Suppress Androgen Receptor Expression Research Articles

Lineage plasticity and treatment resistance in prostate cancer: the intersection of genetics, epigenetics, and evolution.

Androgen deprivation therapy is a cornerstone of treatment for advanced prostate cancer, and the development of castrate-resistant prostate cancer (CRPC) is the primary cause of prostate cancer-related mortality. While CRPC typically develops through a gain in androgen receptor (AR) signaling, a subset of CRPC will lose reliance on the AR. This process involves genetic, epigenetic, and hormonal changes that promote cellular plasticity, leading to AR-indifferent disease, with neuroendocrine prostate cancer (NEPC) being the quintessential example. NEPC is enriched following treatment with second-generation anti-androgens and exhibits resistance to endocrine therapy. Loss of RB1, TP53, and PTEN expression and MYCN and AURKA amplification appear to be key drivers for NEPC differentiation. Epigenetic modifications also play an important role in the transition to a neuroendocrine phenotype. DNA methylation of specific gene promoters can regulate lineage commitment and differentiation. Histone methylation can suppress AR expression and promote neuroendocrine-specific gene expression. Emerging data suggest that EZH2 is a key regulator of this epigenetic rewiring. Several mechanisms drive AR-dependent castration resistance, notably AR splice variant expression, expression of the adrenal-permissive 3βHSD1 allele, and glucocorticoid receptor expression. Aberrant epigenetic regulation also promotes radioresistance by altering the expression of DNA repair- and cell cycle-related genes. Novel therapies are currently being developed to target these diverse genetic, epigenetic, and hormonal mechanisms promoting lineage plasticity-driven NEPC.

Abstract A059: Epigenetic remodelling by class I HDAC inhibitors increases response to androgen signalling inhibitors in prostate cancer

Abstract Prostate cancer (PCa) is one of the most prevalent cancers in the male population with approximately 1 in 8 men diagnosed with the disease during their lifetime. The treatment options available varies depending on individual circumstances, with surgery or radiotherapy employed as first-line treatments for locally advanced PCa whilst hormone therapies are employed as the disease progresses toward castrate-resistant prostate cancer. Despite the advances in therapies and initial treatment effectiveness, tumours develop resistance and overcoming this resistance proves a major challenge for the treatment of the disease. Dysregulation of chromatin regulatory proteins and the epigenetic landscape are key factors contributing to the development, progression and persistence of PCa. Histone deacetylases (HDACs) are a family of chromatin-modifying proteins which assemble into various transcriptional repressor complexes and mediate gene repression through the removal of acetyl groups from histone tails, subsequently compacting the chromatin landscape impacting on cell proliferation, transcription, replication and DNA repair. Overexpression of HDACs in prostate cancer has been shown to shift the balance of acetylation, resulting in improper gene silencing. Therefore, we hypothesise that restoration of the acetylation homeostasis through HDAC inhibition is a potential therapy to overcome resistance in PCa. Here, we show that the class I HDAC inhibitor Entinostat as a single agent promotes anti-tumour effects in PCa by impairing cell proliferation and inducing apoptosis in a panel of PCa cell lines. Interestingly, we found that combination with the androgen signalling inhibitors (ASIs), Enzalutamide or Abiraterone, further enhances these anti-tumour effects. In vitro analysis of matched parental and Enzalutamide-/Abiraterone-resistant C4-2B and CWRR-1 cells further revealed an enhanced reduction in cell survival following combined Entinostat and ASIs treatments in the resistant models compared to parental. Genomic and transcriptomic analyses uncovered distinct molecular signatures of these resistant models. Treatment with Entinostat alone downregulated androgen receptor (AR) expression and combination with ASI further suppressed AR expression and downstream AR target genes. Taken together, these results demonstrate the potential for Entinostat to synergise with current standard-of-care therapies to target ASI resistance in PCa. In conclusion, this study provides strong evidence suggesting the effectiveness of class I HDAC inhibition by Entinostat in the treatment of castration-resistant prostate cancer and opens the door for further research into combinatorial therapies in targeting resistance. Citation Format: Rachel McCole, Ben McCullough, Judith M. Manley, Nicholas Forsythe, Simon S. McDade, Melissa LaBonte Wilson. Epigenetic remodelling by class I HDAC inhibitors increases response to androgen signalling inhibitors in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A059.

