Androgen Receptor Occupancy Research Articles

CYCLIN K down-regulation induces androgen receptor gene intronic polyadenylation, variant expression and PARP inhibitor vulnerability in castration-resistant prostate cancer

Androgen receptor (AR) messenger RNA (mRNA) alternative splicing variants (AR-Vs) are implicated in castration-resistant progression of prostate cancer (PCa), although the molecular mechanism underlying the genesis of AR-Vs remains poorly understood. The CDK12 gene is often deleted or mutated in PCa and CDK12 deficiency is known to cause homologous recombination repair gene alteration or BRCAness via alternative polyadenylation (APA). Here, we demonstrate that pharmacological inhibition or genetic inactivation of CDK12 induces AR gene intronic (intron 3) polyadenylation (IPA) usage, AR-V expression, and PCa cell resistance to the antiandrogen enzalutamide (ENZ). We further show that AR binds to the CCNK gene promoter and up-regulates CYCLIN K expression. In contrast, ENZ decreases AR occupancy at the CCNK gene promoter and suppresses CYCLIN K expression. Similar to the effect of the CDK12 inhibitor, CYCLIN K degrader or ENZ treatment promotes AR gene IPA usage, AR-V expression, and ENZ-resistant growth of PCa cells. Importantly, we show that targeting BRCAness induced by CYCLIN K down-regulation with the PARP inhibitor overcomes ENZ resistance. Our findings identify CYCLIN K down-regulation as a key driver of IPA usage, hormonal therapy-induced AR-V expression, and castration resistance in PCa. These results suggest that hormonal therapy-induced AR-V expression and therapy resistance are vulnerable to PARP inhibitor treatment.

UHRF1 promotes androgen receptor-regulated CDC6 transcription and anti-androgen receptor drug resistance in prostate cancer through KDM4C-Mediated chromatin modifications.

The UHRF1 and CDC6, oncogenes play critical roles in therapeutic resistance. In the present study, we found that UHRF1 mediates androgen receptor (AR)-regulated CDC6 transcription in prostate cancer cells. In prostate cancer tissues and cell lines, levels of UHRF1 and CDC6 were simultaneously upregulated, and this was associated with worse survival. UHRF1 silencing significantly promoted the cytotoxicity and anti-prostate cancer efficacy of bicalutamide in mouse xenografts by inhibiting CDC6 gene expression. UHRF1 promoted AR-regulated CDC6 transcription by binding to the CCAAT motif near the androgen response element (ARE) in the CDC6 promoter. We further found that UHRF1 promoted androgen-dependent chromatin occupancy of AR protein by recruiting the H3K9me2/3-specific demethyltransferase KDM4C and modifying the intense heterochromatin status. Altogether, we found for the first time that UHRF1 promotes AR-regulated CDC6 transcription through a novel chromatin modification mechanism and contributes to anti-AR drug resistance in prostate cancer. Targeting AR and UHRF1 simultaneously may be a novel and promising therapeutic modality for prostate cancer.

MED19 alters AR occupancy and gene expression in prostate cancer cells, driving MAOA expression and growth under low androgen.

Androgen deprivation therapy (ADT) is a mainstay of prostate cancer treatment, given the dependence of prostate cells on androgen and the androgen receptor (AR). However, tumors become ADT-resistant, and there is a need to understand the mechanism. One possible mechanism is the upregulation of AR co-regulators, although only a handful have been definitively linked to disease. We previously identified the Mediator subunit MED19 as an AR co-regulator, and reported that MED19 depletion inhibits AR transcriptional activity and growth of androgen-insensitive LNCaP-abl cells. Therefore, we proposed that MED19 upregulation would promote AR activity and drive androgen-independent growth. Here, we show that stable overexpression of MED19 in androgen-dependent LNCaP cells promotes growth under conditions of androgen deprivation. To delineate the mechanism, we determined the MED19 and AR transcriptomes and cistromes in control and MED19-overexpressing LNCaP cells. We also examined genome-wide H3K27 acetylation. MED19 overexpression selectively alters AR occupancy, H3K27 acetylation, and gene expression. Under conditions of androgen deprivation, genes regulated by MED19 correspond to genes regulated by ELK1, a transcription factor that binds the AR N-terminus to induce select AR target gene expression and proliferation, and genomic sites occupied by MED19 and AR are enriched for motifs associated with ELK1. Strikingly, MED19 upregulates expression of monoamine oxidase A (MAOA), a factor that promotes prostate cancer growth. MAOA depletion reduces androgen-independent growth. MED19 and AR occupy the MAOA promoter, with MED19 overexpression enhancing AR occupancy and H3K27 acetylation. Furthermore, MED19 overexpression increases ELK1 occupancy at the MAOA promoter, and ELK1 depletion reduces MAOA expression and androgen-independent growth. This suggests that MED19 cooperates with ELK1 to regulate AR occupancy and H3K27 acetylation at MAOA, upregulating its expression and driving androgen independence in prostate cancer cells. This study provides important insight into the mechanisms of prostate cancer cell growth under low androgen, and underscores the importance of the MED19-MAOA axis in this process.

