Androgen Receptor Downregulation Research Articles

The andean anticancer herbal product BIRM causes destabilization of androgen receptor and induces caspase-8 mediated-apoptosis in prostate cancer

BIRM is an anticancer herbal formulation from Ecuador. Previous study established its antitumor and antimetastatic activity against prostate cancer models. The activity of BIRM against human prostate cancer (PCa) cells was investigated to uncover its mechanism of antitumor activity. In androgen receptor (AR)-expressing PCa cells BIRM was 2.5-fold (250%) more cytotoxic in presence of androgen (DHT) compared to cells grown in the absence of DHT. In AR-positive cells (LAPC-4 and LNCaP) BIRM caused a dose and time-dependent down-regulation of AR and increased apoptosis. Exposing cells to BIRM did not affect the synthesis of AR and AR promoter activity but increased degradation of AR via proteasome-pathway. BIRM caused destabilization of HSP90-AR association in LAPC-4 cells. It induced apoptosis in PCa cells by activation of caspase-8 via death receptor and FADD-mediated pathways. A synthetic inhibitor of Caspase-8 cleavage (IETD-CHO) aborted BIRM–induced apoptosis. The effect of BIRM on AKT-mediated survival pathway in both AR+ and AR- negative (PC-3 and DU145) showed decreased levels of p-AKTser 473 in all PCa cell lines. BIRM dosed by oral gavage in mice bearing PC-3ML tumors showed selective efficacy on tumor growth; before tumors are established but limited efficacy when treated on existing tumors. Moreover, BIRM inhibited the LNCaP tumor generated by orthotropic implantation into dorsal prostate of nude mice. Partial purification of BIRM by liquid-liquid extraction and further fractionation by HPLC showed 4-fold increased specific activity on PCa cells. These results demonstrate a mechanistic basis of anti-tumor activity of the herbal extract BIRM.

Clinical pharmacology profile of an oral selective androgen receptor down-regulator, AZD3514: Implications on the design of ongoing castrate-resistant prostate cancer clinical studies

Clinical pharmacology profile of an oral selective androgen receptor down-regulator, AZD3514: Implications on the design of ongoing castrate-resistant prostate cancer clinical studies

Abstract 2060: Polymer mediated RNAi-based combination therapy for treating prostate cancer

Abstract Purpose. To determine whether RNAi-based combination therapy targeting androgen receptor (AR) and XIAP signaling pathways using polymeric micelles can treat prostate cancer. Methods. Potency of siRNAs targeting AR and XIAP genes was assessed using real time RT-PCR, Western Blot and ELISA. Cell viability was determined by MTT assay. A library of polymers designed to degrade via hydrolysis and enhance siRNA delivery was synthesized by conjugating hexylamine, pentaethylenehexamine and 3-morpholinopropylamine to poly(ethylene glycol)-block-poly(5-methyl-5-carboxyl-propylene carbonate) backbone using EDC/HOBT coupling chemistry. Polymer structure and molecular weight were verified with proton NMR and Gel Permeation Chromatography, respectively. Results. Three siRNA sequences targeting different regions of the AR and XIAP genes were designed, blasted against the human genome database to eliminate cross-silencing of nontarget genes and their potency screened in LNCaP and C4-2 cells following transfection. All siRNAs targeting AR (start site: 1172, 1648 and 1768) used in this study silenced AR mRNA and protein expression in both cell lines. Furthermore, 1172 was identified as the most potent sequence resulting in approximately 75% knockdown regardless of cell type. Also, the three siRNAs targeting XIAP (start site: 264, 297 and 458) down-regulated XIAP mRNA and protein levels in both cells and were generally more potent in C4-2 cells compared to LNCaP cells. C4-2 cells transfected with siRNA targeting AR (start site: 1172) and XIAP (start site: 264) alone or in combination and assayed after 96 hours revealed XIAP and AR down-regulation resulted in approximately 20% and 50% growth inhibition in C4-2 cells, respectively, compared to control. However, combined silencing of XIAP and AR genes decreased cell growth by 62% and was more potent in inhibiting cell proliferation compared to monotherapy. Additionally, combination of siRNA targeting AR (start site: 1172) and XIAP (start site: 264) significantly increased percentage of cells undergoing apoptosis (85%) compared to control or monotherapy. Poly(ethylene glycol)-block-poly(5-methyl-5-carboxyl-propylene carbonate) copolymer was synthesized by ring opening polymerization and conjugated with various ratios of hexylamine, pentaethylenehexamine and 3-morpholinopropylamine moieties. The resulting copolymer (PEG-b-P(CC-g-PHEXYL-g-PMORPH-g-PPHEA) were non-toxic even at concentration of 1500 μg/mL and successfully condensed siRNA at N/P of 25 with average hydrodynamic diameter of approximately 175 nm. Conclusions. Combination based therapy using siRNA targeting AR and XIAP can potentially treat prostate cancer. Citation Format: Michael Danquah. Polymer mediated RNAi-based combination therapy for treating prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2060.

