs 38 of 100 [38·0%], p=0·0002; median time to palliation of pain interference 1·0 months [95% CI 0·9-1·9] vs 3·7 months [2·7-not estimable], p=0·0004) and median duration of palliation of pain intensity (4·2 months [95% CI 3·0-4·9] vs 2·1 months [1·4-3·7]; p=0·0056) were significantly better with abiraterone acetate and prednisone than with prednisone only. In the overall population, median time to occurrence of first skeletal-related event was significantly longer with abiraterone acetate and prednisone than with prednisone only (25·0 months [95% CI 25·0-not estimable] vs 20·3 months [16·9-not estimable]; p=0·0001). In patients with metastatic castration-resistant prostate cancer previously treated with docetaxel, abiraterone acetate and prednisone offer significant benefits compared with prednisone alone in terms of pain relief, delayed pain progression, and prevention of skeletal-related events. Janssen Research & Development and Janssen Global Services.