Serial [18F]-FDHT-PET to predict bicalutamide efficacy in patients with androgen receptor positive metastatic breast cancer

BackgroundThe androgen receptor (AR) is a potential target in metastatic breast cancer (MBC), and 16β-[18F]-fluoro-5α-dihydrotestosterone positron emission tomography ([18F]-FDHT-PET) can be used for noninvasive visualisation of AR. [18F]-FDHT uptake reduction during AR-targeting therapy reflects AR occupancy and might be predictive for treatment response. We assessed the feasibility of [18F]-FDHT-PET to detect changes in AR availability during bicalutamide treatment and correlated these changes with treatment response.Patients and methodsPatients with AR + MBC, regardless of oestrogen receptor status, received an [18F]-FDHT-PET at baseline and after 4–6 weeks bicalutamide treatment. Baseline [18F]-FDHT uptake was expressed as maximum standardised uptake value. Percentage change in tracer uptake, corrected for background activity (SUVcor), between baseline and follow-up PET scan (% reduction), was assessed per-patient and lesion. Clinical benefit was determined in accordance with Response Evaluation Criteria in Solid Tumours (RECIST) 1.1 or clinical evaluation (absence of disease progression for ≥24 weeks).ResultsBaseline [18F]-FDHT-PET in 21 patients detected 341 of 515 lesions found with standard imaging and 21 new lesions. Follow-up [18F]-FDHT-PET was evaluable in 17 patients with 349 lesions, showing a decrease in median SUVcor from 1.3 to 0.7 per-patient and lesion (P < 0.001). Median % reduction per-patient was −45% and per-lesion −39%. In patients with progressive disease (n = 11), median % reduction was −30% versus −53% for patients who showed clinical benefit (in accordance with RECIST (n = 3) or clinical evaluation (n = 3); P = 0.338).ConclusionIn this feasibility study, a bicalutamide-induced reduction in [18F]-FDHT uptake could be detected by follow-up [18F]-FDHT-PET in patients with AR + MBC. However, this change could not predict bicalutamide response.Clinical trial informationNCT02697032.

Darolutamide antagonizes androgen signaling by blocking enhancer and super-enhancer activation.

Prostate cancer (PCa) is one of the most frequent tumor types in the male Western population. Early‐stage PCa and late‐stage PCa are dependent on androgen signaling, and inhibitors of the androgen receptor (AR) axis represent the standard therapy. Here, we studied in detail the global impact of darolutamide, a newly approved AR antagonist, on the transcriptome and AR‐bound cistrome in two PCa cell models. Darolutamide strongly depleted the AR from gene regulatory regions and abolished AR‐driven transcriptional signaling. Enhancer activation was blocked at the chromatin level as evaluated by H3K27 acetylation (H3K27ac), H3K4 monomethylation (H3K4me1), and FOXA1, MED1, and BRD4 binding. We identified genomic regions with high affinities for the AR in androgen‐stimulated, but also in androgen‐depleted conditions. A similar AR affinity pattern was observed in healthy and PCa tissue samples. High FOXA1, BRD4, H3K27ac, and H3K4me1 levels were found to mark regions showing AR binding in the hormone‐depleted setting. Conversely, low FOXA1, BRD4, and H3K27ac levels were observed at regulatory sites that responded strongly to androgen stimulation, and AR interactions at these sites were blocked by darolutamide. Beside marked loss of AR occupancy, FOXA1 recruitment to chromatin was also clearly reduced after darolutamide treatment. We furthermore identified numerous androgen‐regulated super‐enhancers (SEs) that were associated with hallmark androgen and cell proliferation‐associated gene sets. Importantly, these SEs are also active in PCa tissues and sensitive to darolutamide treatment in our models. Our findings demonstrate that darolutamide is a potent AR antagonist blocking genome‐wide AR enhancer and SE activation, and downstream transcription. We also show the existence of a dynamic AR cistrome that depends on the androgen levels and on high AR affinity regions present in PCa cell lines and also in tissue samples.