Dysregulation of miR-212 Promotes Castration Resistance through hnRNPH1-Mediated Regulation of AR and AR-V7: Implications for Racial Disparity of Prostate Cancer.

The causes of disproportionate incidence and mortality of prostate cancer among African Americans (AA) remain elusive. The purpose of this study was to investigate the mechanistic role and assess clinical utility of the splicing factor heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) in prostate cancer progression among AA men. We employed an unbiased functional genomics approach coupled with suppressive subtractive hybridization (SSH) and custom cDNA microarrays to identify differentially expressed genes in microdissected tumors procured from age- and tumor grade-matched AA and Caucasian American (CA) men. Validation analysis was performed in independent cohorts and tissue microarrays. The underlying mechanisms of hnRNPH1 regulation and its impact on androgen receptor (AR) expression and tumor progression were explored. Aberrant coexpression of AR and hnRNPH1 and downregulation of miR-212 were detected in prostate tumors and correlate with disease progression in AA men compared with CA men. Ectopic expression of miR-212 mimics downregulated hnRNPH1 transcripts, which in turn reduced expression of AR and its splice variant AR-V7 (or AR3) in prostate cancer cells. hnRNPH1 physically interacts with AR and steroid receptor coactivator-3 (SRC-3) and primes activation of androgen-regulated genes in a ligand-dependent and independent manner. siRNA silencing of hnRNPH1 sensitized prostate cancer cells to bicalutamide and inhibited prostate tumorigenesis in vivo Our findings define novel roles for hnRNPH1 as a putative oncogene, splicing factor, and an auxiliary AR coregulator. Targeted disruption of the hnRNPH1-AR axis may have therapeutic implications to improve clinical outcomes in patients with advanced prostate cancer, especially among AA men.

PD32-07 GALETERONE SHOWS ANTI-TUMOR ACTIVITY IN MULTIPLE PRE-CLINICAL MODELS THAT EXPRESS ANDROGEN RECEPTOR SPLICE VARIANTS, SUPPORTING CORRELATIVE PATIENT DATA SEEN IN ARMOR2

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) III1 Apr 2016PD32-07 GALETERONE SHOWS ANTI-TUMOR ACTIVITY IN MULTIPLE PRE-CLINICAL MODELS THAT EXPRESS ANDROGEN RECEPTOR SPLICE VARIANTS, SUPPORTING CORRELATIVE PATIENT DATA SEEN IN ARMOR2 Vincent Njar, Amina Zoubeidi, Eva Corey, Elahe Mostaghel, Andrew Kwegyir-Afful, Senthilmurugan Ramalingam, and Douglas Jacoby Vincent NjarVincent Njar More articles by this author , Amina ZoubeidiAmina Zoubeidi More articles by this author , Eva CoreyEva Corey More articles by this author , Elahe MostaghelElahe Mostaghel More articles by this author , Andrew Kwegyir-AffulAndrew Kwegyir-Afful More articles by this author , Senthilmurugan RamalingamSenthilmurugan Ramalingam More articles by this author , and Douglas JacobyDouglas Jacoby More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.683AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Galeterone is a selective, multitargeted, small molecule that disrupts androgen signaling at multiple points in the pathway by enhancing androgen receptor (AR) degradation, inhibiting androgen biosynthesis, and antagonizing androgen binding to the AR. AR splice variant-7 (AR-V7) is a truncated, constitutively active splice variant of the AR that lacks the ligand binding domain (LBD), and has been implicated in castration resistant prostate cancer (CRPC) progression. AR-V7 expression is detected in approximately 12-26% of men with metastatic CRPC (mCRPC) prior to second-generation anti-androgens or chemotherapy, and AR-V7 expression has been clinically associated with resistance to enzalutamide (Xtandi) and abiraterone (Zytiga). In this study, multiple experimental approaches examined whether, and by what mechanism, galeterone inhibits the growth of prostate cancer cells or tumors expressing AR splice variants, such as AR-V7. METHODS To measure effects of galeterone on prostate tumor cell signaling and growth, we monitored reductions in AR protein levels, and have used in vitro measures of AR-dependent gene expression and tumor cell viability. In addition, we assessed in vivo efficacy of galeterone in mouse xenograft models and clinically by evaluating PSA responses in patients whose CTCs express truncated AR. RESULTS Our findings show that galeterone attenuates tumor cell proliferation, AR signaling and xenograft tumor growth using cells and tumors that express AR splice variants. Galeterone treatment also causes dose-dependent reductions in AR protein. Reductions in AR splice variant protein were observed in cells that co-express wild-type AR, and in AR negative prostate cancer cells that transiently express AR splice variant protein. These findings support the PSA responses seen clinically with galeterone treatment in men with mCRPC whose tumors contain truncated AR. CONCLUSIONS Galeterone's unique mechanism of AR protein downregulation occurs in both splice variant and full-length AR, supporting that galeterone-induced AR downregulation is independent of the AR LBD. Because preclinical and clinical data support an opportunity for treatment in this setting, Tokai is evaluating galeterone in an ongoing global pivotal trial designed to determine whether treatment with galeterone results in a statistically significant increase in radiographic progression-free survival as compared to enzalutamide in approximately 148 treatment-naïve, AR-V7+ mCRPC patients. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e763 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Vincent Njar More articles by this author Amina Zoubeidi More articles by this author Eva Corey More articles by this author Elahe Mostaghel More articles by this author Andrew Kwegyir-Afful More articles by this author Senthilmurugan Ramalingam More articles by this author Douglas Jacoby More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

