Regulation Of Androgen Receptor Activity Research Articles

MED12 and CDK8/19 Modulate Androgen Receptor Activity and Enzalutamide Response in Prostate Cancer.

Prostate cancer progression is driven by androgen receptor (AR) activity, which is a target for therapeutic approaches. Enzalutamide is an AR inhibitor that prolongs the survival of patients with advanced prostate cancer. However, resistance mechanisms arise and impair its efficacy. One of these mechanisms is the expression of AR-V7, a constitutively active AR splice variant. The Mediator complex is a multisubunit protein that modulates gene expression on a genome-wide scale. MED12 and cyclin-dependent kinase (CDK)8, or its paralog CDK19, are components of the kinase module that regulates the proliferation of prostate cancer cells. In this study, we investigated how MED12 and CDK8/19 influence cancer-driven processes in prostate cancer cell lines, focusing on AR activity and the enzalutamide response. We inhibited MED12 expression and CDK8/19 activity in LNCaP (AR+, enzalutamide-sensitive), 22Rv1 (AR-V7+, enzalutamide-resistant), and PC3 (AR-, enzalutamide-insensitive) cells. Both MED12 and CDK8/19 inhibition reduced cell proliferation in all cell lines, and MED12 inhibition reduced proliferation in the respective 3D spheroids. MED12 knockdown significantly inhibited c-Myc protein expression and signaling pathways. In 22Rv1 cells, it consistently inhibited the AR response, prostate-specific antigen (PSA) secretion, AR target genes, and AR-V7 expression. Combined with enzalutamide, MED12 inhibition additively decreased the AR activity in both LNCaP and 22Rv1 cells. CDK8/19 inhibition significantly decreased PSA secretion in LNCaP and 22Rv1 cells and, when combined with enzalutamide, additively reduced proliferation in 22Rv1 cells. Our study revealed that MED12 and CDK8/19 regulate AR activity and that their inhibition may modulate response to enzalutamide in prostate cancer.

Casein Kinase 1α-A Target for Prostate Cancer Therapy?

The androgen receptor (AR) is a key driver of prostate cancer (PCa) and, as such, current mainstay treatments target this molecule. However, resistance commonly arises to these therapies and, therefore, additional targets must be evaluated to improve patient outcomes. Consequently, alternative approaches for indirectly targeting the AR are sought. AR crosstalk with other signalling pathways, including several protein kinase signalling cascades, has been identified as a potential route to combat therapy resistance. The casein kinase 1 (CK1) family of protein kinases phosphorylate a multitude of substrates, allowing them to regulate a diverse range of pathways from the cell cycle to DNA damage repair. As well as its role in several signalling pathways that are de-regulated in PCa, mutational data suggest its potential to promote prostate carcinogenesis. CK1α is one isoform predicted to regulate AR activity via phosphorylation and has been implicated in the progression of several other cancer types. In this review, we explore how the normal biological function of CK1 is de-regulated in cancer, the impact on signalling pathways and how this contributes towards prostate tumourigenesis, with a particular focus on the CK1α isoform as a novel therapeutic target for PCa.

A carboxy-terminal ubiquitylation site regulates androgen receptor activity

Degradation of unliganded androgen receptor (AR) in prostate cancer cells can be prevented by proteasome inhibition, but this is associated with only modest increases in polyubiquitylated AR. An inhibitor (VLX1570) of the deubiquitylases associated with the proteasome did not increase ubiquitylation of unliganded AR, indicating that AR is not targeted by these deubiquitylases. We then identified a series of AR ubiquitylation sites, including a not previously identified site at K911, as well as methylation sites and previously identified phosphorylation sites. Mutagenesis of K911 increases AR stability, chromatin binding, and transcriptional activity. We further found that K313, a previously reported ubiquitylation site, could also be methylated and acetylated. Mutagenesis of K313, in combination with K318, increases AR transcriptional activity, indicating that distinct posttranslational modifications at K313 differentially regulate AR activity. Together these studies expand the spectrum of AR posttranslational modifications, and indicate that the K911 site may regulate AR turnover on chromatin.