Abstract A073: Human prostate-on-chip models to define stromal and epithelial interactions in normal and cancerous prostate

Abstract Background: The secretion of morphogenic factors from the stroma, whose production is dependent on stromal androgen receptor (AR) action, is crucial for maintaining glandular structure and homeostatic function of the prostate gland. However, the precise nature of the factors involved, their regulation, and how they impact homeostasis in the human prostate is not clear. We hypothesize that loss of stromal AR function as seen in prostate cancer progression and during androgen-deprivation therapy likely contribute to tumor progression through disruption of this homeostasis. It is important to understand these interactions in the human context to better aid in improving patient outcomes. Approach: Microfluidic-based technology was used to develop the first human Prostate-on-Chip (PoC) where human prostate fibroblasts are co-cultured with human prostate basal epithelial cells under flow. Addition of androgen to the stromal cells is sufficient to induce luminal cell differentiation on top of the neighboring basal cells – recapitulating the cellular architecture of the human gland. To study prostate cancer in this context, we replaced the normal epithelial cells with tumor cells to interrogate the tumor-stromal interactions; thus, generating a human Prostate Cancer-on-Chip model (PCoC). Cytokine arrays and RT-qPCR was used to identify androgen-induced stromal morphogens and tumor cytokines involved in tumor/stromal interactions. Molecular biology approaches were used to identify the mechanisms involved. Results: Within the PCoC model, tumor cells invaded into the stroma and conversely stromal cells invaded into the tumors. This was accompanied by localized CAF conversion of the stroma. Invasion of tumor cells into the stroma could be blocked with an integrin-α6 binding peptide. Strikingly, the level of AR expression in the stroma was also reduced, recapitulating the loss of stromal AR expression seen in patients. A cytokine array identified TNFα and TGFβ as tumor secreted factors responsible for suppressing stromal AR protein and mRNA expression. TNFα suppressed stromal AR expression in the absence of CAF conversion via NF-κB binding to the AR promoter. On the other hand, p38-MAPK signaling suppressed AR expression independent of TNFα. FGF10 and Wnt16, but not KGF, were identified as androgen-induced stromal secreted morphogens. Loss of stromal AR expression, led to suppression of FGF10 and Wnt16, but not KGF expression. Thus, stromal AR inhibition by tumor secreted TNFα or p38-MAPK signaling suppresses the stromal morphogens required to maintain normal prostate homeostasis. Conclusions: We have developed the first human Prostate-on-Chip and Prostate Cancer-on-Chip models which recapitulate the biology of both the normal human prostate as well as prostate cancer stromal interactions. These models can be used to address a range of clinical problems in cancer development, drug response, and biomarker identification in the context of the tumor microenvironment. This model can easily be adapted for personalized medicine approaches. Citation Format: Shekha K. Tahsin, Linan Jiang, Neha Sane, Kailie Szewczyk, Hunain Khawaja, Yitshak Zohar, Cindy K. Miranti. Human prostate-on-chip models to define stromal and epithelial interactions in normal and cancerous prostate [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A073.

Abstract 2446: Low dose dimethyl sulfoxide (DMSO) downregulates the expression of androgen receptor (AR) and AR-variant 7 (AR-v7) in castration resistant prostate cancer (CRPC) cells

Abstract The highly aggressive prostate cancer (PC) cells utilize androgen receptor (AR) signaling to facilitate their growth and metastasis. Despite androgen deprivation therapy (ADT), recurrence of castration-resistant prostate cancer (CRPC) often occur due to constitutive AR signaling via both full-length AR (AR-FL) and AR splice variants, primarily AR-V7. Therefore, safe strategies to suppress both AR-FL & AR-V7 expression in CRPC cells will be of significant benefit. Several plant-derived organosulfur compounds, e.g. sulforaphane and diallyl-trisulfide, can potently suppress AR expression in CRPC cells, but are yet to be clinically approved. We investigated whether dimethyl sulfoxide (DMSO) an approved organosulfur compound often used as a solvent for pharmaceutical agents, can similarly suppress AR levels in the CRPC cell lines, C4-2B and 22Rv1. Exposure to low dose DMSO (0.1-1%) was not cytotoxic to these CRPC cells, and less than 20% cytotoxicity was seen with 2.5% DMSO even at 96 h post-exposure. Interestingly however, DMSO dose-dependently suppressed AR-FL in C4-2B cells and both AR-FL and AR-V7 in 22Rv1 cells within 24 h post-exposure. Exposure to DMSO also downregulated the expression of hetero-nuclear ribonucleoprotein H1 (hnRNPH1), which is known to regulate AR expression and splicing. Although DMSO exposure showed a dose- and time-dependent effect on reactive oxygen species (ROS) production by PC cells, the AR-suppressive effect of DMSO was not altered by the antioxidant n-acetyl cysteine (NAC). Furthermore, although DMSO did not affect cell viability at these clinically-achievable concentrations, a significant (p<0.01) decrease in the migratory ability of both CRPC cell lines was clearly evident. Our novel findings indicate that safe doses of DMSO may be used to decrease AR expression and metastatic ability in CRPC cells. Citation Format: Namrata Khurana, Hogyoung Kim, Talal Khan, Shohreh Kahhal, Amar Bukvic, Asim B. Abdel-Mageed, Suresh C. Sikka, Debasis Mondal. Low dose dimethyl sulfoxide (DMSO) downregulates the expression of androgen receptor (AR) and AR-variant 7 (AR-v7) in castration resistant prostate cancer (CRPC) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2446.