High throughput microscopy identifies bisphenol AP, a bisphenol A analog, as a novel AR down-regulator.

Prostate cancer remains a deadly disease especially when patients become resistant to drugs that target the Androgen Receptor (AR) ligand binding domain. At this stage, patients develop recurring castrate-resistant prostate cancers (CRPCs). Interestingly, CRPC tumors maintain dependency on AR for growth; moreover, in CRPCs, constitutively active AR splice variants (e.g., AR-V7) begin to be expressed at higher levels. These splice variants lack the ligand binding domain and are rendered insensitive to current endocrine therapies. Thus, it is of paramount importance to understand what regulates the expression of AR and its splice variants to identify new therapeutic strategies in CRPCs. Here, we used high throughput microscopy and quantitative image analysis to evaluate effects of selected endocrine disruptors on AR levels in multiple breast and prostate cancer cell lines. Bisphenol AP (BPAP), which is used in chemical and medical industries, was identified as a down-regulator of both full length AR and the AR-V7 splice variant. We validated its activity by performing time-course, dose-response, Western blot and qPCR analyses. BPAP also reduced the percent of cells in S phase, which was accompanied by a ~60% loss in cell numbers and colony formation in anchorage-independent growth assays. Moreover, it affected mitochondria size and cell metabolism. In conclusion, our high content analysis-based screening platform was used to classify the effect of compounds on endogenous ARs, and identified BPAP as being capable of causing AR (both full-length and variants) down-regulation, cell cycle arrest and metabolic alterations in CRPC cell lines.

The value of integrating pre-clinical data to predict nausea and vomiting risk in humans as illustrated by AZD3514, a novel androgen receptor modulator

Nausea and vomiting are components of a complex mechanism that signals food avoidance and protection of the body against the absorption of ingested toxins. This response can also be triggered by pharmaceuticals. Predicting clinical nausea and vomiting liability for pharmaceutical agents based on pre-clinical data can be problematic as no single animal model is a universal predictor. Moreover, efforts to improve models are hampered by the lack of translational animal and human data in the public domain. AZD3514 is a novel, orally-administered compound that inhibits androgen receptor signaling and down-regulates androgen receptor expression. Here we have explored the utility of integrating data from several pre-clinical models to predict nausea and vomiting in the clinic. Single and repeat doses of AZD3514 resulted in emesis, salivation and gastrointestinal disturbances in the dog, and inhibited gastric emptying in rats after a single dose. AZD3514, at clinically relevant exposures, induced dose-responsive “pica” behaviour in rats after single and multiple daily doses, and induced retching and vomiting behaviour in ferrets after a single dose. We compare these data with the clinical manifestation of nausea and vomiting encountered in patients with castration-resistant prostate cancer receiving AZD3514. Our data reveal a striking relationship between the pre-clinical observations described and the experience of nausea and vomiting in the clinic. In conclusion, the emetic nature of AZD3514 was predicted across a range of pre-clinical models, and the approach presented provides a valuable framework for predicition of clinical nausea and vomiting.

Effect of a new type androgen receptor antagonist, TAS3681, on ligand-independent AR activation through its AR downregulation activity.

