Androgen Synthesis Inhibitors Research Articles

Targeting Metastatic Hormone Sensitive Prostate Cancer: Chemohormonal Therapy and New Combinatorial Approaches.

Androgen deprivation therapy alone has been the standard of care for metastatic hormone sensitive prostate cancer for the last 75 years. This review focuses on recent trials and mechanisms which highlight the new paradigm of combining androgen deprivation therapy with other agents, changing the treatment of patients with prostate cancer who have advanced disease. We searched the peer reviewed literature on the PubMed® and Web of Science® databases through January 2018 using the key words, "metastatic hormone sensitive prostate cancer," "metastatic castration sensitive prostate cancer," "docetaxel," "abiraterone" and "senescence in cancer." ClinicalTrials.gov was queried for ongoing studies. Relevant data recently presented at major urology and medical oncology meetings were also evaluated. Recently published, phase III trials using androgen deprivation therapy combinations for metastatic hormone sensitive prostate cancer can be broadly grouped into chemohormonal studies (docetaxel) or trials of androgen signaling inhibitors. The CHAARTED (Chemohormonal Therapy versus Androgen Ablation Randomized Trial for Extensive Disease in Prostate Cancer) and STAMPEDE (Systemic Therapy in Advancing or Metastatic Prostate Cancer: Evaluation of Drug Efficacy) studies showed a survival advantage when combining androgen deprivation therapy with chemotherapy, as well as increased time to progression to castration resistant status. The abiraterone arm of the STAMPEDE and LATITUDE trials, which analyzed combining androgen deprivation therapy with abiraterone, revealed improved overall and progression-free survival. Androgen deprivation therapy generates a number of phenotypes in resistant cancer cells, including quiescence, autophagy and cellular senescence. Senescent cells represent a metabolic target for synergistic lethality with drugs such as metformin. Ongoing trials are under way to examine the effect of combining newer antiandrogens and novel drugs with androgen deprivation therapy in patients with metastatic hormone sensitive prostate cancer. Combination therapy has evolved as the standard of care for metastatic hormone sensitive prostate cancer. The ideal combination is tailored to patients after individualized counseling taking into account general health and comorbid illness status.

Targeting the turnover of oncoproteins as a new avenue for therapeutics development in castration-resistant prostate cancer

The current therapeutic armamentarium for castration-resistant prostate cancer (CRPC) includes second-generation agents such as the Androgen Receptor (AR) inhibitor enzalutamide and the androgen synthesis inhibitor abiraterone acetate, immunotherapies like sipuleucel-T, chemotherapies including docetaxel and cabazitaxel and the radiopharmaceutical radium 223 dichloride. However, relapse of CRPC resistant to these therapeutic modalities occur rapidly. The mechanisms of resistance to these treatments are complex, including specific mutations or alternative splicing of oncogenic proteins. An alternative approach to treating CRPC may be to target the turnover of these molecular drivers of CRPC. In this review, the mechanisms by which protein stability of several oncoproteins such as AR, ERG, GR, CYP17A1 and MYC, will be discussed, as well as how these findings could be translated into novel therapeutic agents.

Abstract 858: Enzalutamide versus abiraterone as a radiosensitizer in hormone-sensitive prostate cancer cells

Abstract Prostate cancer treatment is based on the estimated risk of recurrence in the U.S. Combined androgen deprivation therapy (ADT) with radiation therapy (XRT) is the standard of care for high-risk localized PCa. However, a large percentage of tumors are resistance to ADT due to continued AR signaling. Abiraterone (ABI), an androgen synthesis inhibitor, and Enzalutamide (ENZA), a potent AR antagonist, are new treatment options for metastatic castration resistance prostate cancer (mCRPC) patients. The aim of this study is to compare the efficacy of ENZA or ABI as a radiosensitizer in XRT therapy on PCa cells. Methods: The effect of ENZA or ABI alone or in combination with XRT was assessed on hormone-sensitive (LNCaP, PC3-AR-T877A) and insensitive (PC3, PC3-AR V7) PCa cells using cell viability (MTT) and also clonogenic assays in different scheduling regimens: A- drug 24 h before XRT, B-drug 2h before XRT, or C- XRT followed by 24h later drug. Results: We first determined the effect of ENZA or ABI on MTT assays in androgen-dependent (AD) and androgen-independent (AI) PCa cell lines. The results of MTT assay showed that ENZA inhibited the growth of the four different cell lines, LNCaP, PC3-T877A, PC3 and PC3 AR-V7 with IC50 values of 20, 22, 50, and 45μmol/L, respectively, after 24 hours of treatment. The same effect was observed on ABI treated cell lines. Radiosensitivity was not significantly increased in AD and AI PCa cell lines by ABI (DEF=1.00, in all cases) while there was a supra-additive dose enhancement factor (DEF= 1.75±0.08) for hormone-sensitive cells treated with ENZA (Table 1). Conclusion: Our data indicates that ENZA acts as a much stronger radiosensitizer compared to ABI through different probable mechanisms of radiosensitivity. Table 1. Dose Enhancement Factors calculations by scheduling ProtocolABCENZAABIENZAABIENZAABIADLNCaP1.35±0.021.05±0.011.75±0.081.001.30±0.051.00PC3-AR T877A1.30±0.031.001.65±0.011.001.35±0.061.05±0.02AIPC31.001.001.001.001.001.00PC3-AR V71.001.001.001.001.001.00 Citation Format: Maryam Ghashghaei, Thierry Muanza, Moulay Alaoui-Jamali, Miltiadis Paliouras, Mohammad Tamim Niazi. Enzalutamide versus abiraterone as a radiosensitizer in hormone-sensitive prostate cancer cells [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 858.

