Androgen Receptor Binding Research Articles

Androgen-Regulated SPARCL1 in the Tumor Microenvironment Inhibits Metastatic Progression.

Prostate cancer is a leading cause of cancer death in men due to the subset of cancers that progress to metastasis. Prostate cancers are thought to be hardwired to androgen receptor (AR) signaling, but AR-regulated changes in the prostate that facilitate metastasis remain poorly understood. We previously noted a marked reduction in secreted protein, acidic and rich in cysteine-like 1 (SPARCL1) expression during invasive phases of androgen-induced prostate growth, suggesting that this may be a novel invasive program governed by AR. Herein, we show that SPARCL1 loss occurs concurrently with AR amplification or overexpression in patient-based data. Mechanistically, we demonstrate that SPARCL1 expression is directly suppressed by androgen-induced AR activation and binding at the SPARCL1 locus via an epigenetic mechanism, and these events can be pharmacologically attenuated with either AR antagonists or HDAC inhibitors. We establish using the Hi-Myc model of prostate cancer that in Hi-Myc/Sparcl1(-/-) mice, SPARCL1 functions to suppress cancer formation. Moreover, metastatic progression of Myc-CaP orthotopic allografts is restricted by SPARCL1 in the tumor microenvironment. Specifically, we show that SPARCL1 both tethers to collagen in the extracellular matrix (ECM) and binds to the cell's cytoskeleton. SPARCL1 directly inhibits the assembly of focal adhesions, thereby constraining the transmission of cell traction forces. Our findings establish a new insight into AR-regulated prostate epithelial movement and provide a novel framework whereby SPARCL1 in the ECM microenvironment restricts tumor progression by regulating the initiation of the network of physical forces that may be required for metastatic invasion of prostate cancer.

The androgen receptor cistrome is extensively reprogrammed in human prostate tumorigenesis.

Master transcription factors interact with DNA to establish cell-type identity and to regulate gene expression in mammalian cells1,2. The genome-wide map of these transcription factor binding sites has been termed the cistrome3. Here we show that the androgen receptor (AR) cistrome undergoes extensive reprogramming during prostate epithelial transformation in man. Using human prostate tissue, we observed a core set of AR binding sites that are consistently reprogrammed in tumors. FOXA1 and HOXB13, co-localized with the reprogrammed AR sites in human tumor tissue. Introduction of FOXA1 and HOXB13 into an immortalized prostate cell line reprogrammed the AR cistrome to resemble that of a prostate tumor, functionally linking these specific factors to AR reprogramming. These findings offer mechanistic insights into a key set of events that drive normal prostate epithelium towards transformation and establish the centrality of epigenetic reprogramming in human prostate tumorigenesis.

The androgen receptor controls expression of the cancer-associated sTn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer.

Patterns of glycosylation are important in cancer, but the molecular mechanisms that drive changes are often poorly understood. The androgen receptor drives prostate cancer (PCa) development and progression to lethal metastatic castration-resistant disease. Here we used RNA-Seq coupled with bioinformatic analyses of androgen-receptor (AR) binding sites and clinical PCa expression array data to identify ST6GalNAc1 as a direct and rapidly activated target gene of the AR in PCa cells. ST6GalNAc1 encodes a sialytransferase that catalyses formation of the cancer-associated sialyl-Tn antigen (sTn), which we find is also induced by androgen exposure. Androgens induce expression of a novel splice variant of the ST6GalNAc1 protein in PCa cells. This splice variant encodes a shorter protein isoform that is still fully functional as a sialyltransferase and able to induce expression of the sTn-antigen. Surprisingly, given its high expression in tumours, stable expression of ST6GalNAc1 in PCa cells reduced formation of stable tumours in mice, reduced cell adhesion and induced a switch towards a more mesenchymal-like cell phenotype in vitro. ST6GalNAc1 has a dynamic expression pattern in clinical datasets, beingsignificantly up-regulated in primary prostate carcinoma but relatively down-regulated in established metastatic tissue. ST6GalNAc1 is frequently upregulated concurrently with another important glycosylation enzyme GCNT1 previously associated with prostate cancer progression and implicated in Sialyl Lewis X antigen synthesis. Together our data establishes an androgen-dependent mechanism for sTn antigen expression in PCa, and are consistent with a general role for the androgen receptor in driving important coordinate changes to the glycoproteome during PCa progression.