199 Background: Two new therapies, enzalutamide and abiraterone, directed at the androgen receptor (AR) signaling axis, represent important advances in the management of castration-resistant prostate cancer (CRPC). However, eventually almost all of patients acquire resistance to these drugs by a variety of mechanisms. Ligand independent AR activation such as induction of AR splice variants and AR overexpression are major issues of current CRPC progression. In the present study, we report the biological characterization of TAS3681, which is a new AR antagonist with AR downregulation activity, and propose this concept as a potential new approach for the treatment of CRPC. Methods: For assay of AR transactivation, prostate cancer (PCa) cells were transiently transfected with androgen-responsive reporter gene construct. The transfected cells were treated with growth factor and cytokine in steroid-depleted media, and luciferase activity was measured. To evaluate the effect of TAS3681 on AR and c-Myc protein expression, PCa cells were treated with TAS3681 in steroid-depleted media. AR and c-Myc protein levels were determined by western blot. Real-time PCR was used to analyze the mRNA levels of c-Myc and c-Myc target gene. Chromatin immunoprecipitation was performed to determine the enrichment of AR at the element. Results: TAS3681 dose-dependently reduced AR protein levels in PCa cells. In contrast to enzalutamide, TAS3681 suppressed androgen-independent AR transactivation by growth factor and cytokine. In PCa cells which express full-length AR and splice variant AR-v7, TAS3681 suppressed AR-v7 target gene expression through downregulation of AR-v7. Moreover, TAS3681 reduced expression of c-Myc, critical driver of androgen-independent mechanisms of PCa progression, via AR downregulation activity. In addition, real-time PCR assay showed the transcriptional suppression of c-Myc and its target gene by TAS3681. Conclusions: TAS3681 exhibits suppressive effects on ligand-independent AR activation via AR decreasing activity. These finding suggest that TAS3681 could be a candidate of breakthrough therapy for resistance to current AR pathway target drugs.

Lin28 induces resistance to anti-androgens via promotion of AR splice variant generation.

Prostate cancer (PCa) is androgen-dependent initially and progresses to a castration-resistant state after androgen deprivation therapy. Treatment options for castration-resistant PCa include the potent second-generation anti-androgen enzalutamide or CYP17A1 inhibitor abiraterone. Recent clinical observations point to the development of resistance to these therapies which may be mediated by constitutively active alternative splice variants of the androgen receptor (AR). Sensitivity of LNCaP cells overexpressing Lin28 (LN-Lin28) to enzalutamide, abiraterone, or bicalutamide was compared to that of control LN-neo cells using cell growth assays, proliferation assays using MTT, anchorage-dependent clonogenic ability assays and soft agar assays. Ability of LN-Lin28 cells to maintain AR activation after treatment with enzalutamide, abiraterone, or bicalutamide was tested using immunofluorescence, Western blotting, ChIP assays, and qRT-PCR. Importance of Lin28 in enzalutamide resistance was assessed by the downregulation of Lin28 expression in C4-2B and 22Rv1 cells chronically treated with enzalutamide. Requirement for sustained AR signaling in LN-Lin28 cells was examined by the downregulation of either full length AR or AR-V7 using siRNA. We show that Lin28 promotes the development of resistance to currently used targeted therapeutics by enhancing the expression of AR splice variants such as AR-V7. PCa cells overexpressing Lin28 exhibit resistance to treatment with enzalutamide, abiraterone, or bicalutamide. Downregulation of Lin28 resensitizes enzalutamide-resistant PCa cells to enzalutamide treatment. We also show that the upregulation of splicing factors such as hnRNPA1 by Lin28 may mediate the enhanced generation of AR splice variants in Lin28-expressing cells. Our findings suggest that Lin28 plays a key role in the acquisition of resistance to AR-targeted therapies by PCa cells and establish the importance of Lin28 in PCa progression.

Study of FN1 and ZNF438 gene expression negatively regulated by androgen receptor in androgen-independent LNCaP cells