Abstract 1798: A detailed comparison between second-generation AR antagonists reveals differences in the overall impact on gene regulation patterns in prostate cancer cells

Abstract Androgen receptor (AR) signaling is essential at all prostate cancer stages and is clinically addressed by surgical or chemical castration, AR antagonists and androgen synthesis inhibitors. Second-generation AR antagonists include the approved drug enzalutamide and the late-stage clinical investigational oral compounds apalutamide and darolutamide. Comparing the gene expression patterns and the overarching epigenetic modifications elicited by AR antagonists will be essential for a detailed understanding of the molecular mode of action of these compounds. Methods: The transcriptome and AR binding patterns were analyzed by RNA-seq and qPCR, respectively. Genome-wide changes of the active transcription histone modification H3K27 acetylation were determined by ChIP-seq. Results: RNA-seq was performed in the androgen-dependent VCaP and LNCaP cell lines for a detailed analysis of the genome-wide transcriptional effects of the AR antagonists darolutamide, enzalutamide and apalutamide. Gene set enrichment analysis indicated that many genes involved in androgen response were up- or down-regulated by androgen treatment with R1881. Principal component analysis showed that treatment with pharmacologically relevant doses of AR antagonists had similar effects at the 8-hour time point, however some differences were visible at the 22-hour time point. A strong expression down-regulation of a number of genes from the Wnt and epithelial-mesenchymal transition (EMT) pathways was observed for darolutamide as compared to enzalutamide and apalutamide treatment in the VCaP cell line. In LNCaP cells the major differences between the AR antagonists were observed in the androgen-regulated genes, which is consistent with varying degrees of inhibition by the drugs in this cell line. Next we determined the global H3K27 acetylation profiles which characterize open chromatin and active transcription. ChIP-seq analysis performed in LNCaP cells revealed a strong impact of androgen treatment on the H3K27 acetylation profiles of numerous genes involved in hormonal regulation. Darolutamide significantly reversed genome-wide H3K27 acetylation patterns, almost to non-androgen stimulated levels. More specifically, a reduction of H3K27 peaks was observed at KLK3 and FKBP5 regulatory elements. In addition, we determined the impact of AR antagonists on AR binding at the KLK2 and KLK3 regulatory elements. Darolutamide and enzalutamide strongly reduced androgen-stimulated AR binding whereas the impact of apalutamide was slightly lower. Conclusions: AR antagonists strongly reversed the effects of the androgen R1881, both at the level of gene expression, and of H3K27 acetylation and AR binding. A detailed comparison between second-generation AR antagonists showed interesting differences in the regulation of genes in the Wnt and EMT pathways. Citation Format: Simon J. Baumgart, Ekaterina Nevedomskaya, Ralf Lesche, Henrik Seidel, Bernard Haendler. A detailed comparison between second-generation AR antagonists reveals differences in the overall impact on gene regulation patterns in prostate cancer cells [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 1798.

HSD3B1(1245A>C) variant regulates dueling abiraterone metabolite effects in prostate cancer.

A common germline variant in HSD3B1(1245A>C) encodes for a hyperactive 3β-hydroxysteroid dehydrogenase 1 (3βHSD1) missense that increases metabolic flux from extragonadal precursor steroids to DHT synthesis in prostate cancer. Enabling of extragonadal DHT synthesis by HSD3B1(1245C) predicts for more rapid clinical resistance to castration and sensitivity to extragonadal androgen synthesis inhibition. HSD3B1(1245C) thus appears to define a subgroup of patients who benefit from blocking extragonadal androgens. However, abiraterone, which is administered to block extragonadal androgens, is a steroidal drug that is metabolized by 3βHSD1 to multiple steroidal metabolites, including 3-keto-5α-abiraterone, which stimulates the androgen receptor. Our objective was to determine if HSD3B1(1245C) inheritance is associated with increased 3-keto-5α-abiraterone synthesis in patients. First, we characterized the pharmacokinetics of 7 steroidal abiraterone metabolites in 15 healthy volunteers. Second, we determined the association between serum 3-keto-5α-abiraterone levels and HSD3B1 genotype in 30 patients treated with abiraterone acetate (AA) after correcting for the determined pharmacokinetics. Patients who inherit 0, 1, and 2 copies of HSD3B1(1245C) have a stepwise increase in normalized 3-keto-5α-abiraterone (0.04 ng/ml, 2.60 ng/ml, and 2.70 ng/ml, respectively; P = 0.002). Increased generation of 3-keto-5α-abiraterone in patients with HSD3B1(1245C) might partially negate abiraterone benefits in these patients who are otherwise more likely to benefit from CYP17A1 inhibition. Prostate Cancer Foundation Challenge Award, National Cancer Institute.