Weekly treatment with SAMiRNA targeting the androgen receptor ameliorates androgenetic alopecia

Androgenetic alopecia (AGA) is the most common type of hair loss in men and women. Dihydrotestosterone (DHT) and androgen receptor (AR) levels are increased in patients with AGA, and DHT-AR signaling correlates strongly with AGA pathogenesis. In this study, treatment with self-assembled micelle inhibitory RNA (SAMiRNA) nanoparticle-type siRNA selectively suppressed AR expression in vitro. Clinical studies with application of SAMiRNA to the scalp and massaging to deliver it to the hair follicle confirmed its efficacy in AGA. For identification of a potent SAMiRNA for AR silencing, 547 SAMiRNA candidates were synthesized and screened. SAMiRNA-AR68 (AR68) was the most potent and could be efficiently delivered to human follicle dermal papilla cells (HFDPCs) and hair follicles, and this treatment decreased the AR mRNA and protein levels. We confirmed that 10 µM AR68 elicits no innate immune response in human PBMCs and no cytotoxicity up to 20 µM with HFDP and HaCaT cells. Clinical studies were performed in a randomized and double-blind manner with two different doses and frequencies. In the low-dose (0.5 mg/ml) clinical study, AR68 was applied three times per week for 24 weeks, and through quantitative analysis using a phototrichogram, we confirmed increases in total hair counts. In the 24-week long high-dose (5 mg/ml) clinical study, AR68 showed average additional hair growth of 1.3-1.9 hairs/cm2 per month, which is comparable to finasteride. No side effects were observed. Therefore, SAMiRNA targeting AR mRNA is a potential novel topical treatment for AGA.

Transcriptome analysis to identify the downstream genes of androgen receptor in dermal papilla cells

BackgroundTestosterone signaling mediates various diseases, such as androgenetic alopecia and prostate cancer. Testosterone signaling is mediated by the androgen receptor (AR). In this study, we fortuitously found that primary and immortalized dermal papilla cells suppressed AR expression, although dermal papilla cells express AR in vivo. To analyze the AR signaling pathway, we exogenously introduced the AR gene via a retrovirus into immortalized dermal papilla cells and comprehensively compared their expression profiles with and without AR expression.ResultsWhole-transcriptome profiling revealed that the focal adhesion pathway was mainly affected by the activation of AR signaling. In particular, we found that caveolin-1 gene expression was downregulated in AR-expressing cells, suggesting that caveolin-1 is controlled by AR.ConclusionOur whole transcriptome data is critical resources for discovery of new therapeutic targets for testosterone-related diseases.

Smoothened loss is a characteristic of neuroendocrine prostate cancer.

PurposeHedgehog (Hh) signaling promotes castration‐resistant prostate cancer by supporting androgen‐independent prostate cancer cell development and growth; however, its role in neuroendocrine prostate cancer (NEPC) has not yet been explored. In this study, we assessed the expression of key genes involved in Hh signaling in prostate cancer and investigated the potential role of smoothened (SMO) in the pathogenesis of NEPC.MethodsSix public datasets, each containing cases of prostate adenocarcinoma (AdPC) and NEPC, were analyzed to compare the differential messenger RNA (mRNA) expression of six classic Hh signaling genes. The SMO, synaptophysin, chromogranin A (CHGA) and androgen receptor (AR) proteins were evaluated in human tissues from 5 cases of NEPC, 2 cases of AdPC mixed with NEPC, 2 cases of AdPC with neuroendocrine differentiation and 22 cases of high‐grade AdPC as determined by an immunohistochemistry assay. Gene set enrichment analysis (GSEA) was performed to identify relevant genetic signatures associated with SMO expression based on the public datasets. Stable SMO‐knockdown LNCaP and C4‐2B cells were established with a lentiviral system, and the expression of SMO, Gli1, AR, prostate‐specific antigen (PSA), and REST was assessed by real‐time polymerase chain reaction and western blot. Secreted PSA in the conditioned medium was assessed by ELISA. Gli1 was ectopically expressed performed by the transfection of Gli1 complementary DNA into SMO‐knockdown LNCaP cells, and western blot was used to assess of AR and PSA expression.ResultsThe mRNA level of SMO was dramatically downregulated in NEPC samples compared with AdPC samples in all 6 public datasets. SMO protein loss was observed in 100% of NEPC samples but in only 9% (2 of 22) of high‐grade AdPC samples. GSEA results showed that SMO loss was closely correlated with AR signaling activity. Stable SMO knockdown significantly attenuated AR signaling activity and suppressed AR expression, while Gli1 overexpression partially reversed the inhibitory effects of SMO knockdown on AR signaling activity and AR expression in LNCaP and C4‐2B cells.ConclusionThese results demonstrate that SMO loss is a characteristic of NEPC and that detecting SMO by IHC could aid pathologists in NEPC diagnosis. SMO loss may promote NEPC pathogenesis by modulating AR signaling.