To verify whether FN1 and ZNF438 are the androgen receptor(AR) target genes in LNCaP-AI cells and investigate the effects of AR exerts on FN1 and ZNF438 gene expression. ChIP-PCR and agarose gel electrophoresis were performed to verify the fact that AR acted on FN1 and ZNF438 gene. Subsequently, LNCaP-AI cells were treated with dihydrotestosterone(DHT)and lentivirus transfection to down-regulate AR expression. The expression of FN1 and ZNF438 gene in mRNA and protein levels were analyzed by RT-qPCR and Western blot. FN1 and ZNF438 gene enrichment of AR-ChIP group were 7.274±0.290 and 6.843±0.078, significantly higher than the IgG-ChIP group of 1.004±0.113 and 1.000±0.014 (t(FN1)=34.91, t(ZNF438)=128.377, P<0.05). Differences were statistical significance. After DHT stimulation, the expression of FN1 and ZNF438 gene in mRNA and protein levels were 0.434±0.050 and 0.069±0.042, significantly lower than the control group of 1.000±0.016 and 1.025±0.277 (t(FN1)=18.532, t(ZNF438)=5.905, P<0.05). Differences were statistical significance. Moreover, after AR down-regulation, the expression of FN1 and ZNF438 gene in mRNA and protein levels were 17.579±4.415 and 1.895±0.424, significantly higher than the control group of 1.028±0.445 and 1.041±0.190 (t(FN1)=6.461, t(ZNF438)=3.184, P<0.05). Differences were statistical significance. FN1 and ZNF438 gene are AR negatively regulated target genes in LNCaP-AI cells. The study might provide a new sight to further explore the mechanism of LNCaP-AI cells grow in androgen-depleted conditions.

MiR-124 and Androgen Receptor Signaling Inhibitors Repress Prostate Cancer Growth by Downregulating Androgen Receptor Splice Variants, EZH2, and Src.

miR-124 targets the androgen receptor (AR) transcript, acting as a tumor suppressor to broadly limit the growth of prostate cancer. In this study, we unraveled the mechanisms through which miR-124 acts in this setting. miR-124 inhibited proliferation of prostate cancer cells in vitro and sensitized them to inhibitors of androgen receptor signaling. Notably, miR-124 could restore the apoptotic response of cells resistant to enzalutamide, a drug approved for the treatment of castration-resistant prostate cancer. We used xenograft models to examine the effects of miR-124 in vivo when complexed with polyethylenimine-derived nanoparticles. Intravenous delivery of miR-124 was sufficient to inhibit tumor growth and to increase tumor cell apoptosis in combination with enzalutamide. Mechanistic investigations revealed that miR-124 directly downregulated AR splice variants AR-V4 and V7 along with EZH2 and Src, oncogenic targets that have been reported to contribute to prostate cancer progression and treatment resistance. Taken together, our results offer a preclinical rationale to evaluate miR-124 for cancer treatment.

Abstract B173: Galeterone shows anti-tumor activity in multiple pre-clinical models that express androgen receptor splice variants, supporting correlative patient data seen in ARMOR2

Abstract Multiple experimental approaches were used to examine whether and by what mechanism galeterone affects prostate cancer growth in cells and tumors expressing androgen receptor (AR) splice variants, such as AR-V7. Galeterone is a selective, multitargeted, small molecule that disrupts androgen signaling at multiple points in the pathway. ARMOR3-SV is a Phase 3, randomized, open-label, multicenter, controlled clinical trial in metastatic castration resistant prostate cancer (mCRPC) patients whose tumors express AR splice variant-7 mRNA (AR-V7). AR-V7 is a truncated, constitutively active splice variant of the AR that lacks the ligand binding domain (LBD) and has been implicated in prostate cancer progression. The expression of AR-V7 occurs in approximately 14-26% of men with mCRPC prior to second-generation anti-androgens or chemotherapy, and AR-V7 expression has been clinically associated with resistance to enzalutamide (Xtandi) or abiraterone (Zytiga). Here we provide the pre-clinical and clinical rationale for the development of galeterone in mCRPC patients with AR-V7. Our findings show that using multiple pre-clinical approaches, galeterone reduces in vitro prostate cancer cell proliferation, androgen receptor signaling and xenograft tumor growth using cells and tumors that express AR splice variants. Reductions in AR splice variant protein were also observed in cells that also co-express wild-type AR, and prostate cancer cells that lack wild type AR, but transiently express AR splice variant protein. Clinical data further support activity of galeterone in patients with truncated androgen receptors. Galeterone's unique mechanism of AR protein downregulation occurs in both splice variant and wild-type AR, supporting that galeterone-induced AR downregulation is independent of the AR LBD. Because preclinical and clinical data support an opportunity for treatment in this setting, Tokai Pharmaceuticals designed ARMOR3-SV to test galeterone vs. enzalutamide in men with metastatic castration-resistant prostate cancer whose tumors express the AR-V7 splice variant. Citation Format: Douglas B. Jacoby, Amina Zoubeidi, Eva B. Corey, Elahe Mostaghel, Andrew K. Kwegyir-Afful, Senthilmurugan Ramalingam, Vincent Njar. Galeterone shows anti-tumor activity in multiple pre-clinical models that express androgen receptor splice variants, supporting correlative patient data seen in ARMOR2. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B173.