Visceral metastases on abiraterone vs. placebo: A post-hoc analysis of mode of radiographic progression in COU-AA-302.

194 Background: Abiraterone prolongs survival in patients with prostate cancer due to its potent inhibition of androgen synthesis. We previously observed increased rates of visceral metastatic disease at the time of progression on abiraterone compared to baseline, a poor prognostic feature associated with non-AR dependent prostate cancer. We hypothesized that the rate of development of visceral disease was increased with abiraterone compared to other treatments without potent androgen signaling inhibition. In order to test this, we examined time to visceral disease among patients treated with abiraterone vs placebo on the COU-AA-302 trial. Methods: We performed a post-hoc analysis of radiographic progression data for the phase 3 study of abiraterone vs placebo, via a data sharing agreement through the Yale Open Data Access Project. Data were censored at end of study treatment. The distribution of cumulative incidence for visceral metastases was calculated by the Kaplan-Meier method and compared with log-rank testing. Multivariable cox regression analysis was performed to assess for independent association of study treatment (abiraterone vs placebo) with the development of visceral metastases. Results: Median follow-up was 12.5mo, and 84 of 1088 patients developed visceral metastases. Univariate log-rank testing showed no difference in time to visceral metastasis between groups (p = 0.97). 1- and 3-yr cumulative incidence of visceral metastases were estimated at 6.4% and 15.6% for abiraterone and 4.6% and 17.0% for placebo, respectively. Study treatment was not a significant predictor for the development of visceral metastasis (HR 0.76; 95% CI 0.47-1.21) after adjustment for baseline metastatic disease burden (soft tissue and bone) and LDH. Conclusions: Abiraterone was not associated with increased visceral metastases at progression compared to placebo. These data give more confidence to oncologists that abiraterone is not driving prostate cancer to an aggressive phenotype characterized by visceral metastases - a finding that is particularly important as abiraterone is increasingly used in early disease states.

Safety and efficacy of abiraterone acetate (AA) in patients aged 75 or more with metastatic castration-resistant prostate cancer (mCRPC) in both pre-chemotherapy or post-chemotherapy settings: Real-life experience from thirteen Italian centers.

209 Background: Prostate cancer affects mainly elderly patients (pts) that have different comorbidities. AA is a selective androgen synthesis inhibitor that showed the efficacy in either chemotherapy (CT) naive pts or those pretreated with docetaxel. Its oral administration and good tolerability make it a manegeable treatment for elderly mCRPC pts. Methods: We collected retrospectively data regarding mCRPC pts aged ≥75 years treated with AA in 13 Italian Centers since April 2013. The median age was 79 years (r. 75-90) with 48% of pts being octagerians. Post CT pts had more extensive disease, higher baseline PSA and ECOG PS. Nearly all the pts had comorbidities, the most frequent being hypertension present in 146 pts (58%), 43 pts (17%) had diabetes type II. We evaluated duration of the AA treatment, overall response rate (ORR), 50% PSA decline, time to progression (TTP) and overall survival (OS). We reported all toxicities observed. Results: A total of 252 pts ,147 pre treated with docetaxel and 105 chemo naive, were included. Median duration of treatment with AA was 8,6 months in post CT and 11,5 in CT naive pts. ORR was 35,3% in pre docetaxel and 27,4% in post docetaxel group. 64 pts (65%) and 51 pts (46%) obtained 50% PSA reduction in pre and post docetaxel group, respectively. Median TTP was 8,6 in post docetaxel and 11,9 in CT naive pts. We observed a median OS of 13,8 months in post CT group while for CT naive pts data were not yet mature. AA was well tolerated with only 8 pts (3,2%) who discontinued treatment due to toxicity, while in 4 pts (1,6%) temporary dose reductions were performed. The most frequent G3 toxicities were hypertension and liver toxicity with 4 pts (1,6%) and 5 pts (2%), respectively. After progressing on AA, 85 pts (34%) received at least one subsequent treatment. 40 pts (15,9%) are still on treatment with AA. Conclusions: Even if almost all the pts reported comorbidities at AA start and 72 pts (28,6%) had PS ECOG 2, only a small proportion of them discontinued the treatment due to toxicity confirming that AA is well tolerated and efficient treatment also for elderly patients.

