Repression Of Androgen Receptor Research Articles

Shikonin inhibits the growth of human prostate cancer cells via modulation of the androgen receptor

Shikonin, a natural naphthoquinone isolated from the traditional Chinese medicine Zi Cao (gromwell), has been shown to possess tumor cell killing activity. The human androgen receptor (AR) is a nuclear transcription factor that serves as a major therapeutic target for prostate cancer. However, AR regulation by shikonin has not been reported. We investigated the effects of shikonin on the growth of prostate cancer cells. We observed that shikonin decreased the expression of AR at both the mRNA and the protein levels in LNCaP and 22RV1 human prostate cancer cells. The results from a luciferase assay showed that shikonin decreased the transcriptional activity of AR. Moreover, shikonin treatment inhibited AR target gene expression, PSA and growth inhibition of prostate cancer cells. In conclusion, the present study shows for the first time that shikonin treatment causes transcriptional repression of AR and inhibition of its nuclear localization in human prostate cancer cells. We propose that shikonin, an anticancer drug extracted from natural sources, induces inhibition of cell growth through modulation of AR in androgen-responsive prostate cancer cells and is a candidate for use in cancer chemotherapy for human prostate cancer.

RANK- and c-Met-mediated signal network promotes prostate cancer metastatic colonization

Prostate cancer (PCa) metastasis to bone is lethal and there is no adequate animal model for studying the mechanisms underlying the metastatic process. Here, we report that receptor activator of NF-κB ligand (RANKL) expressed by PCa cells consistently induced colonization or metastasis to bone in animal models. RANK-mediated signaling established a premetastatic niche through a feed-forward loop, involving the induction of RANKL and c-Met, but repression of androgen receptor (AR) expression and AR signaling pathways. Site-directed mutagenesis and transcription factor (TF) deletion/interference assays identified common TF complexes, c-Myc/Max, and AP4 as critical regulatory nodes. RANKL–RANK signaling activated a number of master regulator TFs that control the epithelial-to-mesenchymal transition (Twist1, Slug, Zeb1, and Zeb2), stem cell properties (Sox2, Myc, Oct3/4, and Nanog), neuroendocrine differentiation (Sox9, HIF1α, and FoxA2), and osteomimicry (c-Myc/Max, Sox2, Sox9, HIF1α, and Runx2). Abrogating RANK or its downstream c-Myc/Max or c-Met signaling network minimized or abolished skeletal metastasis in mice. RANKL-expressing LNCaP cells recruited and induced neighboring non metastatic LNCaP cells to express RANKL, c-Met/activated c-Met, while downregulating AR expression. These initially non-metastatic cells, once retrieved from the tumors, acquired the potential to colonize and grow in bone. These findings identify a novel mechanism of tumor growth in bone that involves tumor cell reprogramming via RANK–RANKL signaling, as well as a form of signal amplification that mediates recruitment and stable transformation of non-metastatic bystander dormant cells.

Mechanism of androgen receptor corepression by CKβBP2/CRIF1, a multifunctional transcription factor coregulator expressed in prostate cancer

The transcription factor coregulator Casein kinase IIβ-binding protein 2 or CR6-interacting factor 1 (CKβBP2/CRIF1) binds the androgen receptor (AR) in prostate cancer cells and in response to dihydrotestosterone localizes with AR on the prostate-specific antigen gene enhancer, but does not bind DNA suggesting CKβBP2/CRIF1 localization in chromatin is determined by AR. In this study we show also that CKβBP2/CRIF1 inhibits wild-type AR and AR N-terminal transcriptional activity, binds to the AR C-terminal region, inhibits interaction of the AR N- and C-terminal domains (N/C interaction) and competes with p160 coactivator binding to the AR C-terminal domain, suggesting CKβBP2/CRIF1 interferes with AR activation functions 1 and 2. CKβBP2/CRIF1 is expressed mainly in stromal cells of benign prostatic hyperplasia and in stroma and epithelium of prostate cancer. CKβBP2/CRIF1 protein is increased in epithelium of androgen-dependent prostate cancer compared to benign prostatic hyperplasia and decreased slightly in castration recurrent epithelium compared to androgen-dependent prostate cancer. The multifunctional CKβBP2/CRIF1 is a STAT3 interacting protein and reported to be a coactivator of STAT3. CKβBP2/CRIF1 is expressed with STAT3 in prostate cancer where STAT3 may help to offset the AR repressor effect of CKβBP2/CRIF1 and allow AR regulation of prostate cancer growth.

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.