Abstract B20: The cholesterol metabolite, 27-hydroxycholesterol induces hyperplasia in an androgen receptor-dependent manner in normal prostate RWPE-1 cells

Abstract For every 6 men, 1 will be diagnosed with prostate cancer (PCa). Moreover, benign prostatic hyperplasia (BPH) also occurs in 50% of men over the age of 50. Although Androgens are known to play an important role in PCa and BPH, some evidence shows estrogens may be involved as well. Recently a cholesterol oxidized metabolite (oxysterol), 27- hydroxycholesterol (27-OHC) was found to bind to estrogen receptor(ER) and act as a selective ER modulator (SERM). 27-OHC is the most prevalent cholesterol metabolite present in the blood with levels increasing with oxidative stress and age, especially in men. The role of 27-OHC in PCa and BPH remains to be investigated. We propose that 27-OHC has deleterious effect in PCa and BPH, whereas the effect on BPH may be androgen dependent. We recently found that incubation with 27-OHC of RWPE-1 (normal epithelial) and LNCaP (cancer cells) increased cell proliferation in both cell lines. Interestingly, upon incubation of RWPE-1 and LNCaP with 27-OHC, we observed an increase in androgen receptor (AR) activity in RWPE-1, not in LNCaP. Furthermore, we found an increase in AR binding to the Androgen Response Element upon exposure of RWPE-1 to 27-OHC. We also found that upon silencing AR gene expression, 27-OHC-induced proliferation in RWPE-1 was markedly attenuated. Additionally, 27-OHC also diminished Docetaxel-mediated apoptosis in RWPE-1. Altogether, our results propose dysregulated levels of 27-OHC as a new risk factor for BPH and PCa. This indicates a novel role for 27-OHC in the etiopathogenesis of BPH and PCa. Citation Format: Shaneabbas Raza, Megan Meyer, Bin Guo, Kimberly Hammer, Othman Ghribi. The cholesterol metabolite, 27-hydroxycholesterol induces hyperplasia in an androgen receptor-dependent manner in normal prostate RWPE-1 cells. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B20.

Androgen and Progesterone Receptors Are Targets for Bisphenol A (BPA), 4-Methyl-2,4-bis-(P-Hydroxyphenyl)Pent-1-Ene--A Potent Metabolite of BPA, and 4-Tert-Octylphenol: A Computational Insight.

Exposure to toxic industrial chemicals that have capacity to disrupt the endocrine system, also known as endocrine disrupting chemicals (EDCs), has been increasingly associated with reproductive problems in human population. Bisphenol A (BPA; 4,4'-(propane-2,2-diyl)diphenol) and 4-tert-octylphenol (OP; 4-(1,1,3,3-tetramethylbutyl)phenol) are among the most common environmental contaminants possessing endocrine disruption properties and are present in plastics, epoxy resins, detergents and other commercial products of common personal and industrial use. A metabolite of BPA, 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) is about 1000 times more biologically active compared to BPA. Epidemiological, clinical, and experimental studies have shown association of BPA and OP with adverse effects on male and female reproductive system in human and animals. The endocrine disruption activity can occur through multiple pathways including binding to steroid receptors. Androgen receptor (AR) and progesterone receptor (PR) are critical for reproductive tract growth and function. Structural binding characterization of BPA, MBP, and OP with AR and PR using molecular docking simulation approaches revealed novel interactions of BPA with PR, and MBP and OP with AR and PR. For BPA, MBP, and OP, five AR interacting residues Leu-701, Leu-704, Asn-705, Met-742, and Phe-764 overlapped with those of native AR ligand testosterone, and four PR interacting residues Leu-715, Leu-718, Met-756, and Met-759 overlapped with those of PR co-complex ligand, norethindrone. For both the receptors the binding strength of MBP was maximum among the three compounds. Thus, these compounds have the potential to block or interfere in the binding of the endogenous native AR and PR ligands and, hence, resulting in dysfunction. The knowledge of the key interactions and the important amino-acid residues also allows better prediction of potential of xenobiotic molecules for disrupting AR- and PR-mediated pathways, thus, helping in design of less potent alternatives for commercial use.

Registered report: the androgen receptor induces a distinct transcriptional program in castration-resistant prostate cancer in man.