Abstract PR09: TAS3681, a new type androgen receptor antagonist, suppresses ligand-independent AR activation through its AR downregulation activity

Abstract Background: Inhibitors of androgen receptor (AR) signaling axis, such as enzalutamide and abiraterone, represent important advances in the management of castration-resistant prostate cancer (CRPC). However, resistance to these drugs is still occurring. Recent reports suggest that there are several mechanisms of resistance. Ligand independent AR activation such as induction of AR splice variants or c-Myc expression is a major emerging mechanism of CRPC progression. There is an urgent need for developing a novel therapy to address these unresolved issues. Methods: For assay of androgen-independent AR transactivation, prostate cancer cells were transiently transfected with androgen-responsive reporter gene construct. The transfected cells were treated with growth factor and cytokine in steroid-depleted media, and luciferase activity was measured. To evaluate the effect of TAS3681 on AR and c-Myc protein expression, prostate cancer cells were treated with TAS3681 in steroid-depleted media. AR and c-Myc protein levels were determined by Western blot. Real-time PCR was used to analyze the mRNA levels of c-Myc and c-Myc target gene. Chromatin immunoprecipitation was performed to determine the enrichment of AR at the element. Results: TAS3681 dose-dependently reduced AR protein levels in prostate cancer cells. In contrast to enzalutamide, TAS3681 suppressed androgen-independent AR transactivation by growth factor and cytokine. In prostate cancer cells which express full-length AR and splice variant AR-v7, TAS3681 suppressed AR-v7 target gene expression through downregulation of AR-v7 occupancy at the enhancer. Moreover, TAS3681 reduced expression of c-Myc, critical driver of androgen-independent mechanisms of prostate cancer progression, via AR downregulation activity. In addition, real-time PCR assay showed the suppression of c-Myc and c-Myc target gene mRNA levels by TAS3681 but not by enzalutamide. Conclusion: Our findings suggest that TAS3681, a new type AR antagonist with AR downregulation activity, has a potential to overcome ligand-independent AR activation and could be a candidate of breakthrough therapy for resistance to current AR pathway target drugs. Citation Format: Daisuke Kajiwara, Kazuhisa Minamiguchi, Masanao Seki, Hiroya Mizutani, Hiroki Aoyagi, Shigeo Okajima, Eiji Sasaki, Teruhiro Utsugi, Yoshikazu Iwasawa. TAS3681, a new type androgen receptor antagonist, suppresses ligand-independent AR activation through its AR downregulation activity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PR09.

Inhibition of IGF-1R diminishes transcriptional activity of the androgen receptor and its constitutively active, C-terminally truncated counterparts Q640X and AR-V7.

Failure of endocrine treatment in castration-resistant prostate cancer (CRPC) is often associated with the emergence of C-terminally truncated androgen receptor variants that function as constitutively active transcription factors (i.e., AR∆LBD). The mechanisms involved in the regulation of AR∆LBD signaling are largely unknown. Since the IGF-1 pathway was repeatedly shown to affect AR function, we studied whether an inhibition of IGF-1R could also affect AR∆LBD signaling. Regulation of androgen receptor (AR) and AR∆LBD signaling was analyzed by reporter gene assays, immunoblotting, ELISA and quantitative RT-PCR. Inhibition of IGF-1R with the small-molecule inhibitor NVP-AEW541 reduced the transcriptional activity of the AR and its truncated counterparts Q640X and AR-V7. As shown in Q640X, the inhibition of transcriptional activity was paralleled by a decreased receptor phosphorylation. Inhibition of IGF-1R leads to a down-regulation of AR∆LBD signaling and provides a rationale for CRPC therapies targeting growth factor receptors.

Methylselenocysteine preventing castration-resistant progression of prostate cancer.

Castration-resistant progression of prostate cancer after androgen deprivation therapy remains a critical challenge in the clinical management of prostate cancer. Resurgent androgen receptor activity is an established driver of castration-resistant progression, and upregulation of androgen receptor expression has been implicated to contribute to the resurgent androgen receptor activity. We reported previously that methylselenocysteine can decrease the expression and activity of androgen receptor. Here we investigated the ability of methylselenocysteine to inhibit castration-resistant progression of prostate cancer. The regrowth of LNCaP prostate cancer xenografts after castration was monitored. The levels of prostate-specific antigen in mouse serum were measured by ELISA. Tumor cell proliferation and apoptosis were analyzed via Ki-67 immunohistochemistry and TUNEL assay, respectively. Intratumoral angiogenesis was assessed by immunohistochemistry staining of vascular endothelial growth factor and CD31. We showed that methylselenocysteine delayed castration-resistant regrowth of LNCaP xenograft tumors after androgen deprivation. This was accompanied by decreased serum levels of prostate-specific antigen, inhibition of prostate cancer cell proliferation and tumor angiogenesis, as well as downregulation of androgen receptor and induction of apoptosis in the relapsed tumors. The present study represents the first to show the preclinical efficacy of methylselenocysteine in delaying castration-resistant progression of prostate cancer. The findings provide a rationale for evaluating the clinical application of combining methylselenocysteine with androgen deprivation therapy for the treatment of advanced prostate cancer.