Nucleolin represses transcription of the androgen receptor gene through a G-quadruplex.

The androgen receptor (AR) is a major driver of prostate cancer development and progression. Men who develop advanced prostate cancer often have long-term cancer control when treated with androgen-deprivation therapies (ADT). Still, their disease inevitably becomes resistant to ADT and progresses to castration-resistant prostate cancer (CRPC). ADT involves potent competitive AR antagonists and androgen synthesis inhibitors. Resistance to these types of treatments emerges, primarily through the maintenance of AR signaling by ligand-independent activation mechanisms. There is a need to find better ways to block AR to overcome CRPC. In the findings reported here, we demonstrate that the nuclear scaffold protein, nucleolin (NCL), suppresses the expression of AR. NCL binds to a G-rich region in the AR promoter that forms a G-quadruplex (G4) structure. Binding of NCL to this G4-element is required for NCL to suppress AR expression, specifically in AR-expressing tumor cells. Compounds that stabilize G4 structures require NCL to associate with the G4-element of the AR promoter in order to decrease AR expression. A newly discovered G4 compound that suppresses AR expression demonstrates selective killing of AR-expressing tumor cells, including CRPC lines. Our findings raise the significant possibility that G4-stabilizing drugs can be used to increase NCL transcriptional repressor activity to block AR expression in prostate cancer. Our studies contribute to a clearer understanding of the mechanisms that control AR expression, which could be exploited to overcome CRPC.

Berberine Ameliorates Prenatal Dihydrotestosterone Exposure-Induced Autism-Like Behavior by Suppression of Androgen Receptor.

Many epidemiology studies have shown that maternal polycystic ovary syndrome (PCOS) results in a greater risk of autism spectrum disorders (ASD) development, although the detailed mechanism remains unclear. In this study, we aimed to investigate the potential mechanism and provide a possible treatment for PCOS-mediated ASD through three experiments: Experiment 1: real-time PCR and western blots were employed to measure gene expression in human neurons, and the luciferase reporter assay and chromatin immunoprecipitation (ChIP) was used to map the responsive elements on related gene promoters. Experiment 2: pregnant dams were prenatally exposed to dihydrotestosterone (DHT), androgen receptor (AR) knockdown (shAR) in the amygdala, or berberine (BBR), and the subsequent male offspring were used for autism-like behavior (ALB) assay followed by biomedical analysis, including gene expression, oxidative stress, and mitochondrial function. Experiment 3: the male offspring from prenatal DHT exposed dams were postnatally treated by either shAR or BBR, and the offspring were used for ALB assay followed by biomedical analysis. Our findings showed that DHT treatment suppresses the expression of estrogen receptor β (ERβ) and superoxide dismutase 2 (SOD2) through AR-mediated hypermethylation on the ERβ promoter, and BBR treatment suppresses AR expression through hypermethylation on the AR promoter. Prenatal DHT treatment induces ERβ suppression, oxidative stress and mitochondria dysfunction in the amygdala with subsequent ALB behavior in male offspring, and AR knockdown partly diminishes this effect. Furthermore, both prenatal and postnatal treatment of BBR partly restores prenatal DHT exposure-mediated ALB. In conclusion, DHT suppresses ERβ expression through the AR signaling pathway by hypermethylation on the ERβ promoter, and BBR restores this effect through AR suppression. Prenatal DHT exposure induces ALB in offspring through AR-mediated ERβ suppression, and both prenatal and postnatal treatment of BBR ameliorates this effect. We conclude that BBR ameliorates prenatal DHT exposure-induced ALB through AR suppression, this study may help elucidate the potential mechanism and identify a potential treatment through using BBR for PCOS-mediated ASD.

Suppressed androgen receptor expression promotes M2 macrophage reprogramming through the STAT3/SOCS3 pathway.

Macrophages are important mediators of inflammatory cardiovascular diseases, and various macrophage phenotypes exert opposite effects during inflammation. In our previous study, we proved that suppressed androgen receptor (AR) alleviated inflammation during experimental autoimmune myocarditis (EAM). As anti-inflammatory cells, whether M2 macrophages are involved in this process remains unclear. Here, we showed that anti-inflammatory cytokines and M2 macrophages were elevated when AR was suppressed during EAM. In IL-4 stimulation-induced M2 macrophages, impaired AR with ASC-J9 increased the expression of M2 macrophage-related factors. Moreover, suppressed AR expression resulted in macrophage M2 polarization by reducing SOCS3 production and enhancing STAT3 activation. Taken together, our data suggest that AR plays a critical role in macrophage polarization and suppressed redundant AR expression promotes anti-inflammatory M2 macrophages reprogramming. This study suggests a potential therapeutic agent for inflammatory cardiomyopathy through the use of ASC-J9.