The Prostate Cancer Foundation-Movember Foundation Reproducibility Initiative (PCFMFRI) seeks to address growing concerns about reproducibility in scientific research by conducting replications of recent papers in the field of prostate cancer. This Registered Report describes the proposed replication plan of key experiments from “The Androgen Receptor Induces a Distinct Transcriptional Program in Castration-Resistant Prostate Cancer in Man” by Sharma and colleagues (2013), published in Cancer Cell in 2013. Of thousands of targets for the androgen receptor (AR), the authors elucidated a subset of 16 core genes that were consistently downregulated with castration and re-emerged with castration resistance. These 16 AR binding sites were distinct from those observed in cells in culture. The authors suggested that cellular context can have dramatic effects on downstream transcriptional regulation of AR binding sites. The present study will attempt to replicate Fig. 7C by comparing gene expression of the 16 core genes identified by Sharma and colleagues in xenograft tumor tissue compared to androgen treated LNCaP cells in vitro. The Prostate Cancer Foundation-Movember Foundation Reproducibility Initiative is a collaboration between the Prostate Cancer Foundation, the Movember Initiative, and Science Exchange, and the results of the replications will be published by PeerJ.

Androgen receptor binding to an androgen-responsive element in the promoter of the Srsf4 gene inhibits its expression in mouse Sertoli cells.

The serine/arginine-rich splicing actor 4 (SRSF4) is essential for pre-mRNA splicing and can influence alternative-splice-site choice. Little is known about the specific function of this gene in the reproductive system, although a recent study identified a SRSF4 polymorphism significantly associated with a decreased risk of non-obstructive azoospermia in Chinese men. We previously found that the expression of Srsf4 was up-regulated in the testes of Sertoli-cell-selective androgen receptor knockout (S-Ar(-/y)) mice compared to wild-type mice using digital gene expression analysis. In this study, we confirmed and extended the selective gene expression data: SRSF4 was mainly located in the nucleus of Sertoli cells, and Srsf4 expression in the Sertoli-cell-derived cell line TM4 is down-regulation by testosterone. Moreover, androgen receptor directly binds the androgen-responsive element of the Srsf4 promoter. Taken together, these results demonstrate that Srsf4 is a direct downstream target of the androgen receptor in mouse Sertoli cells.

Similarities and Distinctions in Actions of Surface-Directed and Classic Androgen Receptor Antagonists.

The androgen receptor (AR) surface-directed antagonist MJC13 inhibits AR function and proliferation of prostate cancer (PC) cells. These effects are related to arrest of an AR/chaperone complex in the cytoplasm. Here, we compared MJC13 and classic AR antagonists such as flutamide and bicalutamide. Microarray analysis and confirmatory qRT-PCR reveals that MJC13 and flutamide inhibit dihydrotestosterone (DHT)-dependent genes in LNCaP PC cells. Both compounds are equally effective on a genome wide basis and as effective as second generation AR antagonists (MDV3100, ARN-509) at selected genes. MJC13 inhibits AR binding to the prostate specific antigen (PSA) promoter more strongly than flutamide, consistent with different mechanisms of action. Examination of efficacy of MJC13 in conditions that reflect aspects castrate resistant prostate cancer (CRPC) reveals that it inhibits flutamide activation of an AR mutant (ART877A) that emerges during flutamide withdrawal syndrome, but displays greatly restricted gene-specific activity in 22Rv1 cells that express a constitutively active truncated AR and is inactive against glucocorticoid receptor (GR), which can co-opt androgen-dependent signaling networks in CRPC. Importantly, MJC13 inhibits AR interactions with SRC2 and β-catenin in the nucleus and, unlike flutamide, strongly inhibits amplification of AR activity obtained with transfected SRC2 and β-catenin. MJC13 also inhibits DHT and β-catenin-enhanced cell division in LNCaP cells. Thus, a surface-directed antagonist can block AR activity in some conditions in which a classic antagonist fails and may display utility in particular forms of CRPC.

Retraction Note to: ChIP-seq analysis of androgen receptor in LNCaP cell line.

The aim of this study was to screen target genes and gene functions of androgen receptor (AR) in LNCaP cell line by ChIP-seq data analysis. We downloaded the gene expression profile GSE14092 from Gene Expression Omnibus database and selected ChIP-seq data (GSM353644) of AR stimulated by androgen R1881 (R1881 stimulation group) and the ChIP-seq data (GSM353643) of AR without R1881 stimulation (control group). MACS peak calling software was used to identify the AR binding cites. After target genes selection and function analysis, motif finding analysis was utilized to predict the AR co-located transcription regulation factors, and we analyzed their functions through GO enrichment analysis. Total 27202 and 2730 AR binding sites were detected in the R1881 stimulation group and the control group, respectively and 398 and 58 target genes were identified in R1881 stimulation group and control group, respectively. Based on GO enrichment analysis, 20 biological processes which AR regulated in the LNCaP cells were enriched, including xenobiotic metabolic process, positive regulation of interleukin-2 production and cellular response to sterol etc. We finally identified 99 transcription factors with high motif enrichment significant levels, and the enrichment of AR co-located transcription factors was significantly enriched in the biological process of regulation of cell proliferation in which 13 transcription factors were involved (FOXJ1, FOXM1, NF1, SOX2, HOXD13, FOXO1, FOXP3, FOXO4, SOX9, PGR, DBP, JUN and TLX1). The analysis of AR target genes and gene functions help us to elucidate the mechanism of the prostate cancer on a molecular level. In addition, it will pave the way to effective therapies for prostatic cancer. However, further experiments are still needed to confirm our results.