Androgen receptor antagonism and impact on inhibitors of androgen synthesis in prostate cancer therapy.

In a recent article by Norris and colleagues, published in high-impact J Clin Invest (1), the authors investigated the potential role of androgen receptor (AR) antagonism and the efficacy of cytochrome P450 17A1 (hereafter called CYP17) inhibitors in prostate cancer models.

New horizons in the management of castrate-resistant prostate cancer

Castrate-resistant prostate cancer is an incurable disease. It continues to be an area of intense research to save prostate cancer patients’ lives. We aim from this review to explore the new lines of treatment that aim at extending disease-specific survival. We conducted a PubMed research on available literature pertaining to key words alone and in combination: prostate cancer, castrate-resistant, immunotherapy, hormonal therapy, chemotherapy, radiation therapy, metastasis, survival and quality of life. We also reviewed the following guidelines on castrate-resistant prostate cancer: National Comprehensive Cancer Network (NCCN), American Urological Association (AUA), the National Institute for Health and Care Excellence (NICE) and the European Association of Urology (EAU). There are four new lines of treatment in castrate-resistant prostate cancer. An immunotherapy, sipuleucel T (Provenge); new hormonal therapies – in the form of a new androgen receptor blocker, enzalutamide (Xtandi) and new adrenal androgen synthesis inhibitor, abiraterone acetate (Zytiga); a new chemotherapy, cabazitaxel (Jevtana) and new radioactive agent, radium 223 (Xofigo). There is an agreement between guidelines that patients with M0 castrate-resistant prostate cancer should not be offered any of these treatments. Similarly, patients with castrate-resistant prostate cancer and poor performance status should be offered only palliative care. There are several new lines of treatment that can be used in castrate-resistant prostate cancer, and hopefully in the near future castrate-resistant prostate cancer will be changing from a lethal disease to more of a chronic disease.

Resveratrol, piceatannol and analogs inhibit activation of both wild-type and T877A mutant androgen receptor

Prostate cancer growth and progression are mainly dependent on androgens and many current prostate cancer treatment options target the synthesis or function of androgens. We have previously reported that resveratrol and synthetic analogs of resveratrol with a higher bioavailability inhibit the synthesis of androgens in human adrenocortical H295R cells. Now we have studied the antiandrogenic properties of resveratrol, piceatannol and analogs in two different prostate cell lines; LNCaP and RWPE. LNCaP carry a T877A mutation in the androgen receptor while RWPE has a wild-type androgen receptor. We found that resveratrol, piceatannol and all studied analogs were able to inhibit a dihydrotestosterone-induced activation of the androgen receptor, showing that they act as antiandrogens. In LNCaP cells, all studied compounds were able to statistically significantly decrease the androgenic signaling in concentrations ≥1μM and the synthetic analogs trimethylresveratrol (RSVTM) and tetramethylpiceatannol (PICTM) were the most potent compounds. RWPE cells were not as responsive to the studied compounds as the LNCaP cells. A statistically significant decrease in the androgenic signaling was observed at concentrations ≤5μM for most compounds and RSVTM was found to be the most potent compound. Further, we studied the effects of resveratrol, piceatannol and analogs on the levels of prostate-specific antigen (PSA) in LNCaP cells and found that all studied compounds decreased the level of PSA and that the synthetic analogs diacetylresveratrol (RSVDA), triacetylresveratrol (RSVTA) and RSVTM were the most potent compounds, decreasing the PSA level by approx. 50% at concentrations ≥10μM. In a cell-free receptor binding assay we were unable to show binding of resveratrol or analogs to the ligand binding domain of the androgen receptor, indicating that the observed effects are mediated via other mechanisms than direct ligand competition. We conclude that the resveratrol, piceatannol and analogs are highly interesting for chemoprevention of prostate cancer, since they have a high potency both as inhibitors of androgen synthesis and androgen receptor activation.

Differential proteome expression analysis of androgen-dependent and -independent pathways in LNCaP prostate cancer cells