Abstract 4809: Bardoxolone-methyl suppresses both androgen receptor and its splice-variant ARv7 in prostate cancer cells to enhance the anti-cancer efficacy of enzalutamide

Abstract Prostate cancer (PCa) is a leading cause of morbidity and mortality in elderly men in the United States. Despite androgen deprivation therapy (ADT), the recurrence of castration resistant prostate cancer (CRPC) remains a significant challenge. Augmented signaling via the full-length androgen receptor (AR) and the constitutively active AR splice variants, especially ARv7, is associated with ADT-resistance. Therefore, a proper understanding of augmented AR signaling and adjunct strategies to suppress both AR and ARv7 levels are critically needed. ADT-induced oxidative stress (Ox-stress) can facilitate CRPC outgrowth. We previously documented that the Ox-stress induced transcription factor, Nrf2 can suppress AR expression and AR-transactivation function in PCa cells. Recently, we also showed that the Nrf2-inducing natural compound, sulforaphane (SFN) can decrease both AR and ARv7 levels in PCa cells, and co-exposure to SFN enhanced the efficacy of anti-androgens. In this study, we examined whether a synthetic triterpenoid drug, Bardoxolone-methyl (CDDO-me), a potent enhancer of Nrf2, can similarly decrease AR and ARv7 expression. Exposure to nanomolar (nM) concentrations of CDDO-me rapidly downregulated AR levels in the androgen-dependent LNCaP cells and androgen-independent C4-2B cells. Exposure to CDDO-me also suppressed both AR and ARv7 levels in the CRPC line, CWR22Rv1. A biphasic effect of CDDO-me on reactive oxygen species (ROS) levels was observed. Pre-exposure to the antioxidant, n-acetyl cysteine (NAC) blocked the AR-suppressive effect of CDDO-me. Molecular mechanistic studies using actinomycin-D (transcription inhibitor), cycloheximide (translation inhibitor), and MG132 (proteasomal inhibitor) suggested that CDDO-me may decrease AR gene expression and increase degradation of AR and ARv7 proteins. Furthermore, exposure to CDDO-me increased proteasomal activity in PCa cells and suppressed the levels of several AR splicing factors, i.e. the heteronuclear ribonucleoproteins A1 and H1 (hnRNP-A1 and hnRNP-H1). Co-exposure to CDDO-me increased the anti-cancer efficacy of enzalutamide (ENZ) as evident by decreased cell-viability (MTT), migration (scratch-wound) and colony forming unit (CFU) assays. Therefore, adjunct treatment with low-dose CDDO-me may be an effective strategy to suppress AR and ARv7 levels and augment the efficacy of anti-androgens. Most importantly, since CDDO-me is in late-stage clinical trials for chronic kidney disease (CKD), its repositioning as a potent AR suppressive agent may be of significant translational value in PCa patients undergoing ADT. Citation Format: Namrata Khurana, Partha Chandra, Hogyoung Kim, Asim B Abdel-Mageed, Suresh Sikka, Debasis Mondal. Bardoxolone-methyl suppresses both androgen receptor and its splice-variant ARv7 in prostate cancer cells to enhance the anti-cancer efficacy of enzalutamide [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 4809.

Analysis of the androgen receptor-regulated lncRNA landscape identifies a role for ARLNC1 in prostate cancer progression.

The androgen receptor (AR) plays a critical role in the development of the normal prostate as well as prostate cancer. Using an integrative transcriptomic analysis of prostate cancer cell lines and tissues, we identified ARLNC1 (AR-regulated long non-coding RNA 1) as an important long non-coding RNA that is strongly associated with AR signaling in prostate cancer progression. Not only was ARLNC1 induced by AR protein, ARLNC1 stabilized the AR transcript via RNA-RNA interaction. ARLNC1 knockdown suppressed AR expression, global AR signaling, and prostate cancer growth in vitro and in vivo. Taken together, these data support a role for ARLNC1 in maintaining a positive feedback loop that potentiates AR signaling during prostate cancer progression, and identifies ARLNC1 as a novel therapeutic target.

Heat shock protein 70 inhibitors suppress androgen receptor expression in LNCaP95 prostate cancer cells

Heat shock protein 70 inhibitors suppress androgen receptor expression in LNCaP95 prostate cancer cells

Heat shock protein 70 inhibitors suppress androgen receptor expression in LNCaP95 prostate cancer cells.