Abstract 1849: Androgen receptor transcriptionally represses genes mediating DNA synthesis and repair in prostate cancer

Abstract In the U.S., prostate cancer (PCa) ranked second for cancer related deaths for men. It has been well established that androgen receptor (AR) plays an essential role in the development of primary PCa and its activity is restored in castration resistant prostate cancers (CRPC). While AR has been extensively characterized as a transcriptional activator, it can also function by direct or indirect mechanisms to suppress gene expression. Despite the critical role AR plays in PCa, mechanisms by which it functions as a transcriptional repressor are poorly understood. Using VCaP and VCaP derived CRPC cell lines, we have reported that androgen-repressed genes were functionally enriched for DNA synthesis and repair, while AR stimulated genes were associated with lipid/sterol synthesis and other aspects of cellular metabolism. Significantly, a subset of the genes that were androgen-repressed in VCaP cells were consistently increased in CRPC clinical samples, and this 53-gene subset was also highly enriched for genes mediating DNA synthesis and repair. Using AR ChIP-seq and transcriptome profiling in VCaP cells, we found that the majority of this AR-suppressed gene subset (39/53) have one or more AR binding sites within the gene locus. The expression level of these direct AR-suppressed genes is associated with poor survival of disease in clinical cohorts. Interestingly, although the suppression activity of AR on this subset of genes is direct, in other PCa cell lines such as LNCaP, there is a secondary mechanism that allows AR to indirectly stimulate the expression of these genes. Importantly, the direct AR mediated repression of these genes is enhanced in PCa cells expressing higher levels of AR, and overexpression of AR in LNCaP cells resulted in increased and more persistent AR binding to these genes, and also enhanced the direct AR repression of this gene subset. Mechanistically, we found that the indirect activation of these DNA synthesis genes by AR is mediated through transcriptionally stimulating lipid synthesis, which could subsequently drive the G1/S cell cycle progression and RB1 phosphorylation. Indeed, this indirect stimulatory effect of AR on these genes could be suppressed by cyclin dependent kinase inhibition or by preventing the AR stimulation of mTOR (rapamycin) or sterol synthesis (statins). Taken together, these findings indicate that the ability of AR to repress this gene set mediating DNA synthesis may persist in CRPC, and may be exploited therapeutically by combination therapies that stimulate AR while repressing its metabolic functions. Citation Format: Yanfei Gao, Shuai Gao, Housheng He, Xiaming Liu, Sen Chen, Fen Ma, X. Shirley Liu, Myles Brown, Steven P. Balk, Shaoyong Chen, Changmeng Cai. Androgen receptor transcriptionally represses genes mediating DNA synthesis and repair in 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 1849. doi:10.1158/1538-7445.AM2015-1849

Abstract 5050: Proteome-based combined treatment strategies synergizing antagonism of androgen receptor function in prostate cancer