Abstract 5210: Darolutamide impairs prostate cancer growth by altering chromatin conformation and transcriptional activity of genes involved in cell proliferation and survival

Abstract Androgen signalling is essential for early and late stage prostate cancer (PCa), as demonstrated by the efficacy of androgen receptor (AR) antagonists. The AR gene and its regulatory element are frequently amplified in castration-resistant tumors for which improved treatments are highly needed. Recent findings underscore that gene regulatory elements play an important role in tumor development due to their impact on chromatin conformation and gene regulation. Here we describe the genome-wide effects of darolutamide, a novel AR antagonist which just completed a pivotal clinical phase III trial, by analyzing AR occupancy and its impact on chromatin looping, histone H3K27 acetylation and downstream gene regulation. Genome-wide RNA-seq and ChIP-seq were used to analyze the androgen-sensitive cell lines VCaP, LAPC4 and LNCaP treated with R1881, or with R1881 and darolutamide. Binding of AR and of the pioneer factor FOXA1, as well as histone H3K27 acetylation were determined. Chromatin conformation was detected using the HiChIP method with an AR-specific antibody. Differential gene expression was analyzed by DESeq2 and Gene Set Enrichment Analyses. ChIP-seq peak identification was done by MACS2. HiChIP analysis was performed with the hichipper pipeline. Darolutamide efficiently blocked AR signalling at the level of transcriptional regulation and altered chromatin landscape interaction. Analysis of transcriptomic data showed a strong reduction of the androgen response, as well as down-regulation of proliferation and cell cycle genes by darolutamide. Concordantly, AR occupancy was decreased genome-wide compared to androgen stimulation. Importantly, no binding cluster was identified which showed increased AR occupancy after darolutamide treatment, as reported for other AR antagonists. Interestingly, we observed in darolutamide-treated samples a parallel reduction of FOXA1 occupancy at AR sites, which argues for a co-dependence between this pioneer factor and the AR. Also, we found that H3K27 acetylation, a histone mark linked to active regions, was markedly down-regulated at specific enhancer and gene regions following darolutamide treatment, further pointing to reduced activity of enhancer regions recognized by the AR. Finally, determination of chromatin looping mediated by the AR between enhancers and gene promoters showed a dense network at genes involved in prostate cancer survival. The loss of AR binding at these regions was in line with the significantly reduced transcriptional response observed following darolutamide treatment. In conclusion, we found that darolutamide strongly prevented genome-wide AR binding in multiple hormone-sensitive cellular PCa models, importantly at genes involved in cell proliferation and survival. We furthermore highlight the cross-talk between the pioneer factor FOXA1 and the AR. Citation Format: Simon J. Baumgart, Ekaterina Nevedomskaya, Ralf Lesche, Bernard Haendler. Darolutamide impairs prostate cancer growth by altering chromatin conformation and transcriptional activity of genes involved in cell proliferation and survival [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 5210.

MRGBP promotes AR-mediated transactivation of KLK3 and TMPRSS2 via acetylation of histone H2A.Z in prostate cancer cells

The androgen receptor (AR) promotes growth of prostate cancer cells by controlling the expression of target genes. This study showed that MRG domain binding protein (MRGBP) accelerated AR-mediated transactivation. We first showed that MRGBP promoted growth of AR-positive prostate cancer cells. MRGBP increased the expression of certain AR target genes, including KLK3 and TMPRSS2, and it associated with AR binding regions of these genes during androgen treatment. Furthermore, MRGBP interacted with MRG15 and TIP60 in prostate cancer cells. Androgen-stimulated AR enhanced histone H3K4me1 or H3K4me3 levels at AR binding regions. MRGBP was recruited to active gene regions through its binding with H3K4me1/3 by MRG15. Then, MRGBP promoted recruitment of TIP60 and acetylation of histone variant H2A.Z at the location of AR binding. Accordingly, AR occupancy of the AR binding regions was increased by MRGBP. Together, these results suggest that MRGBP promotes activation of AR-associated enhancer and promoter regions through an epigenetic mechanism.