THU550 The Splicing Factor SRSF6 Regulates Androgen Receptor Activity And Represents A Potential Therapeutic Target In Prostate Cancer

Abstract Disclosure: R.M. Luque: None. A.J. Montero-Hidalgo: None. E. Gomez-Gomez: None. D. Olmos: None. M.D. Gahete: None. J.M. Jimenez-Vacas: None. Background: Prostate cancer (PCa) is the fifth leading cause of cancer-related death worldwide. Finding novel therapeutic strategies to tackle PCa, especially its most advanced phenotype [i.e., castration-resistant prostate cancer (CRPC)], is urgently needed. In this sense, although the dysregulation of the splicing process has emerged as a distinctive feature of advanced PCa, the potential role that splicing regulators may play in this disease remain poorly understood. In this study, we aimed to analyse the levels, pathophysiological role and associated molecular landscape of the splicing factor SRSF6 in PCa. Methods: SRSF6 levels (mRNA, protein and CNA) were interrogated in seven well-characterized cohorts of PCa patients and in the Hi-MYC transgenic model. The effect of SRSF6 overexpression and silencing was tested in vitro (proliferation, migration, colony- and tumorspheres-formation) and in vivo (tumor growth of xenograft tumours). RNAseq was performed in 22Rv1 cells to analyse splicing and gene expression changes in response to SRSF6 silencing. Findings: Our results showed that SRSF6 levels (mRNA/protein) are upregulated in PCa vs non-tumour prostate samples, linked to clinical parameters of tumour-aggressiveness (e.g., Gleason score, T-stage, perineural infiltration, metastasis at diagnosis), and associated with poor prognosis (i.e., shorter disease-free survival time) in PCa patients. Moreover, SRSF6-overexpression increased, while its silencing decreased, functional parameters of aggressiveness in vitro and tumour growth in vivo. Mechanistically, SRSF6 modulation resulted in dysregulation of Androgen-Receptor (AR) activity through transcriptional control of APPBP2 and TOP2B. Discussion: SRSF6 could represent a new therapeutic target to inhibit persistent AR-signalling in advanced PCa. Funding: Spanish Ministry of Science, Innovation, and Universities (PID2019-105564RB-I00, FPU18/02485), Health Institute Carlos III (DTS20-00050), and CIBERobn. Presentation: Thursday, June 15, 2023

Abstract B066: Genome-wide CRISPR screens identify PTGES3 as a druggable AR modulator

Abstract BACKGROUND The androgen receptor (AR) is the central therapeutic target in advanced prostate cancer (PCa). While treatments that directly target AR are effective in prolonging patient survival, aggressive prostate cancers invariably find a way to evade these therapies. Frequently, evasion mechanisms reestablish AR expression and therefore AR-driven oncogenic pathways are restored. Comprehensively understanding the protein regulation machinery of AR and identifying alternative therapeutic targets are crucial for effective treatment of this disease. METHODS To study regulators of AR, we utilized CRISPR-Cas9 technology to knock-in a split neon green fluorescent protein onto AR in prostate cancer cells. We evaluated the AR expression and activity in these AR reporter cell lines by multiple approaches including ARE-luciferase assay, ChIP, and RNA-seq. We further built a unique loss-of-function CRISPRi screening system in this AR reporter cell line where we performed a genome-wide fluorescence-activated cell sorting based screen and functionally interrogated hits that modulate AR protein levels. RESULTS We established the first prostate cancer cell line models harboring an endogenous AR reporter. Extensive genotypic and phenotypic characterization of this system demonstrated that the reporter is effective and does not alter AR activity. These models have enabled us to precisely measure the dynamic endogenous AR protein levels by its fluorescence signal. By utilizing a genome-wide CRISPRi screening method in this system, we identified classic AR regulators such as HOXB13, GATA2 and GRHL2, as well as many novel genes that have not been characterized in the context of prostate cancer. Among these novel hits, Prostaglandin E synthase 3 (PTGES3) is the highest ranked druggable target. We validated this finding by establishing PTGES3 knockdown cell lines and demonstrated that loss of PTGES3 downregulated AR and its downstream genes. Follow-up studies show that beyond the canonical chaperone function, PTGES3 physically interacts with AR in the nucleus, where it promotes AR activity. PTGES3 is required for cell proliferation in vitro and in vivo in multiple AR-driven PCa models but not in AR-independent PCa models, such as PC3 cells. From our preliminary data, this unique interaction further suggests that high tumor PTGES3 expression is associated with resistance to first-line androgen deprivation therapies in a retrospective clinical cohort. These results collectively confirm that PTGES3 regulates AR activity and is a potential therapeutic target. Lastly, using a disulfide tethering fragment screen, we developed a PTGES3 inhibitor that blocks the PTGES3/AR interaction and represses AR signaling in PCa cells. We aim to further optimize this PTGES3-specific inhibitor as a therapeutic alternative for patients with aggressive prostate cancer. CONCLUSIONS Together, these genetic, biochemical and chemical biology experiments point to a new class of AR-directed PTGES3 inhibitor therapies that may overcome known mechanisms of ARSI resistance in mCRPC. Citation Format: Haolong Li, James E. Melnyk, Becky Xu Hua Fu, Siyu Feng, Martin Sjöström, Raunak Shrestha, Meng Zhang, Lisa N. Chesner, Hyun Jin Shin, Marsha Calvert, Jonathan Chou, Rajdeep Das, Emily A. Egusa, Aidan Winters, Jun Zhu, Ashutosh Maheshwari, Junjie T. Hua, Mohammed Alshalalfa, William S. Chen, Bradley A. Stohr, Javed Siddiqui, Bo Huang, Eric J. Small, David A. Quigley, Kevan M. Shokat, Luke A. Gilbert, Felix Y. Feng. Genome-wide CRISPR screens identify PTGES3 as a druggable AR modulator [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 B066.