Androgen deprivation therapy is initially effective for treating patients with advanced prostate cancer; however, the prostate cancer gradually becomes resistant to androgen deprivation therapy, which is termed castration‐resistant prostate cancer (CRPC). Androgen receptor splice variant 7 (AR‐V7), one of the causes of CRPC, is correlated with resistance to a new‐generation AR antagonist (enzalutamide) and poor prognosis. Heat shock protein 70 (Hsp70) inhibitor is known to decrease the levels of full‐length AR (AR‐FL), but little is known about its effects against CRPC cells expressing AR‐V7. In this study, we investigated the effect of the Hsp70 inhibitors quercetin and VER155008 in the prostate cancer cell line LNCaP95 that expresses AR‐V7, and explored the mechanism by which Hsp70 regulates AR‐FL and AR‐V7 expression. Quercetin and VER155008 decreased cell proliferation, increased the proportion of apoptotic cells, and decreased the protein levels of AR‐FL and AR‐V7. Furthermore, VER155008 decreased AR‐FL and AR‐V7 mRNA levels. Immunoprecipitation with Hsp70 antibody and mass spectrometry identified Y‐box binding protein 1 (YB‐1) as one of the molecules regulating AR‐FL and AR‐V7 at the transcription level through interaction with Hsp70. VER155008 decreased the phosphorylation of YB‐1 and its localization in the nucleus, indicating that the involvement of Hsp70 in AR regulation might be mediated through the activation and nuclear translocation of YB‐1. Collectively, these results suggest that Hsp70 inhibitors have potential anti‐tumor activity against CRPC by decreasing AR‐FL and AR‐V7 expression through YB‐1 suppression.

Abstract LB-268: Androgen signaling in cancer-associated fibroblasts contributes to progression of prostate cancer

Abstract Interactions between epithelial and stromal cells are important in the development of prostate cancer (PCa). Cancer-associated fibroblasts (CAFs) have been to support tumor progression, metastasis, and differentiation. Androgen receptor (AR) and related pathways are known to support the growth and survival of prostate epithelial cancer cells, the roles of AR-dependent processes in cancerous stroma are less clear. We sought to investigate if AR-dependent pathways present in CAF cells influence the growth and tumorogencity of epithelial cancer cells in relation to androgen-deprivation therapy in prostate cancer. Murine CAFs were isolated from a well-described PTEN-dependent cancer mouse model. A co-culture system was developed based on multiple lines of murine CAFs grown along with human prostate cancer epithelial cells, and a murine-specific anti-sense oligonucleotide (ASO) against murine AR was used to specifically suppress AR expression in murine CAFs in this system. Using this co-culture system, we found that murine CAFs promoted cell proliferation and colony formation in several human prostate cancer cell lines. Further, these processes were decreased by suppression of AR-expression in CAFs. Expression of genes related to tumorogenicity in epithelial cells were investigated by real-time quantitative PCR. Markers associated with epithelial-mesenchymal transition (EMT) and stemness were increased in human prostate cancer cells grown with low-AR CAFs. Our data indicates that suppression of AR in CAFs results in down-regulation in the growth and tumorogenecity of prostate cancer cells through pathways related to EMT and “cell reprograming”. As such, development of therapies which inhibit the tumor-promoting pathways present in stromal cells may be one approach to improve the treatment of prostate cancer. Citation Format: Chun-Peng Liao, Leng-Ying Chen, Andrea Luethy, Youngsoo Kim, Robert MacLeod, Mitchell Gross. Androgen signaling in cancer-associated fibroblasts contributes to progression of 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 LB-268.

Abstract 2990: Sulforaphane enhances the anti-cancer efficacy of anti-androgens in prostate cancer cells (LNCaP and C4-2B) by increasing androgen receptor (AR) degradation