A novel transcriptional network for the androgen receptor in human epididymis epithelial cells.

What is the transcriptional network governed by the androgen receptor (AR) in human epididymis epithelial (HEE) cells from the caput region and if the network is tissue-specific, how is this achieved? About 200 genes are differentially expressed in the caput HEE cells after AR activation; the AR transcriptional network is tissue-specific and may be mediated in part by distinct AR co-factors including CAAT-enhancer binding protein beta (CEBPB) and runt-related transcription factor 1 (RUNX1). Little is known about the AR transcriptional program genome wide in HEE cells, nor its co-factors in those cells. AR has been best studied in the prostate gland epithelium and prostate cancer cell lines, due to the important role of this factor in prostate cancer. However AR-associated differentially expressed genes (DEGs) and AR co-factors have not yet been compared between human epididymis and prostate epithelial cells. Caput HEE cells from two donors were exposed to the synthetic androgen R1881 at 1 nM for 12-16 h after 72 h of hormone starvation. Chromatin was prepared from R1881-treated and vehicle control HEE cells. AR-associated chromatin was purified by chromatin immunoprecipitation (ChIP) and AR occupancy genome wide was revealed by deep sequencing (ChIP-seq). Two independent biological replicates were performed. Total RNA was prepared from R1881 and control-treated HEE cells and gene expression profiles were documented by RNA-seq. The interaction of the potential novel AR co-factors CEBPB and RUNX1, identified through in-silico motif analysis of AR ChIP-seq data, was examined by ChIP-qPCR after siRNA-mediated depletion of each co-factor individually or simultaneously. The results identify about 200 genes that are differentially expressed (DEGs) in HEE cells after AR activation. Some of these DEGs show occupancy of AR at their promoters or cis-regulatory elements suggesting direct regulation. However, there is little overlap in AR-associated DEGs between HEE and prostate epithelial cells. Inspection of over-represented motifs in AR ChIP-seq peaks identified CEBPB and RUNX1 as potential co-factors, with no evidence for FOXA1, which is an important co-factor in the prostate epithelium. CEBPB and RUNX1 ChIP-seq in HEE cells showed that both these factors often occupied AR-binding sites, though rarely simultaneously. Further analysis at a single AR-regulated locus (FK506-binding protein 5, FKPB5) suggests that CEBPB may be a co-activator. These data suggest a novel AR transcriptional network governs differentiated functions of the human epididymis epithelium. AR ChIP-seq and RNA-seq data are deposited at GEO: GSE109063. There is substantial donor-to-donor variation in primary HEE cells cultures. We applied stringent statistical tests with a false discovery rate (FDR) of 0.1% for ChIP-seq and standard pipelines for RNA-seq so it is possible that we have missed some AR-regulated genes that are important in caput epididymis function. Our data suggest that a novel AR transcriptional network governs differentiated functions of the human epididymis epithelium. Since this cell layer has a critical role in normal sperm maturation, the results are of broader significance in understanding the mechanisms underlying the maintenance of fertility in men. This work was funded by the National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Development: R01 HD068901 (PI: Harris). The authors have no competing interests to declare.

The phytochemical 3,3′-diindolylmethane decreases expression of AR-controlled DNA damage repair genes through repressive chromatin modifications and is associated with DNA damage in prostate cancer cells

Androgen receptor (AR) is a transcription factor involved in normal prostate physiology and prostate cancer (PCa) development. 3,3′-Diindolylmethane (DIM) is a promising phytochemical agent against PCa that affects AR activity and epigenetic regulators in PCa cells. However, whether DIM suppresses PCa via epigenetic regulation of AR target genes is unknown. We assessed epigenetic regulation of AR target genes in LNCaP PCa cells and showed that DIM treatment led to epigenetic suppression of AR target genes involved in DNA repair (PARP1, MRE11, DNA-PK). Decreased expression of these genes was accompanied by an increase in repressive chromatin marks, loss of AR occupancy and EZH2 recruitment to their regulatory regions. Decreased DNA repair gene expression was associated with an increase in DNA damage (γH2Ax) and up-regulation of genomic repeat elements LINE1 and α-satellite. Our results suggest that DIM suppresses AR-dependent gene transcription through epigenetic modulation, leading to DNA damage and genome instability in PCa cells.