Lysine Acetyltransferases and Their Role in AR Signaling and Prostate Cancer.

The development and growth of a normal prostate gland, as well as its physiological functions, are regulated by the actions of androgens through androgen receptor (AR) signaling which drives multiple cellular processes including transcription, cellular proliferation, and apoptosis in prostate cells. Post-translational regulation of AR plays a vital role in directing its cellular activities via modulating its stability, nuclear localization, and transcriptional activity. Among various post-translational modifications (PTMs), acetylation is an essential PTM recognized in AR and is governed by the regulated actions of acetyltransferases and deacetyltransferases. Acetylation of AR has been identified as a critical step for its activation and depending on the site of acetylation, the intracellular dynamics and activity of the AR can be modulated. Various acetyltransferases such as CBP, p300, PCAF, TIP60, and ARD1 that are known to acetylate AR, may directly coactivate the AR transcriptional function or help to recruit additional coactivators to functionally regulate the transcriptional activity of the AR. Aberrant expression of acetyltransferases and their deregulated activities have been found to interfere with AR signaling and play a key role in development and progression of prostatic diseases, including prostate cancer (PCa). In this review, we summarized recent research advances aimed at understanding the role of various lysine acetyltransferases (KATs) in the regulation of AR activity at the level of post-translational modifications in normal prostate physiology, as well as in development and progression of PCa. Considering the critical importance of KATs in modulating AR activity in physiological and patho-physiological context, we further discussed the potential of targeting these enzymes as a therapeutic option to treat AR-related pathology in combination with hormonal therapy.

The Role of Heat Shock Protein 70 Subfamily in the Hyperplastic Prostate: From Molecular Mechanisms to Therapeutic Opportunities.

Benign prostatic hyperplasia (BPH) is one of the most common causes of lower urinary tract symptoms (LUTS) in men, which is characterized by a noncancerous enlargement of the prostate. BPH troubles the vast majority of aging men worldwide; however, the pathogenetic factors of BPH have not been completely identified. The heat shock protein 70 (HSP70) subfamily, which mainly includes HSP70, glucose-regulated protein 78 (GRP78) and GRP75, plays a crucial role in maintaining cellular homeostasis. HSP70s are overexpressed in the course of BPH and involved in a variety of biological processes, such as cell survival and proliferation, cell apoptosis, epithelial/mesenchymal transition (EMT) and fibrosis, contributing to the development and progress of prostate diseases. These chaperone proteins also participate in oxidative stress, a cellular stress response that takes place under stress conditions. In addition, HSP70s can bind to the androgen receptor (AR) and act as a regulator of AR activity. This interaction of HSP70s with AR provides insight into the importance of the HSP70 chaperone family in BPH pathogenesis. In this review, we discuss the function of the HSP70 family in prostate glands and the role of HSP70s in the course of BPH. We also review the potential applications of HSP70s as biomarkers of prostate diseases for targeted therapies.

Poly-glutamine-dependent self-association as a potential mechanism for regulation of androgen receptor activity.