Abstract Introduction: Prostate Cancer (PC) cells utilize androgen for their growth. Therefore, androgen deprivation therapy (ADT) that blocks both systemic androgen production and androgen receptor (AR) signaling remains the mainstay of PC treatment. However, despite the efficacy of AR antagonists such as bicalutamide (BIC) and enzalutamide (ENZ), their sub-therapeutic efficacy in tumor microenvironments enable the selection and outgrowth of castration resistant PC (CRPC). Interestingly, CRPC cells continue to express AR and exploit the intratumoral androgen. Hence, alternate strategies to suppress AR expression in PC cells will be needed to enhance the efficacy of BIC and ENZ. The phytochemical, sulforaphane (SFN) is known to decrease AR protein levels. We hypothesize that co-exposure to SFN will increase the anti-cancer efficacy of BIC/ENZ. Methods: The AR expressing PC lines, both androgen-dependent (AD) LNCaP cells and androgen-independent CRPC cells (C4-2B), were used to investigate the effects of BIC or ENZ, alone and in combination with SFN. The following cellular parameters were evaluated: (a) cell proliferation by MTT-assay; (b) clonogenic ability by colony forming unit (CFU); (c) AR expression by immunoblot analysis; (d) subcellular AR localization by immunofluorescence microscropy (IFM); (e) AR and prostate specific antigen (PSA) gene expression by qRT-PCR; and (e) PSA protein secretion by ELISA. Studies were carried out under hormone deprived (charcoal-stripped FBS) conditions and/or in presence of AR agonist, R1881. Results: Co-exposure to SFN (10μM) significantly (p<0.05) enhanced the anti-proliferative effects of BIC (50μM) or ENZ (20μM) at 48 h post treatment, and suppressed R1881 stimulated PC growth, as well. At much lower concentrations, SFN (0.2μM) co-exposure enhanced the suppressive effects of BIC (0.5μM) or ENZ (0.2μM) on both the number and size of CFUs at 14 days. Western blot analysis revealed that SFN decreases AR protein levels in a time- and dose-dependent manner. IFM analysis indicated that SFN suppresses R1881-stimulated AR nuclear localization. Cycloheximide treatment suggested that SFN's effect on AR is primarily regulated at the translational level. Experiments with MG-132, a proteasomal inhibitor indicated that the SFN-induced AR degradation is partially regulated by the proteasomal pathway. Studies with qRT-PCR showed that SFN enhances the efficacy of BIC in suppressing PSA mRNA levels and decreased PSA protein secretion was also corroborated by ELISA. Conclusion: Sulforaphane decreases AR levels in both LNCaP and C4-2B cells to increase the anti-cancer efficacy of AR antagonists in a synergistic manner. This implies that adjuvant therapy with this safe phytochemical may be very promising. Citation Format: Namrata Khurana, Sudha Talwar, Partha Chandra, Asim B. Abdel-Mageed, Debasis Mondal, Suresh Sikka. Sulforaphane enhances the anti-cancer efficacy of anti-androgens in prostate cancer cells (LNCaP and C4-2B) by increasing androgen receptor (AR) degradation. [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 2990.

Cisplatin enhances NK cells immunotherapy efficacy to suppress HCC progression via altering the androgen receptor (AR)-ULBP2 signals

The aim of this study is to investigate the influence of cisplatin on the efficacy of natural killer (NK) cells immunotherapy to suppress HCC progression, and provide valuable information on better application of cisplatin in clinical settings. By using in vitro cell cytotoxicity test and in vivo liver orthotopic xenograft mice model, we identified the role of cisplatin in modulating NK cells cytotoxicity. Luciferase report assay and chromatin immunoprecipitation assay were applied for mechanism dissection. Immunohistochemistry is performed for sample staining. We found cisplatin could enhance the efficacy of NK cells immunotherapy to better suppress HCC progression via altering the androgen receptor (AR)-UL16-binding protein 2 (ULBP2) signals both in vitro and in vivo. Mechanism dissection revealed that cisplatin could suppress AR expression via two distinct ways: increasing miR-34a-5p to suppress AR expression and altering the ubiquitination to accelerate the AR protein degradation. The suppressed AR might then function through up-regulating ULBP2, a natural-killer group 2 member D ligand, to enhance the cytotoxicity of NK cells. Together, these results indicated an unrecognized favoring effect of cisplatin in HCC treatment. By suppressing AR in HCC, cisplatin could up-regulate cytotoxicity of NK cells to better target HCC. This finding may provide a potential new approach to control HCC by combining traditional chemotherapy with immunotherapy.

Effect of androgen signaling in stromal cells on growth of prostate cancer.

297 Background: Interactions between epithelial and stroma cells are important in the development of prostate cancer (PCa). Cancer-associated fibroblasts (CAFs) have been to support tumor progression, metastasis, and differentiation. Androgen receptor (AR) and related pathways are known to support the growth and survival of prostate epithelial cancer cells, the roles of AR-dependent processes in cancerous stroma are less clear. We sought to investigate if AR-dependent pathways present in CAF cells influence the growth and tumorogencity of epithelial cancer cells in relation to androgen-deprivation therapy in prostate cancer. Methods: Murine CAFs were isolated from a well-described PTEN-dependent cancer mouse model (Liao, et al Cancer Res, 2010. 70(18):7294). A co-culture system was developed based on multiple lines of murine CAFs grown along with human prostate cancer epithelial cells, and a murine-specific anti-sense oligonucleotide (ASO) against murine AR was used to specifically suppress AR expression in murine CAFs in this system. RT-PCR was used to investigate changes in gene expression. Results: Using this co-culture system, we found that murine CAFs promoted cell proliferation and colony formation in several human prostate cancer cell lines. Further, these processes were decreased by suppression of AR-expression in CAFs. Expression of genes related to tumorigenicity in epithelial cells were investigated. Markers associated with epithelial-mesenchymal transition (EMT, N-Cad) and “stemness” (OCT4, Sox2, Nanog) were increased in human prostate cancer cells grown with low-AR CAFs. Conclusions: Our data indicates that suppression of AR in CAFs results in down-regulation in the growth and tumorigenicity of prostate cancer cells through pathways related to EMT and “cell reprograming”. As such, development of therapies which inhibit the tumor-promoting pathways present in stromal cells may be one approach to improve the treatment of prostate cancer.