Abstract 3452: Conversion of abiraterone to D4A drives antitumor activity in castration-resistant prostate cancer

Abstract Castration resistant prostate cancer (CRPC) still relies on androgens for progression. Abiraterone, an FDA approved CYP17A inhibitor, blocks androgen synthesis and prolongs survival. Abiraterone metabolites are generally thought to be inactive. We have identified a novel abiraterone metabolite, D4A, which inhibits three steroidogenic enzymes and androgen receptor (AR) itself. After abiraterone acetate administration, plasma from mice and patients were analyzed with liquid chromatography-mass spectrometry (LC-MS). The LC-MS results indicate that abiraterone is converted to D4A at detectable concentrations in both mice and patients. To test the function of D4A, the androgen synthesis pathway was analyzed in cell lines and xenografts. After incubation with [3H] labeled androgens, metabolites from cell lines and xenografts were analyzed by HPLC. We found that D4A inhibits activity of the steroidogenic enzymes CYP17A, 3β-hydroxysteroid dehydrogenase (3[[Unsupported Character - Symbol Font ]]HSD) and 5α-reductase. Furthermore D4A competes with the AR agonist R1881 and binds to AR directly. D4A inhibits DHT-induced AR occupancy on the PSA enhancer as detected by chromatin immunoprecipitation and reduces PSA expression. Our findings suggest that D4A is an abiraterone metabolite and a potent inhibitor of the androgen pathway. Citation Format: Zhenfei Li, Nima Sharifi. Conversion of abiraterone to D4A drives antitumor activity in castration-resistant prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3452. doi:10.1158/1538-7445.AM2015-3452

Inhibition of Stat5a/b Enhances Proteasomal Degradation of Androgen Receptor Liganded by Antiandrogens in Prostate Cancer.

Although poorly understood, androgen receptor (AR) signaling is sustained despite treatment of prostate cancer with antiandrogens and potentially underlies development of incurable castrate-resistant prostate cancer. However, therapies targeting the AR signaling axis eventually fail when prostate cancer progresses to the castrate-resistant stage. Stat5a/b, a candidate therapeutic target protein in prostate cancer, synergizes with AR to reciprocally enhance the signaling of both proteins. In this work, we demonstrate that Stat5a/b sequesters antiandrogen-liganded (MDV3100, bicalutamide, flutamide) AR in prostate cancer cells and protects it against proteasomal degradation in prostate cancer. Active Stat5a/b increased nuclear levels of both unliganded and antiandrogen-liganded AR, as demonstrated in prostate cancer cell lines, xenograft tumors, and clinical patient-derived prostate cancer samples. Physical interaction between Stat5a/b and AR in prostate cancer cells was mediated by the DNA-binding domain of Stat5a/b and the N-terminal domain of AR. Moreover, active Stat5a/b increased AR occupancy of the prostate-specific antigen promoter and AR-regulated gene expression in prostate cancer cells. Mechanistically, both Stat5a/b genetic knockdown and antiandrogen treatment induced proteasomal degradation of AR in prostate cancer cells, with combined inhibition of Stat5a/b and AR leading to maximal loss of AR protein and prostate cancer cell viability. Our results indicate that therapeutic targeting of AR in prostate cancer using antiandrogens may be substantially improved by targeting of Stat5a/b.

SUMOylation modulates the transcriptional activity of androgen receptor in a target gene and pathway selective manner.

Androgen receptor (AR) plays an important regulatory role in prostate cancer. AR's transcriptional activity is regulated by androgenic ligands, but also by post-translational modifications, such as SUMOylation. To study the role of AR SUMOylation in genuine chromatin environment, we compared androgen-regulated gene expression and AR chromatin occupancy in PC-3 prostate cancer cell lines stably expressing wild-type (wt) or doubly SUMOylation site-mutated AR (AR-K386R,K520R). Our genome-wide gene expression analyses reveal that the SUMOylation modulates the AR function in a target gene and pathway selective manner. The transcripts that are differentially regulated by androgen and SUMOylation are linked to cellular movement, cell death, cellular proliferation, cellular development and cell cycle. Fittingly, SUMOylation mutant AR cells proliferate faster and are more sensitive to apoptosis. Moreover, ChIP-seq analyses show that the SUMOylation can modulate the chromatin occupancy of AR on many loci in a fashion that parallels their differential androgen-regulated expression. De novo motif analyses reveal that FOXA1, C/EBP and AP-1 motifs are differentially enriched at the wtAR- and the AR-K386R,K520R-preferred genomic binding positions. Taken together, our data indicate that SUMOylation does not simply repress the AR activity, but it regulates AR's interaction with the chromatin and the receptor's target gene selection.