The androgen receptor (AR) plays a central role in prostate cancer. Development of castration resistant prostate cancer (CRPC) requires androgen-independent activation of AR, which involves its large N-terminal domain (NTD) and entails extensive epigenetic changes depending in part on histone lysine demethylases (KDMs) that interact with AR. The AR-NTD is rich in low-complexity sequences, including a polyQ repeat. Longer polyQ sequences were reported to decrease transcriptional activity and to protect against prostate cancer, although they can lead to muscular atrophy. However, the molecular mechanisms underlying these observations are unclear. Using NMR spectroscopy, here we identify weak interactions between the AR-NTD and the KDM4A catalytic domain, and between the AR ligand-binding domain and a central KDM4A region that also contains low-complexity sequences. We also show that the AR-NTD can undergo liquid-liquid phase separation in vitro, with longer polyQ sequences phase separating more readily. Moreover, longer polyQ sequences hinder nuclear localization in the absence of hormone and increase the propensity for formation of AR-containing puncta in the nucleus of cells treated with dihydrotestosterone. These results lead us to hypothesize that polyQ-dependent liquid-liquid phase separation may provide a mechanism to decrease the transcriptional activity of AR, potentially opening new opportunities to design effective therapies against CRPC and muscular atrophy.

Enzalutamide-induced Proteolytic Degradation of the Androgen Receptor in Prostate Cancer Cells Is Mediated Only to a Limited Extent by the Proteasome System.

Androgen receptor (AR) degradation is the primary regulator of androgen receptor activity. This study was designed to investigate the influence of the proteasome on AR protein stability after enzalutamide (Enz) treatment. Cell counting after treatment was utilized to assess the effect of Enz on cell proliferation. Changes in mRNA levels were evaluated using reverse transcription-polymerase chain reaction (RT-PCR). Proteasome activity was assessed by measurement of the chymotrypsin-like activity of the beta-5 subunit of the proteasome. Changes in protein levels after treatment with Enz, MG132 (MG), bortezomib (Bor), or their combination were assessed using western blot analysis. Treatment with Enz led to a significant reduction of cell proliferation and AR protein levels. However, AR mRNA levels were unchanged. Inhibition of proteasome activity by MG counteracts the Enz-mediated AR degradation transiently, whereas Bor showed no inhibition of the Enz-mediated AR degradation. Enz-mediated change in AR stability as an early and essential event after treatment was shown. However, investigations of the ubiquitin/proteasome system indicate involvement of several proteases in the Enz-mediated AR degradation process.

FKBP51 and FKBP52 regulate androgen receptor dimerization and proliferation in prostate cancer cells.

The growth of prostate cancer is dependent on the androgen receptor (AR), which serves as a ligand‐specific transcription factor. Although two immunophilins, FKBP51 and FKBP52, are known to regulate AR activity, the precise mechanism remains unclear. We found that depletion of either FKBP51 or FKBP52 reduced AR dimer formation, chromatin binding, and phosphorylation, suggesting defective AR signaling. Furthermore, the peptidyl‐prolyl cis/trans isomerase activity of FKBP51 was found to be required for AR dimer formation and cancer cell growth. Treatment of prostate cancer cells with FK506, which binds to the FK1 domain of FKBPs, or with MJC13, an inhibitor of FKBP52–AR signaling, also inhibited AR dimer formation. Finally, elevated expression of FKBP52 was associated with a higher rate of prostate‐specific antigen recurrence in patients with prostate cancer. Collectively, these results suggest that FKBP51 and FKBP52 might be promising targets for prostate cancer treatment through the inhibition of AR dimer formation.

Proliferating cell nuclear antigen directly interacts with androgen receptor and enhances androgen receptor‑mediated signaling.