TAS3681: New class of androgen receptor antagonist with androgen receptor downregulating activity.

266 Background: The persistent activation of the androgen receptor (AR) signaling axis plays a major role in castration-resistant prostate cancer (CRPC). This knowledge has driven the clinical development of new inhibitors of androgen production (e.g., abiraterone) and AR signaling (e.g., enzalutamide). However, in many patients, the efficacy is short-lived and eventually resistance appears. Reactivation of AR signaling occurs by several mechanisms in CRPC. In this study, we report the biological properties of TAS3681, a new AR antagonist with AR downregulating activity, and propose this concept as a potential new approach for the treatment of CRPC. Methods: For assay of AR transactivation, COS-7 cells were transiently transfected with androgen-responsive reporter gene construct and expression vectors encoding wt or mu ARs. The transfected cells were treated with TAS3681 and androgen in steroid depleted media for 24 h, and luciferase activity was measured. For an in vivo pharmacodynamic assay to confirm AR downregulation in tumor, TAS3681 was orally dosed in CRPC tumor xenograft model. Tumor was isolated and AR level in tumor was determined by Western blot. Results: TAS3681 suppressed the growth of AR positive prostate cancer (PCa) cells but did not affect that of AR-negative DU145 PCa cells, indicating a dependency on AR for efficacy. TAS3681 did not stimulate AR nuclear translocation and suppressed wt and mu ARs (including F876L) transactivation in cells, indicative of its pure AR antagonist profile. In contrast to enzalutamide, TAS3681 effectively suppressed androgen-independent AR transactivation by growth factors and cytokines via AR downregulating activity. Interestingly, in PCa cells which express full-length and splice variant AR, TAS3681 reduced the expression of both ARs. Moreover, TAS3681 treatment effectively decreased AR level in CRPC tumors in vivo. Conclusions: TAS3681 exerts an anti-androgenic effect via two mechanisms of action: pure AR antagonism and AR decreasing activity. It is expected that TAS3681 has a potential to overcome the resistance to current and 2nd-generation therapies targeting AR signaling.

Beyond the HPA-axis: The role of the gonadal steroid hormone receptors in modulating stress-related responses in an animal model of PTSD

The hypothalamic–pituitary–adrenal (HPA) axis, which plays a major role in the response to stress, and the hypothalamic–pituitary–gonadal (HPG) axis are closely linked with the ability to inhibit the other. Testosterone, a product of the HPG, has many beneficial effects beyond its functions as a sex hormone including anti-anxiety properties. In this study we examined the effect of stress exposure on gonadal hormones, and their efficacy in modulating anxiety-like response in an animal model of PTSD. Male rats were exposed to predator scent stress, followed by analysis of brain expression of androgen receptor (AR) receptor and estrogen receptor α (ERα). The behavioral effects of immediate treatment with testosterone, testosterone receptor antagonist (flutamide) or vehicle were evaluated using the elevated plus-maze, acoustic startle response and trauma-cue response. Levels of circulating corticosterone and testosterone were also measured after treatment. The behavioral effects of delayed testosterone treatment were explored in the same manner. We report that animals whose behavior was extremely disrupted (EBR) selectively displayed significant down-regulation of AR and ERα in the hippocampus. Immediate treatment with flutamide or delayed treatment with testosterone significantly increased prevalence rates of minimal behavioral response (MBR) and decreased prevalence of EBR with favorable behavioral results. Testosterone levels were higher in control un-exposed animals, while corticosterone was higher in control exposed animals. This study suggests that gonadal steroid hormones are involved in the neurobiological response to predator scent stress and thus warrant further study as a potential therapeutic avenue for the treatment of anxiety-related disorders.

MicroRNA-185 downregulates androgen receptor expression in the LNCaP prostate carcinoma cell line.