Abstract 675: Dual inhibition of the androgen receptor and PI-3K/mTor pathways has significant antitumor activity in castrate-resistant prostate cancer

Abstract Inhibition of the androgen receptor (AR) results in activation of the PI-3K/mTOR pathway in PTEN deficient prostate cancer cells by reciprocal feedback inhibition (Carver BS, 2011). Enzalutamide or PI-3K/mTor inhibition result in feedback activation and combined inhibition abrogated this effect. We explored AR inhibition in CRPC cell lines with low, intermediate and high AR expression in the context of PI-3K/mTor inhibition to identify biomarkers of response and resistance. We utilized no/low (DU145, PC3), intermediate (LNCaP) and high (22Rv1,VCap) AR expressing prostate cancer (PC) cell lines to evaluate anti-tumor activity with 4 pan-PI3K/mTOR inhibitors (GDC-0980, GDC-0941, LY294002, PF-04691502), enzalutamide and abiraterone as single agents or various in combinations at clinically relevant doses. Cytotoxic activity of enzalutamide was more potent than abiraterone (IC50's was < 1µM and > 10µM) in LNCaP cells, however, the opposite was seen in DU145 and PC-3 cell lines. High AR expressing cells (22Rv1, VCap) were more sensitive to AR inhibition. PI-3K/mTor SMIs GDC-0980, GDC-0941 or PF-04691502 had IC50's in the range 50-500nM while LY294002 had an IC50 16-24 µM except for DU145. This cell line had increased IC50 values comparatively for PI-3K/mTor SMIs GDC-0980, GDC-0941 or PF-04691502 (1.5-3.75 µM) and LY29002 (48.5 µM). The combination index (CI) isobologram method indicated significant synergism for PI-3K/mTOR SMIs plus enzalutamide or abiraterone in all PC cell lines irrespective of AR status. Combination treatments were associated with increased apoptosis (Annexin-V staining and PARP-cleavage assay) in LNCaP and VCaP > 22Rv1 > DU145 and PC3. Cell cycle analysis by flow cytometry demonstrated that enzalutamide induces G1 arrest in LNCaP and DU145 cells and G2/M arrest in PC3 cells. PI-3K inhibition induces S arrest in LNCaP and DU145 and G2/M arrest in PC3 cells. The combination of enzalutamide plus PI-3K inhibition enhanced S arrest in LNCaP while in DU145 and PC3, G0/G1 arrest was prominent. PI-3K SMI plus enzalutamide completely inhibited pAkt308/473, pS6 and p4EBP1 in LNCaP cells, pAkt473 in PC3 cells and pS6 and p4EBP1 in 22Rv1 cells. Enzalutamide alone or in combination with PI-3K inhibition also suppressed AR expression in 22Rv1 cells. A 22Rv1 mouse xenograft model is evaluating safety and efficacy of the combination. This is a novel therapeutic strategy for CRPC patients to be evaluated in a clinical trial. Citation Format: Daruka Mahadevan, Carla Morales, Laurence S. Cooke, Bradley Somer, Wenqing Qi. Dual inhibition of the androgen receptor and PI-3K/mTor pathways has significant antitumor activity in castrate-resistant prostate cancer. [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 675. doi:10.1158/1538-7445.AM2014-675

Epigenetic Repression of miR-31 Disrupts Androgen Receptor Homeostasis and Contributes to Prostate Cancer Progression

Androgen receptor signaling plays a critical role in prostate cancer pathogenesis. Yet, the regulation of androgen receptor signaling remains elusive. Even with stringent androgen deprivation therapy, androgen receptor signaling persists. Here, our data suggest that there is a complex interaction between the expression of the tumor suppressor miRNA, miR-31, and androgen receptor signaling. We examined primary and metastatic prostate cancer and found that miR-31 expression was reduced as a result of promoter hypermethylation, and importantly, the levels of miR-31 expression were inversely correlated with the aggressiveness of the disease. As the expression of androgen receptor and miR-31 was inversely correlated in the cell lines, our study further suggested that miR-31 and androgen receptor could mutually repress each other. Upregulation of miR-31 effectively suppressed androgen receptor expression through multiple mechanisms and inhibited prostate cancer growth in vivo. Notably, we found that miR-31 targeted androgen receptor directly at a site located in the coding region, which was commonly mutated in prostate cancer. In addition, miR-31 suppressed cell-cycle regulators including E2F1, E2F2, EXO1, FOXM1, and MCM2. Together, our findings suggest a novel androgen receptor regulatory mechanism mediated through miR-31 expression. The downregulation of miR-31 may disrupt cellular homeostasis and contribute to the evolution and progression of prostate cancer. We provide implications for epigenetic treatment and support clinical development of detecting miR-31 promoter methylation as a novel biomarker.