Androgen receptor (AR) and/or its constitutively active splicing variants (AR‑Vs), such as AR‑V7 and ARv567es, is required for prostate cancer cell growth and survival, and cancer progression. Proliferating cell nuclear antigen (PCNA) is preferentially overexpressed in all cancers and executes its functions through interaction with numerous partner proteins. The aim of the present study was to investigate the potential role of PCNA in the regulation of AR activity. An identical consensus sequence of the PCNA‑interacting protein‑box (PIP‑box) was identified at the N‑terminus of human, mouse and rat AR proteins. It was found that PCNA complexes with the full‑length AR (AR‑FL) and AR‑V7, which can be attenuated by the small molecule PIP‑box inhibitor, T2AA. PCNA also complexes with ARv567es and recombinant AR protein. The PCNA inhibitors, PCNA‑I1S and T2AA, inhibited AR transcriptional activity and the expression of AR target genes in LNCaP‑AI and 22Rv1 cells, but not in AR‑negative PC‑3 cells. The knockdown of PCNA expression reduced dihydrotestosterone‑stimulated AR transcriptional activity and abolished the inhibitory effect of PCNA‑I1S on AR activity. The PCNA inhibitor, PCNA‑I1, exerted additive growth inhibitory effects with androgen deprivation and enzalutamide in cells expressing AR‑FL or AR‑FL/AR‑V7, but not in AR‑negative PC‑3 cells. Finally, R9‑AR‑PIP, a small peptide mimicking AR PIP‑box, was found to bind to GFP‑PCNA at Kd of 2.73µM and inhibit the expression of AR target genes, AR transcriptional activity and the growth of AR‑expressing cells. On the whole, these data strongly suggest that AR is a PCNA partner protein and interacts with PCNA via the PIP‑box and that targeting the PCNA‑AR interaction may represent an innovative and selective therapeutic strategy against prostate cancer, particularly castration‑resistant prostate cancers overexpressing constitutively active AR‑Vs.

Epithelial Transcription Factor FOXA1 Regulates Prostate Cancer Immune Response

Background: While localized prostate cancer (PCa) can be mitigated by surgery and radiation, metastatic PCa remains a challenge to treat. Androgen deprivation therapies and androgen receptor (AR) pathway inhibitors are mainstay treatments for advanced PCa. Yet, resistance often develops leading to castration-resistant prostate cancer (CRPC). Forkhead Box A1 (FOXA1) is a pioneer transcription factor that plays pivotal roles in regulating AR activity and promoting epithelial differentiation. Studies have shown that FOXA1 is frequently downregulated in CRPC tumors. Congruently, FOXA1 loss is reported to induce aberrant AR signaling, epithelial-mesenchymal transition, and PCa de-differentiation. However, the role of FOXA1 in regulating PCa immune response, an area of much interest recently, has not been reported. CRPC has shown poor response to immune checkpoint inhibitors, due to its immunosuppressive nature. A better understanding of the tumor intrinsic mechanisms regulating PCa tumor immunity will inform the design of better targeted immunotherapeutic approaches. Methods: We performed RNA-seq, ChIP-seq, qPCR, western blot, and ELISA analyses to evaluate how FOXA1 regulates inflammatory response genes. We utilized an in vitro macrophage infiltration transwell assay, in which M2-like macrophages were added to the upper chamber and PCa cells were plated in the lower chamber, to examine how perturbations to PCa cells affect macrophage migration. Finally, we performed bioinformatic analyses of patient datasets to confirm the clinical relevance of FOXA1 repression of inflammatory genes in PCa. Results: Through integration of RNA-seq and ChIP-seq data, we uncovered a novel function of FOXA1 in suppressing inflammatory response pathways. In accordance, patient data analyses revealed that inflammatory response genes were upregulated in FOXA1-low PCa tumors. Mechanistically, we showed that FOXA1 proteins bound an intragenic enhancer of Hypoxia-inducible factor 1-alpha (HIF1A) gene to directly repress its expression, such that FOXA1 loss induced HIF1A upregulation. We further showed that Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) became upregulated upon FOXA1 depletion in a HIF1A-dependent manner. This led to infiltration by immunosuppressive, tumor promoting M2-like macrophages. Inhibiting this HIF1A-CCL2 axis with a HIF1A inhibitor or CCL2 neutralizing antibody blocked macrophage infiltration. Future studies using immunocompetent mouse models are needed to confirm the effect of FOXA1 on macrophage infiltration in vivo and evaluate the preclinical potential of targeting the FOXA1-HIF1A-CCL2 axis in CRPC. Conclusion: This study proposes a novel role for FOXA1 loss in promoting macrophage infiltration via the HIF1A-CCL2 axis. Moreover, our findings suggest that targeting this axis may be a promising approach for the treatment of FOXA1-low CRPC tumors.

Targeting the p300/CBP Axis in Lethal Prostate Cancer.