Abstract Acquired resistance to second-generation androgen receptor (AR)-targeted therapies compels the development of novel therapeutic agents for castration-resistant prostate cancer (CRPC). We have recently reported a novel function of endostatin (ES) through direct binding and inhibition of AR in human prostate cancer (PCa) cells. Molecular characterization of the putative mechanisms of ES function on AR-positive PCa cells by global proteomic analysis revealed dramatic upregulation of CYP51A1 expression in LNCaP cells, suggesting that the increases in cholesterol biosynthesis and the resultant steroidogenesis may provide survival mechanism for PCa cells through the augmentation of androgen-AR axis. To validate this hypothesis, anti-proliferative effects of combined treatment of ES with Lovastatin or Fluconazole, inhibitor of HMG-CoA reductase or CYP5A1, were analyzed in vitro using AR-positive LNCaP cells. Results indicated a significantly enhanced ES-mediated growth inhibition on LNCaP cells by targeting both the ligand biosynthesis and receptor function. Proteomic profiling further indicated that ES significantly decreased the levels of AR, coactivator Ran (ARA24) and downregulated AR-driven expression of MCM complex 2, 3, 4 and 6, MDC1 and STMN1, suggesting that AR-targeted effects of ES include disruption of DNA replication, cell cycle propagation and cell division. We found that dual inhibition of AR nuclear transport and AR-mediated cell division by co-treatment of ES and Taxol greatly decreased PCa cell proliferation. Together, this interdisciplinary approach integrating proteomics with PCa cell function possibly suggests that antagonism of AR, which is the principal drug target for the PCa treatment, can be potentiated by the combination of ES with currently used chemotherapy agents and adjuvants. These findings further provide new insights into proteome-based approach as a useful tool to design and optimize effective therapeutic regimens for individual patients with CRPC. Citation Format: Joo Hyoung Lee, James A. Mobley, Selvarangan Ponnazhagan. Proteome-based combined treatment strategies synergizing antagonism of androgen receptor function in 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 5050. doi:10.1158/1538-7445.AM2015-5050

The Deubiquitinating Enzyme USP7 Regulates Androgen Receptor Activity by Modulating Its Binding to Chromatin

The androgen receptor (AR), a nuclear receptor superfamily transcription factor, plays a key role in prostate cancer. AR signaling is the principal target for prostate cancer treatment, but current androgen-deprivation therapies cannot completely abolish AR signaling because of the heterogeneity of prostate cancers. Therefore, unraveling the mechanism of AR reactivation in androgen-depleted conditions can identify effective prostate cancer therapeutic targets. Increasing evidence indicates that AR activity is mediated by the interplay of modifying/demodifying enzymatic co-regulators. To better understand the mechanism of AR transcriptional activity regulation, we used antibodies against AR for affinity purification and identified the deubiquitinating enzyme ubiquitin-specific protease 7, USP7 as a novel AR co-regulator in prostate cancer cells. We showed that USP7 associates with AR in an androgen-dependent manner and mediates AR deubiquitination. Sequential ChIP assays indicated that USP7 forms a complex with AR on androgen-responsive elements of target genes upon stimulation with the androgen 5α-dihydrotestosterone. Further investigation indicated that USP7 is necessary to facilitate androgen-activated AR binding to chromatin. Transcriptome profile analysis of USP7-knockdown LNCaP cells also revealed the essential role of USP7 in the expression of a subset of androgen-responsive genes. Hence, inhibition of USP7 represents a compelling therapeutic strategy for the treatment of prostate cancer.

Abstract 83: Comparison of surface-directed and classic androgen receptor antagonists

Abstract Background: The androgen receptor (AR) surface-directed antagonist MJC13 inhibits AR function and proliferation of prostate cancer (PC) cells and these effects are related to arrest of an AR/chaperone complex in the cytoplasm. Here, we compared activities of MJC13 and the classic AR antagonist flutamide. Method and Results: MJC13 and flutamide inhibit all dihydrotestosterone (DHT)-dependent genes in LNCaP PC cells and both compounds are equally effective. Control ChIP assays confirm that MJC13 inhibits AR binding to the prostate specific antigen (PSA) promoter more strongly than flutamide, consistent with different mechanisms of action. Examination of efficacy of MJC13 in conditions that reflect aspects of castrate resistant prostate cancer (CRPC) reveals that it inhibits a flutamide-dependent AR mutant (ART877A), but displays greatly restricted gene-specific activity in 22Rv1 cells that express a constitutively active truncated AR and is inactive against glucocorticoid receptor (GR), which can co-opt androgen-dependent signaling networks in CRPC. While flutamide inhibits AR interactions with the coactivator SRC2, it does not affect β-catenin binding. In contrast, MJC13 inhibits AR interactions with both coregulators and, unlike flutamide, strongly inhibits amplification of AR activity obtained with SRC2 and β-catenin. MJC13 also preferentially inhibits β-catenin-enhanced cell division in LNCaP cells relative to flutamide. Conclusion: Our findings suggest that a surface-directed antagonist can block AR activity in some conditions in which a classic antagonist fails and may display utility in some forms of CRPC. Citation Format: Ji Ho Suh, Arundhati Chattopadhyay, Douglas H. Sieglaff, Cheryl L. Storer, Marc B. Cox, Paul Webb. Comparison of surface-directed and classic androgen receptor antagonists. [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 83. doi:10.1158/1538-7445.AM2015-83