Role of Hes6 in castration-resistant prostate cancer

BackgroundCastration-resistant prostate cancer is a heterogeneous conditon that is poorly characterised. Although the androgen receptor (AR) is of particular importance, other factors such as c-Myc and the E2F family also have a role in later stage disease. Hes6 is a transcription cofactor associated with stem-cell characteristics in neural tissue, but its role in cancer remains uncertain. We aimed to assess the role of Hes6 in castration-resistant prostate cancer and to identify its prognostic and therapeutic relavance. MethodsWe transduced an androgen-sensitive cell line (LNCaP) to constitutively overexpress Hes6 and luciferase to permit xenografting in NOD-SCID gamma mice and bioluminescent monitoring of tumour growth. We used microarray genomics and chromatin immunoprecipitation-sequencing to study the molecular environment underpinning the cellular effects of Hes6. We generated a 61-patient hormone relapsed prostate cancer tissue microarray and assessed 285 publicly available human prostate cancers in a meta-analysis to profile Hes6 and its associated regulome in aggressive human disease. FindingsWe found that Hes6 was upregulated in aggressive human prostate cancer with its expression controlled by c-Myc and AR. Hes6 drove castration-resistant tumour growth by enhancing AR transcriptional activity in the absence of ligand binding. The AR was preferentially directed to a regulatory network enriched for other transcription factors including E2F1. We found a physical interaction between E2F1 and both Hes6 and AR, with increased occupancy of AR at E2F1 target sites in the presence of Hes6. In the clinical setting, we uncovered a Hes6-associated signature that predicts poor outcome in prostate cancer on longitudinal follow-up of men after prostatectomy. We found, in our model of castration resistance, that this process can be pharmacologically targeted by inhibition of PLK1, part of the Hes6 and E2F1 regulome, with restoration of sensitivity to castration. InterpretationWe have shown for the first time, to our knowledge, the crucial role of Hes6 in development of castration-resistant prostate cancer and identified its potential in patient-specific therapeutic strategies. FundingCambridge Biomedical Research Centre, GlaxoSmithKline, Raymond and Beverley Sackler Studentship.

Scaffold attachment factor B1: an intrinsic inhibitor of androgen receptor downregulated in prostate cancer

The androgen receptor (AR) is a pleiotrophic transcription factor that regulates expression of a large number of genes involved in many diverse cellular processes. The AR activation pathways have been studied extensively. However, the molecular mechanism and biological significance of AR inhibitory signals remain poorly understood. Mukhopadhyay et al. have now identified the nuclear matrix protein scaffold attachment factor B1 (SAFB1) as a novel AR corepressor. The authors found that SAFB1 physically associates with AR protein and inhibits AR transcriptional activity and androgen-sensitive gene expression. SAFB1 had no effect on chromatin occupancy of AR, but silencing of SAFB1 abolished recruitment MST1, a known AR repressor, at AR target loci. They also showed that SAFB1 interacts with EZH2, SUZ12 and EED, three core components of the Polycomb repressive complex 2 (PRC2), which catalyzes the gene repression histone modification H3 lysine 27 trimethylation (H3K27me3). The authors further showed that forced expression of SAFB1 increases H3K27me3 at AR target loci and this effect requires EZH2. Finally, the authors demonstrated that expression of SAFB1 is downregulated in human prostate cancer (PCa) specimens and SAFB1 knockdown results in an aggressive phenotype of PCa. These findings identify SAFB1 as an important node for integration of multiple inhibitory signals of AR, which represents a viable pathway for therapeutic intervention of PCa.