Prostate cancer (PC) is one of the leading causes of cancer death in men. It commonly develops in older males, but the number of younger men diagnosed with the disease has increased in recent years. Hormone therapies, such as chemical and surgical methods that inhibit androgen synthesis or androgen receptor (AR) activation, have been used for advanced disease. However, castration-resistant PC (CRPC), which exhibits androgen-independent mechanisms for activating AR, develops after a few years of such treatment and no therapy is available. In this study, we examined differences in the proteomes associated with the androgen-dependent (DHT treatment) and -independent (FSK, forskolin treatment) AR signaling conditions in LNCaP prostate cancer cells. Moreover, we used EPI-001, which inhibits AR-mediated transcriptional activity, to examine whether the observed differences in protein expression were directly affected by AR-mediated mechanisms. A total of 213 protein spots were matched in our 2-dimensional gel electrophoresis (2DE) analysis and 8 proteins with significant expression changes in our 5 different treatment groups were identified by mass spectrometry. Among these proteins, expression levels of PEPCK-M, catalase, tubulin alpha chain, alpha-enolase, and endoplasmic reticulum resident protein 29 were significantly altered by DHT and the levels of HSP 90 and EF-Tu were changed by FSK. These changes were blocked by EPI-001 in DHT-regulated proteins, PEPCK-M, catalase, and tubulin alpha chain and FSK-regulated EF-Tu protein. The results from our immunohistochemical analysis using in vivo xenograft models were consistent with the 2DE data. We therefore report the first identification of differences in proteins that are significantly regulated under androgen-dependent and -independent AR signaling conditions. Our findings could suggest a possible molecular mechanism through which AR is activated in the absence and/or presence of androgen, and might help explain the inhibitory action of EPI-001 on CRPC.

Abstract 1579: Bicalutamide-induced expression of T-type Ca2+ channels in prostate cancer cells undergoing neuroendocrine differentiation in vitro

Abstract The growth of prostate cancer cells depends on the activation of androgen receptors. Anti-androgen therapies that inhibit androgen synthesis or receptor activation are effective in limiting tumor growth. However, prolonged treatment with anti-androgen therapies result in the transition of prostate cancers into an androgen refractory state. Neuroendocrine differentiation (NED) has been associated with the progression of prostate cancers to an androgen resistant phenotype. In this work we investigated the effect of the androgen receptor blocker bicalutamide in promoting NED of LNCaP cells and whether it is accompanied by increased T-type Ca2+ channel expression. The ability of bicalutamide to evoke morphological and biochemical changes associated with NED was assessed by PCR, western blot and immuno-histochemical analysis. Changes in the Cav3.2 T-type Ca2+ channel subunit expression was studied using PCR analysis, western blot and whole cell recordings. The role of T-type Ca2+ channels in promoting the differentiation of LNCaP cells was assessed by a cell viability assay and changes in cell morphology, specifically the development of neurite-like processes. PCR analysis of bicalutamide-stimulated cells indicates no significant changes in Cav3.2 mRNA, yet results in Cav3.2 protein expression and functional channels. Western blot and immuno-histochemical analysis of LNCaP cells simulated with bicalutamide for 4-10 day reveals biochemical changes consistent with NED including the expression of tubulin IIIbeta and neuron-specific enolase. Pharmacological inhibition of T-type Ca2+ channel function with nickel ions and NNC 33-0936 disrupts the morphological differentiation and cell viability of LNCaP cells treated with bicalutamide. These results suggest that bicalutamide treatment of LNCaP cells evokes significant morphological and biochemical changes associated with NED and results in increased expression of T-type Ca2+ channels, which can significantly alter Ca2+ homeostasis. Citation Format: Miguel Martin-Caraballo, Megan Hall, Bryan Todd, Yoon Jung Kwon, Vu Nguyen, Jennifer L. Hearne. Bicalutamide-induced expression of T-type Ca2+ channels in prostate cancer cells undergoing neuroendocrine differentiation in vitro [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 1579. doi:10.1158/1538-7445.AM2017-1579

Abstract 1114: Combining anti-androgen therapy and PARP inhibition results in synergistic cytotoxicity of metastatic castration-resistant prostate cancer (mCRPC) cells

Abstract Androgen deprivation therapy is an efficient first line therapy for advanced prostate cancer. However, in most cases resistance inevitably develops resulting in metastatic castration-resistant prostate cancer (mCRPC). Abiraterone and enzalutamide are approved to treat mCRPC and act by inhibiting androgen biosynthesis and androgen receptor (AR) signaling, respectively. Poly (ADP-ribose) polymerase 1 (PARP1) is a key enzyme in the DNA damage repair pathway and has also been implicated in AR signaling cascade. Recent reports suggest significant efficacy combining anti-androgens and inhibitors of Poly (ADP-ribose) polymerase (PARP) particularly against cells harboring mutations in the DNA repair pathway. Here, we provide evidence of synergistic cytotoxicity combining PARP inhibition and androgen signaling ablation in the AR positive human CRPC cell line 22RV1 harboring mutation in the DNA repair gene BRCA2. The PARP inhibitor talazoparib (IC50 - 0.05 μM) was upto 20 times more potent than olaparib (IC50 – 1.0 μM) in inducing cell death in 22RV1 as seen by cell viability and clonogenic assays. When combined with AR antagonist enzalutamide (IC50 – 0.25 μM) or androgen synthesis inhibitor abiraterone (IC50 – 1.25 μM) both PARP inhibitors showed significant synergy, although talazoparib was more prominent owing to its superior potency. Higher concentrations of enzalutamide and abiraterone (>10μM) induced PARP cleavage and increased expression of γ-H2AX, a marker for DNA damage. Our results suggest that AR signaling inhibition involves PARP mediated apoptosis and highlight the crosstalk between PARP and AR pathways. In summary, combining anti-androgen therapy and PARP inhibitors carry significant therapeutic potential for CRPC patients harboring mutations in DNA repair pathway. Citation Format: Sahithi Pamarthy, Vinay Sagar, Rajita J. Vatapalli, Zachary Reinstein, Muhammad Zayd Ansari, Benedito A. Carneiro Filho, Francis J. Giles, Sarki A. Abdulkadir. Combining anti-androgen therapy and PARP inhibition results in synergistic cytotoxicity of metastatic castration-resistant prostate cancer (mCRPC) cells [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 1114. doi:10.1158/1538-7445.AM2017-1114