The present study aimed to investigate whether microRNA (miR)‑185 downregulated androgen receptor expression in the LNCaP prostate carcinoma cell line. Human prostate cancer (PCa) LNCaPcells were cultured and transfected with synthetic has‑miR‑185 mimic or inhibitor. The transfected cells were subsequently evaluated with a viability assay, nuclear staining, reverse transcription quantitative polymerase chain reaction (RT‑qPCR), dual luciferase assay and western blot analysis. The results of the western blot analysis and RT‑qPCR indicated that transfection with an miR‑185 mimic markedly reduced the androgen receptor (AR) protein expression levels in LNCaPcells, whereas transfection with an miR‑185 inhibitor increased the protein expression of AR in the LNCaPcells. The results of the luciferase reporter assay demonstrated that the predicted target site in the AR 3' untranslated regions was a specific functional binding site for miR‑185, and that AR was a direct target of miR‑185. In addition, downregulation of AR by miR‑185 impaired the interaction between AR and androgen response element, and downregulated the expression of the AR target gene prostate specific antigen. Data also suggested that the downregulation of AR mediated by miR‑185, inhibited the proliferation and induced the apoptosis of the LNCaPcells. Therefore, the results of the present study suggested that miR‑185 may be a potential negative modulator of AR‑mediated signaling and may act as a tumor suppressor in prostate cancer cells.

Active ERK2 is sufficient to mediate growth arrest and differentiation signaling.

Although extracellular signal-regulated kinases (ERK1/2) have been shown to be required in Raf/MEK/ERK pathway signaling, its sufficiency for mediating the pathway signaling has not been firmly established. In an effort to address this, we evaluated previously described ERK2 mutants that exhibit enhanced autophosphorylation of TEY sites in the activation loop in terms of their ability to induce growth arrest and differentiation in LNCaP and PC12 cells. We demonstrate that expression of ERK2-L73P/S151D, containing Lys73Pro and Ser151Asp substitutions that synergistically promote ERK autophosphorylation, is sufficient to induce growth arrest and differentiation, whereas expression of ERK2-I84A and ERK2-R65S/D319N is not as effective. When compared to the constitutively active MEK1-ΔN3/S218E/S222D, expression of ERK2-L73P/S151D only mildly increased ERK kinase activity in cells, as assessed using the ERK substrates p90(RSK) and ETS domain-containing protein (ELK1). However, ERK2-L73P/S151D expression effectively induced down-regulation of androgen receptors, Retinoblastoma (Rb) protein and E2F1 transcription factor, and up-regulation of p16(INK4A) and p21(CIP1), accompanied by cell-cycle arrest and morphological differentiation in LNCaP cells and neurite-like processes in PC12 cells. These effects and the TEY site phosphorylation of ERK2-L73P/S151D were abrogated upon introduction of the active site-disabling Lys52Arg mutation, suggesting that its autoactivation drives this signaling. Moreover, introduction of mutations Asp316/319Ala or Asp319Asn, which impair the common docking site/D-domain-based physical interaction of ERK, did not significantly affect ERK2-L73P/S151D signaling, suggesting that ERK2 mediates growth arrest and differentiation independently of the conventional ERK-target interaction mechanism. Thus, our study presents convincing evidence of ERK sufficiency for Raf/MEK/ERK signaling.

Androgens inhibit aromatase expression through DAX-1: insights into the molecular link between hormone balance and Leydig cancer development.

Leydig cell tumors (LCTs) of the testis are steroid-secreting tumors associated with various steroid biosynthetic abnormalities and endocrine dysfunctions. Despite their overall rarity, LCTs are still of substantial interest owing to the paucity of information regarding their exact nature and malignant potential. In the present study, we disclose the ability of androgens to inhibit Leydig tumor cell proliferation by opposing to self-sufficient in situ estrogen production. In rat Leydig tumor cells, R2C, androgen treatment significantly decreases the expression and the enzymatic activity of cytocrome P450 aromatase, responsible for the local conversion of androgens into estrogens. This inhibitory effect relies on androgen receptor (AR) activation and involves negative regulation of the CYP19 gene transcriptional activity through the nuclear orphan receptor DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1). Ligand-activated AR up-regulates the expression of DAX-1 and promotes its increased recruitment within the steroidogenic factor-1 site-containing region of the aromatase proximal promoter II in association with the nuclear receptor corepressor. The biological relevance in LCTs of the newly highlighted functional interplay between AR, DAX-1, and aromatase is underlined by our in vivo observations, revealing a marked down-regulation of AR and DAX-1 expression and a strong increase in aromatase levels in testes tissues from old Fischer rats with spontaneously developed Leydig cell neoplasia, compared with normal testes tissues from younger animals. In elucidating a mechanism by which androgens modulate the growth of Leydig tumor cells, our finding support the hypothesis that maintaining the adequate balance between androgen and estrogens may represent the key for blocking estrogen-secreting Leydigioma development, opening new prospects for therapeutic intervention.