CCAR1 promotes chromatin loading of androgen receptor (AR) transcription complex by stabilizing the association between AR and GATA2

Androgen receptor (AR), a ligand-dependent transcription factor, plays a critical role in prostate cancer onset and progression, and its transcriptional function is mediated largely by distinct nuclear receptor co-regulators. Here, we show that cell cycle and apoptosis regulator 1 (CCAR1) functions as an AR co-activator. CCAR1 interacted with and enhanced the transcriptional activity of AR. Depletion of CCAR1 caused reduction in androgen-dependent expression of a subset of AR target genes. We further showed that CCAR1 is required for recruitment of AR, MED1 and RNA polymerase II to the enhancers of AR target genes and for androgen-induced long-range prostate specific antigen enhancer–promoter interaction. The molecular mechanism underlying CCAR1 function in AR-mediated transcription involves CCAR1-mediated enhanced recruitment of GATA2, a pioneer factor for AR, to AR-binding sites. CCAR1 stabilized the interaction between AR and GATA2 by interacting directly with both proteins, thereby facilitating AR and GATA2 occupancy on the enhancers. Furthermore, CCAR1 depletion inhibited the growth, migration, invasion of prostate cancer cells and reduced the tumorigenicity of prostate cancer cells in vivo. Our results firmly established CCAR1 as an AR co-activator that plays a key role in AR transcription complex assembly and has an important physiological role in androgen signaling and prostate tumorigenesis.

Dual Roles of PARP-1 Promote Cancer Growth and Progression

PARP-1 is an abundant nuclear enzyme that modifies substrates by poly(ADP-ribose)-ylation. PARP-1 has well-described functions in DNA damage repair and also functions as a context-specific regulator of transcription factors. With multiple models, data show that PARP-1 elicits protumorigenic effects in androgen receptor (AR)-positive prostate cancer cells, in both the presence and absence of genotoxic insult. Mechanistically, PARP-1 is recruited to sites of AR function, therein promoting AR occupancy and AR function. It was further confirmed in genetically defined systems that PARP-1 supports AR transcriptional function, and that in models of advanced prostate cancer, PARP-1 enzymatic activity is enhanced, further linking PARP-1 to AR activity and disease progression. In vivo analyses show that PARP-1 activity is required for AR function in xenograft tumors, as well as tumor cell growth in vivo and generation and maintenance of castration resistance. Finally, in a novel explant system of primary human tumors, targeting PARP-1 potently suppresses tumor cell proliferation. Collectively, these studies identify novel functions of PARP-1 in promoting disease progression, and ultimately suggest that the dual functions of PARP-1 can be targeted in human prostate cancer to suppress tumor growth and progression to castration resistance. These studies introduce a paradigm shift with regard to PARP-1 function in human malignancy, and suggest that the dual functions of PARP-1 in DNA damage repair and transcription factor regulation can be leveraged to suppress pathways critical for promalignant phenotypes in prostate cancer cells by modulation of the DNA damage response and hormone signaling pathways. The combined studies highlight the importance of dual PARP-1 function in malignancy and provide the basis for therapeutic targeting.

Role of the HSP90-Associated Cochaperone p23 in Enhancing Activity of the Androgen Receptor and Significance for Prostate Cancer

Prostate tumor growth initially depends on androgens, which act via the androgen receptor (AR). Despite androgen ablation therapy, tumors eventually progress to a castrate-resistant stage in which the AR remains active. The mechanisms are poorly understood but it may be that changes in levels or activity of AR coregulators affect trafficking and activation of the receptor. A key stage in AR signaling occurs in the cytoplasm, where unliganded receptor is associated with the heat shock protein (HSP)90 foldosome complex. p23, a key component of this complex, is best characterized as a cochaperone for HSP90 but also has HSP90-independent activity and has been reported as having differential effects on the activity of different steroid receptors. Here we report that p23 increases activity of the AR, and this appears to involve steps both in the cytoplasm (increasing ligand-binding capacity, possibly via direct interaction with AR) and the nucleus (enhancing AR occupancy at target promoters). We show, for the first time, that AR and p23 can interact, perhaps directly, when HSP90 is not present in the same complex. The effects of p23 on AR activity are at least partly HSP90 independent because a mutant form of p23, unable to bind HSP90, nevertheless increases AR activity. In human prostate tumors, nuclear p23 was higher in malignant prostate cells compared with benign/normal cells, supporting the utility of p23 as a therapeutic target in prostate cancer.