An androgen response element driven reporter assay for the detection of androgen receptor activity in prostate cells.

The androgen receptor (AR) transcription factor plays a key role in the development and progression of prostate cancer, as is evident from the efficacy of androgen-deprivation therapy, AR is also the most frequently mutated gene, in castration resistant prostate cancer (CRPC). AR has therefore become an even more attractive therapeutic target in aggressive and disseminated prostate cancer. To investigate mechanisms of AR and AR target gene activation in different subpopulations of prostate cancer cells, a toolkit of AR expressor and androgen response element (ARE) reporter vectors were developed. Three ARE reporter vectors were constructed with different ARE consensus sequences in promoters linked to either fluorescence or luciferase reporter genes in lentiviral vector backbones. Cell lines transduced with the different vectors expressed the reporters in an androgen-dependent way according to fluorescence microscopy, flow cytometry and multi-well fluorescent and luminescence assays. Interestingly, the background reporter activity in androgen-depleted medium was significantly higher in LNCaP cells compared to the prostate transit amplifying epithelial cell lines, EP156T-AR and 957E/hTERT-AR with exogenous AR. The androgen-induced signal to background was much higher in the latter benign prostate cells than in LNCaP cells. Androgen-independent nuclear localization of AR was seen in LNCaP cells and reduced ARE-signaling was seen following treatment with abiraterone, an androgen synthesis inhibitor. The ARE reporter activity was significantly stronger when stimulated by androgens than by β-estradiol, progesterone and dexamethasone in all tested cell types. Finally, no androgen-induced ARE reporter activity was observed in tumorigenic mesenchymal progeny cells of EP156T cells following epithelial to mesenchymal transition. This underscores the observation that expression of the classical luminal differentiation transcriptome is restricted in mesenchymal type cells with or without AR expression, and presence of androgen.

Serum androgens and survival in metastatic castration resistant prostate cancer (mCRPC) patients treated with docetaxel and prednisone: Results from CALGB 90401 (Alliance).

5067 Background: Higher baseline androgens have been previously shown to be associated with an improved overall survival (OS) in mCRPC patients treated with the androgen synthesis inhibitors, ketoconazole or abiraterone. The purpose of this analysis was to determine whether baseline serum androgen levels (Testosterone (T), Androstenedione (A) and DHEA (D) are associated with OS in mCRPC patients treated with docetaxel-based chemotherapy. Methods: Data from 1,050 men treated on CALGB 90401 with docetaxel, prednisone and either bevacizumab or placebo were used. Eligibility required progressive mCRPC and no prior chemotherapy. Pre-treatment serum assays for T, A and D were performed via tandem Liquid Chromatography-Mass Spectrometry (LC-MS/MS) at NMS labs. The proportional hazards model was used to assess the prognostic significance of T, A, and D in predicting OS adjusting for known prognostic factors. Results: Median values for T, A, and, D were 1.00, 13.00 and 8.12, ng/dL respectively.Values above the median were defined as low, above as high. Median OS for low vs high levels was 22.7 and 23.1 month for T, 22.4 and 21.7 month for A and 21.8 and 24.0 month for D, respectively, all NS. In multivariable analysis adjusting for 10 known prognostic values and prior keto use in mCRPC (Halabi JCO 2014), A (p-value = 0.013) levels were associated with OS. The HR for A was = 0.99 (95% CI = 0.98-0.99). Conclusions: In multivariate analysis, baseline androstenedione levels are prognostic factors for OS in mCRPC patients receiving chemotherapy. Low or undetectable levels of other androgens are associated with shorter OS, consistent with prior results in androgen synthesis inhibitor treated pts in both the chemotherapy naive and post chemotherapy settings. This relationship may reflect more aggressive tumor biology that evolves in an extreme androgen deprived milieu. Clinical trial information: NCT00110214.

A phase III trial comparing atezolizumab with enzalutamide vs enzalutamide alone in patients with metastatic castration-resistant prostate cancer (mCRPC).