Abstract 5051: Androgen receptor-mediated regulation of p14ARF transcription in prostate tumor cells

Abstract The p14ARF tumor suppressor is often deleted or silenced in malignancies. Prostate tumors are an exception, where p14 expression is elevated. To understand this phenomenon, we assessed the expression of p53 pathway members, which are most effected by p14ARF. The expression of androgen receptor (AR), a pivotal prostate cancer regulator, which is also affected by p14arf and MDM2 was analyzed as well. The studies used archival prostate tumor tissues obtained from prostatectomies performed at the Veterans Affairs-Northern California Health Care System in Mather California between 1996 and 2002 to better define the relationship between these interrelated networks. A prostate tumor tissue array consisting of 78 tumors of differing stages and grades was constructed to evaluate correlations between multiple parameters. Immunohistochemical studies assessed expression of the proliferation marker Ki67, p53, MDM2, MDM4, p14ARF, and the AR in the nuclear and cytoplasmic compartments of tumor and adjacent cells. p53, MDM4, p14ARF and AR were detected in nuclear and cytoplasmic compartments of tumor and non-tumor cells, but were predominantly nuclear. MDM2 expression was primarily cytoplasmic in tumor cells. Multivariate analysis of the immunohistochemical markers identified a strong correlation between expression of p14ARF and AR. Studies utilizing the prostate CWR22 xenograft and LNCaP cell line models revealed that castration or androgen deprivation resulted in reduced p14arf levels and that this effect correlated with a precipitous decline in E2F1-3a levels. In a reciprocal analysis, RB ablation enhanced p14ARF transcription, arguing that the E2F/RB pathway mediates AR-dependent p14ARF expression. However, we also identified an AR binding site located ∼40 KB upstream of the p14ARF gene. Chromatin immunoprecipitation (ChIP) studies showed that in prostate cells this site was bound by AR. ChIP studies also revealed E2F1 and E2F3 were present at the p14ARF promoter. Together, the studies argue p14ARF is a direct transcriptional target of AR and that AR and E2F collaborate to promote p14ARF expression. . Citation Format: Maria Mudryj, Salma Siddiqui, Stephen J. Libertini, Alan P. Lombard, Benjamin Mooso, Leandro D'Abronzo, Frank Melgoza, Alexander Borowsky, Christiana Drake, LiHong Qi, Paramita M. Ghosh. Androgen receptor-mediated regulation of p14ARF transcription in prostate tumor cells. [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 5051. doi:10.1158/1538-7445.AM2015-5051

Abstract 79: An androgen response element-based reporter assay for the detection of androgen receptor activity in prostate cells

Abstract Background: The androgen receptor (AR) transcription factor plays pivotal roles in the development and progression of prostate cancer. AR is activated and maintained throughout prostate cancer progression even in castration-resistant prostate cancer (CRPC). AR and its classical target genes such as PSA (KLK3) are, however, efficiently shut off in basal epithelial prostate cells and possibly in prostate cancer stem cells (CSCs). There is limited information on the mechanisms that keep the AR OFF in these cells, and the conditions that turn the AR and target genes ON. Results: Fluorescent and luminescent reporters were constructed to develop a functional assay for AR activity. These reporters contain the repeated AR binding promoter sequences and were designed in a way that the AR can activate these AR element (ARE) reporters. The 241B reporter was selected among the constructed ARE reporters on the basis of higher activity. The AR positive prostate cancer cell line, LNCaP, was transduced with the lentiviral 241B-mCherry fluorescence reporter. FACS analysis quantitated the ARE reporter activity when reporter cells were treated with androgen and with the AR antagonist, enzalutamide, or the anti-androgen, abiraterone. AR expression vectors were also constructed with the AR open reading frames cloned into lentiviral expression vectors. All constructs were sequenced to verify the correct AR DNA sequence and their activity were tested using ARE reporter system. Conclusion: The developed ARE reporter system is useful to understand the mechanisms of AR and AR target gene activation in different subpopulations of prostate cancer cells. It will help us to investigate the role of the AR in differentiation and proliferation of prostate cells and to study the AR activity in two and three dimensional cell cultures. This system can also be useful in screening for drugs with activity against the AR. Citation Format: Waqas Azeem, Margrete R. Hellem, Jan R. Olsen, Yaping Hua, Kristo Marvyin, Lisha Li, Yi Qu, Biaoyang Lin, Xisong Ke, Anne M. Oyan, Karl-Henning Kalland. An androgen response element-based reporter assay for the detection of androgen receptor activity in prostate cells. [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 79. doi:10.1158/1538-7445.AM2015-79

Androgen receptor modulates Foxp3 expression in CD4+CD25+Foxp3+ regulatory T-cells.

CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells are able to inhibit proliferation and cytokine production in effector T-cells and play a major role in immune responses and prevention of autoimmune disease. A master regulator of Treg cell development and function is the transcription factor Foxp3. Several cytokines, such as TGF-β and IL-2, are known to regulate Foxp3 expression as well as methylation of the Foxp3 locus. We demonstrated previously that testosterone treatment induces a strong increase in the Treg cell population both in vivo and in vitro. Therefore we sought to investigate the direct effect of androgens on expression and regulation of Foxp3. We show a significant androgen-dependent increase of Foxp3 expression in human T-cells from women in the ovulatory phase of the menstrual cycle but not from men and identify a functional androgen response element within the Foxp3 locus. Binding of androgen receptor leads to changes in the acetylation status of histone H4, whereas methylation of defined CpG regions in the Foxp3 gene is unaffected. Our results provide novel evidence for a modulatory role of androgens in the differentiation of Treg cells.

MiR-22 and miR-29a Are Members of the Androgen Receptor Cistrome Modulating LAMC1 and Mcl-1 in Prostate Cancer.

The normal prostate as well as early stages and advanced prostate cancer (PCa) require a functional androgen receptor (AR) for growth and survival. The recent discovery of microRNAs (miRNAs) as novel effector molecules of AR disclosed the existence of an intricate network between AR, miRNAs and downstream target genes. In this study DUCaP cells, characterized by high content of wild-type AR and robust AR transcriptional activity, were chosen as the main experimental model. By integrative analysis of chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray expression profiling data, miRNAs putatively bound and significantly regulated by AR were identified. A direct AR regulation of miR-22, miR-29a, and miR-17-92 cluster along with their host genes was confirmed. Interestingly, endogenous levels of miR-22 and miR-29a were found to be reduced in PCa cells expressing AR. In primary tumor samples, miR-22 and miR-29a were less abundant in the cancerous tissue compared with the benign counterpart. This specific expression pattern was associated with a differential DNA methylation of the genomic AR binding sites. The identification of laminin gamma 1 (LAMC1) and myeloid cell leukemia 1 (MCL1) as direct targets of miR-22 and miR-29a, respectively, suggested a tumor-suppressive role of these miRNAs. Indeed, transfection of miRNA mimics in PCa cells induced apoptosis and diminished cell migration and viability. Collectively, these data provide additional information regarding the complex regulatory machinery that guides miRNAs activity in PCa, highlighting an important contribution of miRNAs in the AR signaling.

Estrogen induces androgen-repressed SOX4 expression to promote progression of prostate cancer cells.

The sex determing region Y-box 4 (SOX4) gene is a critical developmental transcriptional factor that is overexpressed in prostate cancer (PCa). While we and others have investigated the role of SOX4 overexpression in PCa, the molecular mechanism underlying its aberrant expression remains unclear. Immunohistochemistry were utilized to detect SOX4 expression and the correlation between estrogen receptor β (ERβ), androgen receptor (AR) and SOX4 in a cohort of 94 clinical specimens. Real-time quantitative PCR and Western blotting were used to study the transcript and protein expression levels. Immunofluorescence staining and co-immunoprecipitation were performed to assess the interaction and subcellular location of ERβ and AR. Chromatin immunoprecipitation (ChIP) assays and Luciferase reporter assays were performed to explore the binding and transcriptional activities of ERβ and AR to the SOX4 promoter. Cellular function was evaluated by MTS, invasion and wound healing assays. SOX4 expression is up-regulated in Castration-Resistant Prostate Cancer (CRPC) tumors compared to hormone-dependent PCa (HDPC) cases. Increased expression was also observed in PCa cells after long-term androgen-deprivation treatment (ADT). In vitro data indicated that SOX4 is an AR transcriptional target and down-regulated by dihydrotestosterone (DHT) via AR. 17β-estradiol (E2) up-regulates SOX4 expression in the absence of androgen through the formation of a protein complex between ERβ and AR. Knockdown of AR or ERβ blocks the E2-induced SOX4 expression. ChIP assays confirmed that both ERβ and AR bind to the SOX4 promoter in response to E2. Functionally, silencing SOX4 significantly attenuates the proliferative effect, as well as the capacity of migration and invasion of E2 on PCa cells. Clinically, overexpression of SOX4 is significantly associated with ERβ expression in PCa. In addition, this association is still retained in CRPC patients with poor prognosis. These findings suggest that SOX4 is a novel DHT-repressed AR-target gene. E2 could promote proliferation of PCa cells through the up-regulation of SOX4 under androgen-depleted environment. Our data provides a possible molecular basis for the overexpression of SOX4 in CRPC and may facilitate the detection and prevention of the emergence of CRPC.