Abstract LB-273: Transcriptional regulation by androgen receptor in prostate cancer

Abstract Transcriptional regulation by the androgen receptor (AR) is critical to prostate cancer development and progression. Through integrative analysis of genome-wide AR occupancy and androgen-regulated gene expression, we have recently reported AR as a globally acting transcriptional repressor. This repression is mediated by androgen responsive elements (ARE) and dictated by Polycomb group protein EZH2 and repressive chromatin remodeling. In addition, AR-repressed genes are enriched for developmental regulators controlling cell differentiation. They tend to get down-regulated in advanced prostate cancer probably accountable for stem cell-like de-differentiation and thus tumor progression. However, critical AR-repressed genes of functional relevance are yet to be identified and their roles to be carefully characterized. Through meta-analysis of several expression microarray data, we nominate one top candidate AR-repressed gene. Using this gene as a model, we present further evidence that AR directly inhibits gene expression through physically binding to the enhancer, establishing promoter-enhancer DNA looping, and inducing repressive chromatin remodeling. Further, overexpression of this gene inhibits prostate cancer cell growth, migration, and invasion in vitro and xenograft tumor growth in mice, thus exhibiting a tumor suppressive role. In addition, the loss of this gene drives castration-resistant prostate cancer. Therefore, our findings support that AR-mediated inhibition of tumor suppressors plays important roles during prostate cancer progression. Our study provides an innovative mechanism for AR in castration resistance, which may eventually lead to novel therapeutic targets for treatment of advanced prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-273. doi:1538-7445.AM2012-LB-273

Inhibition of p53 expression modifies the specificity of chromatin binding by the androgen receptor

The androgen receptor (AR) is known to play a critical role in prostate cancer (PC). p53 likely also plays a role given that p53 mutations are commonly found in advanced PC, and loss of wild-type protein function contributes to the phenotype of castration-resistant prostate cancer (CRPC). Nevertheless, the extent of the contribution of p53 dysfunction to PC remains unclear. Here we analyze the effects of p53 inhibition in PC cells and show that it has significant consequences for both the interaction between AR, and chromatin and the proliferative capacity of these cells. Inhibition of p53 expression enabled LNCaP cells to proliferate independently of androgens. Moreover, it modified the genome-wide binding pattern of AR. ChIP-sequnce analyis (ChIP-seq) revealed that fewer AR-binding sites were present in the context of p53 inhibition, suggesting that wild-type p53 is required for stable binding of AR to certain chromatin regions. Further analysis revealed that a lower AR occupancy was accompanied by a reduction in FoxA1 binding at regulatory regions of AR-dependent genes. Our study also identifies a pool of genes that may be transcriptionally regulated by AR only in the absence of p53, and that may contribute to the CRPC phenotype. Overall, our results point to p53 playing an important role in regulating AR activity across the genome.

DNA Demethylation-Dependent AR Recruitment and GATA Factors Drive Rhox5 Homeobox Gene Transcription in the Epididymis

Mammalian male fertility depends on the epididymis, a highly segmented organ that promotes sperm maturation and protects sperm from oxidative damage. Remarkably little is known about how gene expression is controlled in the epididymis. A candidate to regulate genes crucial for epididymal function is reproductive homeobox gene on X chromosome (RHOX)5, a homeobox transcription factor essential for optimal sperm motility that is expressed in the caput region of the epididymis. Here, we report the identification of factors that control Rhox5 gene expression in epididymal cells in a developmentally regulated and region-specific fashion. First, we identify GATA transcription factor-binding sites in the Rhox5 proximal promoter (Pp) necessary for Rhox5 expression in epididymal cells in vitro and in vivo. Adjacent to the GATA sites are androgen-response elements, which bind to the nuclear hormone receptor androgen receptor (AR), and are responsible for the AR-dependent expression of Rhox5 in epididymal cells. We provide evidence that AR is recruited to the Pp in a region-specific and developmentally regulated manner in the epididymis that is dictated not only by differential AR availability but differential methylation of the Pp. Site-specific methylation of the Pp cytosine and guanine separated by one phosphate, most of which overlap with androgen-response elements, inhibited both AR occupancy at the Pp and Pp-dependent transcription in caput epididymal cells. Together, our data support a model in which DNA methylation, AR, and GATA factors collaborate to dictate the unique developmental and region-specific expression pattern of the RHOX5 homeobox transcription factor in the caput epididymis, which in turn controls the expression of genes critical for promoting sperm motility and function.