TPS5090 Background: In the past decade, several therapies have been approved for patients (pts) with mCRPC, including the androgen receptor (AR) antagonist enzalutamide (enza) and the androgen synthesis inhibitor abiraterone acetate (abi). Despite these advances, most pts experience disease progression and there are inadequate data to guide the sequencing of agents to optimize outcomes. Pts with mCRPC who progress on enza have increased circulating PD-L1/PD-L2–positive dendritic cells compared with enza-naive pts or pts who are still responding to treatment (Bishop et al. Oncotarget. 2014). In two recent studies, PSA and radiographic responses were observed in mCRPC pts treated with a PD-L1/PD-1 pathway inhibitor with or without enza (Graff et al. Oncotarget. 2016; Hansen et al. Ann Oncol. 2016). Atezolizumab (atezo) is an anti–PD-L1 monoclonal antibody that inhibits the interaction between PD-L1 and its receptors, PD-1 and B7.1, enhancing T-cell responses and improving anti-tumor activity. Taken together, this suggests that the combination of atezo and enza may provide an effective treatment option for mCRPC pts. Methods: A Phase III randomized, multicenter, clinical trial (NCT03016312) is being conducted to evaluate the efficacy and safety of atezo with enza compared with enza alone in mCRPC pts who have received prior abi treatment and have progressed on, are ineligible for, or have refused a taxane regimen. Eligibility criteria include mCRPC or locally advanced, incurable CRPC and ECOG PS 0-1. Exclusion criteria include CNS metastasis, autoimmune disease, history of seizures, prior immunotherapy and prior treatment with enza or any other newer AR antagonists. Pts will be randomized 1:1 to receive atezo 1200 mg q3w and enza 160 mg qd or enza alone. The primary endpoint is OS, and secondary endpoints include PSA response rate, rPFS, ORR and safety. Exploratory biomarkers associated with responses to atezo and enza will be evaluated in tumor tissue collected at baseline and progression. Approximately 550 pts will be enrolled at 150 sites globally. Clinical trial information: NCT03016312.

A phase IV, randomized, double-blind, placebo (PBO)-controlled study of continued enzalutamide (ENZA) post prostate-specific antigen (PSA) progression in men with chemotherapy-naive metastatic castration-resistant prostate cancer (mCRPC).

5004 Background: We hypothesized resistance to the androgen receptor inhibitor ENZA is due to increases in androgens and can be overcome by combination with the androgen synthesis inhibitor abiraterone (abi). The phase 4 PLATO trial (NCT01995513) is evaluating the safety and efficacy of continued ENZA + abi/prednisone (abi/P) vs PBO + abi/P after PSA progression on ENZA. Methods: In Period (P) 1, men with chemotherapy-naïve mCRPC received ENZA (160 mg); men with no PSA increase from baseline at wk 13 and 21 continued treatment until PSA progression (≥ 25% increase and ≥ 2 ng/mL above nadir). Eligible men were then randomized 1:1 in P2 to ENZA + abi/P (1000 mg/10 mg) or PBO + abi/P. The primary endpoint (EP) was progression-free survival (PFS; radiographic or unequivocal clinical progression, or death on study) in P2, with a prespecified sensitivity analysis of radiographic PFS (rPFS); protected secondary EPs were time to PSA progression (TTPP) and PSA response ≥ 50% in P2. Results: 509 men enrolled in P1. At data cutoff (Oct 7, 2016), 84 were active, 174 discontinued, and 251 were randomized in P2 (ENZA + abi/P, n = 126; PBO + abi/P, n = 125). Median treatment duration in P2 was 5.6 mo for both arms. PFS event by radiographic/clinical/death was 38%/25%/2% for ENZA + abi/P and 55%/18%/1% for PBO + abi/P. Median PFS was 5.7 mo for ENZA + abi/P and 5.6 mo for PBO + abi/P (hazard ratio [HR], 0.83; 95% confidence interval [CI], 0.61, 1.12; P = 0.22). Median TTPP was 2.8 mo for both arms (HR, 0.87; 95% CI, 0.62, 1.24; P = 0.45). PSA response rate was 0.8% for ENZA + abi/P and 2.5% for PBO + abi/P ( P = 0.31). Median rPFS was 10.0 mo for ENZA + abi/P and 7.0 mo for PBO + abi/P (HR, 0.67; 95% CI, 0.47, 0.94; P = 0.02). The most common (≥ 15%) adverse events for ENZA + abi/P vs PBO + abi/P were back pain (21% vs 23%), hypertension (20% vs 7%), nausea (17% vs 9%), and fatigue (14% vs 15%). Conclusions: ENZA + abi/P post PSA progression on ENZA was associated with increased hypertension and nausea and did not result in a statistically significant improvement in composite PFS. The signal seen in rPFS needs further evaluation. Clinical trial information: NCT01995513.