Resistance to androgen receptor (AR) blockade in castration-resistant prostate cancer (CRPC) is associated with sustained AR signaling, including through alternative splicing of AR (AR-SV). Inhibitors of transcriptional coactivators that regulate AR activity, including the paralog histone acetyltransferase proteins p300 and CBP, are attractive therapeutic targets for lethal prostate cancer. Herein, we validate targeting p300/CBP as a therapeutic strategy for lethal prostate cancer and describe CCS1477, a novel small-molecule inhibitor of the p300/CBP conserved bromodomain. We show that CCS1477 inhibits cell proliferation in prostate cancer cell lines and decreases AR- and C-MYC-regulated gene expression. In AR-SV-driven models, CCS1477 has antitumor activity, regulating AR and C-MYC signaling. Early clinical studies suggest that CCS1477 modulates KLK3 blood levels and regulates CRPC biopsy biomarker expression. Overall, CCS1477 shows promise for the treatment of patients with advanced prostate cancer. SIGNIFICANCE: Treating CRPC remains challenging due to persistent AR signaling. Inhibiting transcriptional AR coactivators is an attractive therapeutic strategy. CCS1477, an inhibitor of p300/CBP, inhibits growth and AR activity in CRPC models, and can affect metastatic CRPC target expression in serial clinical biopsies.See related commentary by Rasool et al., p. 1011.This article is highlighted in the In This Issue feature, p. 995.

MLL5, a histone modifying enzyme, regulates androgen receptor activity in prostate cancer cells by recruiting co-regulators, HCF1 and SET1

In prostate cancer, the androgen receptor (AR) transcription factor is a major regulator of cell proliferation and metastasis. To identify new AR regulators, we focused on Mixed lineage leukemia 5 (MLL5), a histone-regulating enzyme, because significantly higher MLL5 expression was detected in prostate cancer tissues than in matching normal tissues. When we expressed shRNAs targeting MLL5 gene in prostate cancer cell line, the growth rate and AR activity were reduced compared to those in control cells, and migration ability of the knockdown cells was reduced significantly. To determine the molecular mechanisms of MLL5 on AR activity, we proved that AR physically interacted with MLL5 and other co-factors, including SET-1 and HCF-1, using an immunoprecipitation method. The chromatin immunoprecipitation analysis showed reduced binding of MLL5, co-factors, and AR enzymes to AR target gene promoters in MLL5 shRNA-expressing cells. Histone H3K4 methylation on the AR target gene promoters was reduced, and H3K9 methylation at the same site was increased in MLL5 knockdown cells. Finally, xenograft tumor formation revealed that reduction of MLL5 in prostate cancer cells retarded tumor growth. Our results thus demonstrate the important role of MLL5 as a new epigenetic regulator of AR in prostate cancer.

Histone Demethylase KDM7A Regulates Androgen Receptor Activity, and Its Chemical Inhibitor TC-E 5002 Overcomes Cisplatin-Resistance in Bladder Cancer Cells.

Histone demethylase KDM7A regulates many biological processes, including differentiation, development, and the growth of several cancer cells. Here, we have focused on the role of KDM7A in bladder cancer cells, especially under drug-resistant conditions. When the KDM7A gene was knocked down, bladder cancer cell lines showed impaired cell growth, increased cell death, and reduced rates of cell migration. Biochemical studies revealed that KDM7A knockdown in the bladder cancer cells repressed the activity of androgen receptor (AR) through epigenetic regulation. When we developed a cisplatin-resistant bladder cancer cell line, we found that AR expression was highly elevated. Upon treatment with TC-E 5002, a chemical inhibitor of KDM7A, the cisplatin-resistant bladder cancer cells, showed decreased cell proliferation. In the mouse xenograft model, KDM7A knockdown or treatment with its inhibitor reduced the growth of the bladder tumor. We also observed the upregulation of KDM7A expression in patients with bladder cancer. The findings suggest that histone demethylase KDM7A mediates the growth of bladder cancer. Moreover, our findings highlight the therapeutic potential of the KMD7A inhibitor, TC-E 5002, in patients with cisplatin-resistant bladder cancer.

Increased nuclear factor I/B expression in prostate cancer correlates with AR expression.