The lncRNA DRAIC/PCAT29 Locus Constitutes a Tumor-Suppressive Nexus.

Long noncoding RNAs (lncRNA) are emerging as major regulators of cellular phenotypes and implicated as oncogenes or tumor suppressors. Here, we report a novel tumor-suppressive locus on human chromosome 15q23 that contains two multiexonic lncRNA genes of 100 kb each: DRAIC (LOC145837) and the recently reported PCAT29. The DRAIC lncRNA was identified from RNA-seq data and is downregulated as prostate cancer cells progress from an androgen-dependent (AD) to a castration-resistant (CR) state. Prostate cancers persisting in patients after androgen deprivation therapy (ADT) select for decreased DRAIC expression, and higher levels of DRAIC in prostate cancer are associated with longer disease-free survival (DFS). Androgen induced androgen receptor (AR) binding to the DRAIC locus and repressed DRAIC expression. In contrast, FOXA1 and NKX3-1 are recruited to the DRAIC locus to induce DRAIC, and FOXA1 specifically counters the repression of DRAIC by AR. The decrease of FOXA1 and NKX3-1, and aberrant activation of AR, thus accounts for the decrease of DRAIC during prostate cancer progression to the CR state. Consistent with DRAIC being a good prognostic marker, DRAIC prevents the transformation of cuboidal epithelial cells to fibroblast-like morphology and prevents cellular migration and invasion. A second tumor-suppressive lncRNA PCAT29, located 20 kb downstream of DRAIC, is regulated identically by AR and FOXA1 and also suppresses cellular migration and metastasis. Finally, based on TCGA analysis, DRAIC expression predicts good prognosis in a wide range of malignancies, including bladder cancer, low-grade gliomas, lung adenocarcinoma, stomach adenocarcinoma, renal clear cell carcinoma, hepatocellular carcinoma, skin melanoma, and stomach adenocarcinoma. This study reveals a novel tumor-suppressive locus encoding two hormone-regulated lncRNAs, DRAIC and PCAT29, that are prognostic for a wide variety of cancer types.

Genome-wide analysis of androgen receptor binding sites in prostate cancer cells.

The transformation of prostate cancer from an androgen-dependent state to an androgen-independent state is a lethal progression. Alterations in transcriptional programs are the basis of prostate cancer deterioration. The androgen receptor (AR), a member of the nuclear hormone receptor superfamily, mediates prostate cancer progression by functioning primarily through the ligand-activated transcription of target genes. Therefore, a detailed map of AR-regulated genes and AR genomic binding sites is required for hormone-naive and castration-resistant prostate cancer. Through the use of chromatin immunoprecipitation in combination with direct sequencing, 4,143 AR binding sites were defined in the LNCaP androgen-sensitive prostate cancer cell line. Using the same method, 2,380 AR binding regions were identified in the LNCaP-AI long-term androgen-deprived cell line. Approximately 8.5% (354/4,143) of the binding regions were mapped to within 2 kb of the transcription start site (TSS) in the LNCaP cells, while ∼12.6% (299/2,380) were mapped to within 2 kb of the TSS in the LNCaP-AI cells. In total, the study mapped 2,796 genes in LNCaP cells and 1,854 genes in LNCaP-AI cells. The cell lines shared 789 mutual genes. In addition, gene ontology (GO) analysis of the genes revealed that there was a notable overlap between the GO terms in the LNCaP cells and LNCaP-AI cells. However, GO terms within the biological process domain that were only observed in the LNCaP-AI cells included the reproduction process, death, immune system process, multi-organism process, pigmentation and viral reproduction. The major genes in the different GO terms were TNFAIP8, RTN4, APP and SYNE1. Through analyzing the AR binding sites in the two cell types, the present study aimed to map potential AR-regulated genes, identify their associated transcription factors and provide a new perspective on the biological processes in the development of prostate cancer. The results provided a valuable data set that furthered the understanding of the genome-wide analysis of AR binding sites in prostate cancer cells, which may be exploited for the development of novel prostate cancer therapeutic strategies.