Transcriptional profile of ethylene glycol monomethyl ether-induced testicular toxicity in rats

To clarify the molecular mechanism of ethylene glycol monomethyl ether (EGME)-induced testicular toxicity, the potential for EGME-related changes in transcript levels of genes including spermatocyte-specific genes was evaluated in the testis of rats given single dosing of EGME at 200, 600, or 2000 mg/kg. Furthermore, the contribution of decreased testicular testosterone on EGME-induced spermatocyte toxicity was investigated by comparing to transcriptional profile due to a testosterone synthesis inhibitor, ketoconazole (KET), at 30 or 300 mg/kg. EGME at 600 mg/kg or more dose-dependently caused testicular toxicity characterized by degeneration and necrosis of spermatocytes at stage VII–XIV seminiferous tubules. The spermatocyte injury was well correlated with decreased spermatocyte-specific gene expression. Analysis of upstream regulators by the Ingenuity Pathways Analysis system suggested that up-regulation of oxidative stress, protein kinase activation, and histone acetylation was involved in EGME-induced spermatocyte toxicity. Interestingly, KET decreased testicular testosterone to a similar extent compared to the EGME treatment, but KET at up to 300 mg/kg did not show any histopathological abnormality or change in the expression of spermatocyte-specific genes. These results suggested that the decreased testicular testosterone have little impact on EGME-induced spermatocyte injury. In contrast, KET showed trends toward increases in Hsd3b2 and Hsd17b2 mRNAs, presumably resulting from inhibition of androgen synthesis. Transcriptome analysis clearly demonstrated the differential effects of EGME and KET on androgen synthesis. In conclusion, EGME caused spermatocyte toxicity correlated with decreased expression of spermatocyte-specific genes. Furthermore, oxidative stress, protein kinase activation, and histone acetylation were suggested to be involved in EGME-induced testicular toxicity.

Chemotherapy With Androgen Deprivation for Hormone-Naïve Prostate Cancer

Research regarding the treatment of metastatic prostate cancer has been undergoing dramatic progress. Treatment of hormone-naïve metastatic prostate cancer includes surgical castration and medical castration that lowers androgen level in the blood using drugs. Although these androgen deprivation therapies are very effective, hormone-naïve metastatic prostate cancer finally leads to castration-resistant prostate cancer because resistance to surgical or medical castration occurs. The treatment at this stage includes not only docetaxel, but also new androgen synthesis inhibitor or androgen receptor inhibitors such as abiraterone or enzalutamide, new cytotoxic anticancer agents such as carbazitaxel, and radioisotope treatment such as radium-223. Recently, studies on the effect of chemotherapy on hormone-naïve metastatic prostate cancer before the development of castration-resistant prostate cancer have been actively published. As a result, various guidelines have recommended docetaxel as the first-line therapy for hormone-naïve metastatic prostate cancer. In this manuscript, we will summarize the basic concepts of androgen deprivation therapy for hormone-naïve metastatic prostate cancer and the main results of research on chemotherapy for hormone-naïve metastatic prostate cancer. Key Words: Prostate neoplasms; Drug therapy.

Androgen Receptor Splice Variants Are Not Substrates of Nonsense-Mediated Decay.

Androgen receptor (AR) splice variants have been clinically associated with progressive cancer, castration-resistance, and resistance to AR antagonists and androgen synthesis inhibitors. AR variants can be generated by genomic alterations and alternative splicing, and their expression is androgen-regulated. There has been a suggestion that AR variants bearing premature termination codons and coding for truncated proteins should be regulated by the nonsense-mediated decay (NMD) mRNA surveillance pathway, suggesting that either the NMD pathway is dysfunctional in variant-expressing cell lines or that variants are somehow able to evade degradation by NMD. We first used siRNA knockdown of the NMD regulator, UPF1, in an NMD reporter assay to determine if this surveillance pathway is functioning normally in AR variant-expressing cell lines. We then used UPF1 knockdown to determine if expression of the AR variants ARV3 and ARV7 is affected by inhibition of NMD. Next, we analyzed androgen regulation of UPF1 and used transcript expression analysis to determine if there is any association between UPF1 expression, resistance, and ARV3 or ARV7 expression. We found that the NMD pathway functions normally in the AR variant-expressing cell line 22Rv1 and that inhibition of NMD does not increase expression of ARV3 or ARV7. Furthermore, we found that expression of UPF1 is not androgen-regulated. We also found that UFP1 expression levels do not differentiate castration-sensitive from resistant cell line and that UPF1 expression does not correlate with expression of ARV3 or ARV7 in cells in which these variants are highly expressed. This study eliminates a possible mechanism of regulation of certain AR variants. Future research into the regulation of AR variants should focus on other mechanisms to better understand the origin of these variants and to possibly inhibit their expression for the resensitization of resistant cancers. Prostate 77:829-837, 2017. © 2017 Wiley Periodicals, Inc.