Most prostate cancers express androgen receptor (AR), and our previous studies have focused on identifying transcription factors that modify AR function. We have shown that nuclear factor I/B (NFIB) regulates AR activity in androgen-dependent prostate cancer cells in vitro. However, the status of NFIB in prostate cancer was unknown. We immunostained a tissue microarray including normal, hyperplastic, prostatic intraepithelial neoplasia, primary prostatic adenocarcinoma, and castration-resistant prostate cancer tissue samples for NFIB, AR, and synaptophysin, a marker of neuroendocrine differentiation. We interrogated publically available data sets in cBioPortal to correlate NFIB expression and AR and neuroendocrine prostate cancer (NEPCa) activity scores. We analyzed prostate cancer cell lines for NFIB expression via Western blot analysis and used nuclear and cytoplasmic fractionation to assess where NFIB is localized. We performed co-immunoprecipitation studies to determine if NFIB and AR interact. NFIB increased in the nucleus and cytoplasm of prostate cancer samples versus matched normal controls, independent of Gleason score. Similarly, cytoplasmic AR and synaptophysin increased in primary prostate cancer. We observed strong NFIB staining in primary small cell prostate cancer. The ratio of cytoplasmic-to-nuclear NFIB staining was predictive of earlier biochemical recurrence in prostate cancer, once adjusted for tumor margin status. Cytoplasmic AR was an independent predictor of biochemical recurrence. There was no statistically significant difference between NFIB and synaptophysin expression in primary and castration-resistant prostate cancer, but cytoplasmic AR expression was increased in castration-resistant samples. In primary prostate cancer, nuclear NFIB expression correlated with cytoplasmic NFIB and nuclear AR, while cytoplasmic NFIB correlated with synaptophysin, and nuclear and cytoplasmic AR. In castration-resistant prostate cancer samples, NFIB expression correlated positively with an AR activity score, and negatively with the NEPCa score. In prostate cancer cell lines, NFIB exists in several isoforms. We observed NFIB predominantly in the nuclear fraction of prostate cancer cells with increased cytoplasmic expression seen in castration-resistant cell lines. We observed an interaction between AR and NFIB through co-immunoprecipitation experiments. We have described the expression pattern of NFIB in primary and castration-resistant prostate cancer and its positive correlation with AR. We have also demonstrated AR interacts with NFIB.

SAT-712 Computational Study of the Effect of Androgen Receptor BF 3 Site Mutations on DDE Binding

Exposure to endocrine disrupting chemicals (EDCs) affects the function of the androgen receptor (AR) causing reproductive system problems such as reduced sperm counts, increased infertility, testicular dysgenesis syndrome, and testicular and prostate cancers, as well as reduced bone mass and diabetes mellitus in males. Experimental results have shown that the presence of EDCs such as the diphenyl compound DDT and its analogue DDE, allosterically cause the release of the stably bound dihydrotestosterone (DHT) from the steroid binding site of the AR ligand binding domain. It was hypothesized that EDCs mediate this effect via binding to the Binding Function 3 (BF 3) surface binding site. Five mutations of three BF 3 amino acids (F673K, F673W, G724R, G724M, and L830D) showed that the ability of DDE to inhibit AR activity was reduced, suggesting that DDE binds to the BF 3 site and allosterically regulates AR activity. In this study, the Induced Fit Docking protocol of the Schrodinger software was used to dock DDE into the BF 3 site of the wild type AR ligand binding domain as well as the five mutant BF 3 sites. The docking poses generated for each receptor were clustered and representative structures were selected. The receptor-ligand complexes were energy minimized using the Schrodinger module Macromodel. Finally, the energy of interaction between DDE and the BF 3 site amino acids was evaluated for each of the selected docks of the wild type and mutant receptors. The relationship between the energies of interaction and the experimental results for DDE inhibition of the mutant AR activities will be discussed.

Abstract 3061: Role of ID4 (inhibitor of DNA binding protein 4) in FKBP52-AR pathway

Abstract Introduction: Inhibitor of DNA binding/differentiation protein 4 (ID4), acts as a dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors. In normal prostate epithelial cells, ID4 collaborates with androgen receptor (AR) and p53 to exert its tumor suppressor activity. Previous studies have shown that ID4 promotes tumor suppressive function of AR whereas loss of ID4 results in tumor promoter activity of AR through selective interaction with FKBP52. Pharmacological inhibition of FKBP52, by MJC13, attenuated the tumor growth, weight, and volume of LNCaP cells lacking ID4(L(-)ID4) xenografts. The L(-)ID4 cells also gains CRPC phenotype through FKBP52-mediated AR signaling. In this study we investigated the role of ID4 in pharmacological inhibition of FKBP52 with GMC1 (Initial hit molecule by in-silico screening with the most potent inhibition of FKBP52) in regulating the AR pathway. Experimental procedures: GMC1 drug treatments in LNCaP(-)ID4 and CW22RV1 generated xenograft tumors were further analyzed for tumor volume and tumor weight. We also performed immunohistochemistry on GMC1 treated xenografts to determine AR, PSA, FKBP51 and FKBP52 protein expression. Our in vivo studies with GMC1 drug treatments in castrated male SCID mice injected with LNCaP-ID4 and CW22RV1 cells showed decreased tumor size and volume when compared to untreated mice tumors. Results: IHC results on GMC1 treated LNCaP(-)ID4 and CW22RV1 generated xenograft tumors showed no change in ID4 expression with decrease expression of AR, PSA, FKBP51, FKBP52 and KI67 when compared to untreated xenograft tumors. These results suggest that inhibition of FKBP52-regulated AR activity (via GMC1) demonstrated a significant reduction in the tumor volume of subcutaneous xenografts in vivo. Overall, ID4 appears as a potential tumor suppressor gene that selectively regulates AR activity through direct interaction with FKBP52, and its loss, promotes CRPC through FKBP52-mediated AR signaling. Conclusions: In conclusion, we demonstrate that loss of ID4 in LNCaP cells elevates FKBP52 levels, transcriptionally potentiates AR activity which in turn then leads to progression of androgen-independent prostate cancer. GMC1 is a novel inhibitor that inhibits the binding of FKBP52 to AR thereby prevents the AR transcriptional hyperactivity in PCa Acknowledgement: These studies were supported by DOD, Grant # W81XWH-17-1-0437 Citation Format: Shravan Kumar Komaragiri. Role of ID4 (inhibitor of DNA binding protein 4) in FKBP52-AR pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3061.

PARP-1 regulation of DNA repair factor availability.

269 Background: PARP-1 holds major functions on chromatin, DNA damage repair and transcriptional regulation, both of which are relevant in the context of cancer. Previously, it was determined that PARP-1 ins involved in regulation of androgen receptor activity. Methods: Here, unbiased transcriptional profiling revealed the downstream transcriptional profile of PARP-1 enzymatic activity.Results: Further investigation of the PARP-1-regulated transcriptome and secondary strategies for assessing PARP-1 activity in patient tissues revealed that PARP-1 activity was unexpectedly enriched as a function of disease progression and was associated with poor outcome independent of DNA double-strand breaks, suggesting that enhanced PARP-1 activity may promote aggressive phenotypes. Mechanistic investigation revealed that active PARP-1 served to enhance E2F1 transcription factor activity, and specifically promoted E2F1-mediated induction of DNA repair factors involved in homologous recombination (HR). Conversely, PARP-1 inhibition reduced HR factor availability and thus acted to induce or enhance “BRCA-ness”. Conclusions: These observations bring new understanding of PARP-1 function in cancer and have significant ramifications on predicting PARP-1 inhibitor function in the clinical setting.

Inositol polyphosphate 4-phosphatase type II regulation of androgen receptor activity.

Activation and transcriptional reprogramming of AR in advanced prostate cancer frequently coincides with the loss of two tumor suppressors, INPP4B and PTEN, which are highly expressed in human and mouse prostate epithelium. While regulation of AR signaling by PTEN has been described by multiple groups, it is not known whether the loss of INPP4B affects AR activity. Using prostate cancer cell lines we showed that INPP4B regulates AR transcriptional activity and the oncogenic signaling pathways Akt and PKC. Analysis of gene expression in prostate cancer patient cohorts showed a positive correlation between INPP4B expression and both AR mRNA levels and AR transcriptional output. Using an Inpp4b-/- mouse model, we demonstrated that INPP4B suppresses Akt and PKC signaling pathways and modulates AR transcriptional activity in normal mouse prostate. Remarkably, PTEN protein levels and phosphorylation of S380 were the same in Inpp4b-/- and WT males, suggesting that the observed changes were due exclusively to the loss of INPP4B. Our data show that INPP4B modulates AR activity in normal prostate and its loss contributes to the AR-dependent transcriptional profile in prostate cancer.