Hypoxia-Independent Downregulation of Hypoxia-Inducible Factor 1 Targets by Androgen Deprivation Therapy in Prostate Cancer

We explored changes in hypoxia-inducible factor 1 (HIF1) signaling during androgen deprivation therapy (ADT) of androgen-sensitive prostate cancer xenografts under conditions in which no significant change in immunostaining of the hypoxia marker pimonidazole had occurred. Gene expression profiles of volume-matched androgen-exposed and androgen-deprived CWR22 xenografts, with similar pimonidazole-positive fractions, were compared. Direct targets of androgen receptor (AR) and HIF1 transcription factors were identified among the differentially expressed genes by using published lists. Biological processes affected by ADT were determined by gene ontology analysis. HIF1α protein expression in xenografts and biopsy samples from 35 patients receiving neoadjuvant ADT was assessed by immunohistochemistry. A total of 1344 genes showed more than 2-fold change in expression by ADT, including 35 downregulated and 5 upregulated HIF1 targets. Six genes were shared HIF1 and AR targets, and their downregulation was confirmed with quantitative RT-PCR. Significant suppression of the biological processes proliferation, metabolism, and stress response in androgen-deprived xenografts was found, consistent with tumor regression. Nineteen downregulated HIF1 targets were involved in those significant biological processes, most of them in metabolism. Four of these were shared AR and HIF1 targets, including genes encoding the regulatory glycolytic proteins HK2, PFKFB3, and SLC2A1. Most of the downregulated HIF1 targets were induced by hypoxia in androgen-responsive prostate cancer cell lines, confirming their role as hypoxia-responsive HIF1 targets in prostate cancer. Downregulation of HIF1 targets was consistent with the absence of HIF1α protein in xenografts and downregulation in patients by ADT (P<.001). AR repression by ADT may lead to downregulation of HIF1 signaling independently of hypoxic fraction, and this may contribute to tumor regression. HIF1α expression is probably not a useful hypoxia biomarker during ADT in prostate cancer.

Scaffold attachment factor B1 regulates the androgen receptor in concert with the growth inhibitory kinase MST1 and the methyltransferase EZH2

The androgen receptor (AR) is a transcription factor that employs many diverse interactions with coregulatory proteins in normal physiology and in prostate cancer (PCa). The AR mediates cellular responses in association with chromatin complexes and kinase cascades. Here we report that the nuclear matrix protein, scaffold attachment factor B1 (SAFB1), regulates AR activity and AR levels in a manner that suggests its involvement in PCa. SAFB1 mRNA expression was lower in PCa in comparison with normal prostate tissue in a majority of publicly available RNA expression data sets. SAFB1 protein levels were also reduced with disease progression in a cohort of human PCa that included metastatic tumors. SAFB1 bound to AR and was phosphorylated by the MST1 (Hippo homolog) serine-threonine kinase, previously shown to be an AR repressor, and MST1 localization to AR-dependent promoters was inhibited by SAFB1 depletion. Knockdown of SAFB1 in androgen-dependent LNCaP PCa cells increased AR and prostate-specific antigen (PSA) levels, stimulated growth of cultured cells and subcutaneous xenografts and promoted a more aggressive phenotype, consistent with a repressive AR regulatory function. SAFB1 formed a complex with the histone methyltransferase EZH2 at AR-interacting chromatin sites in association with other polycomb repressive complex 2 (PRC2) proteins. We conclude that SAFB1 acts as a novel AR co-regulator at gene loci where signals from the MST1/Hippo and EZH2 pathways converge.

Abstract A21: Orphan nuclear receptor TLX recruits lysine-specific demethylase 1 to repress androgen receptor gene transcription and functions to promote hormone-resistant growth of prostate cancer cells

Abstract Prostate cancer (PCa) is the most frequently diagnosed type of cancer and the second leading cause of death from cancer in males in the United States. The initial growth of most localized PCa is slow and androgen-dependent. Thus, surgical treatment (prostatectomy) and hormone therapy (androgen deprivation) are effective initially. However, many patients relapse and progress to a fatal androgen-independent and bone-metastatic stage shortly when both primary and secondary hormone therapies fail. Unfortunately, the current options for the metastatic hormone-refractory PCa are still limited and mostly ineffective, resulting in high mortality of this disease. Although dysregulated androgen receptor (AR) signaling pathway has been proposed to contribute to the development of hormone-resistance, the involved mechanisms are still not fully defined. Our present findings revealed that the orphan nuclear receptor TLX (NR2E1) exhibited increased expression patterns in androgen-independent and antiandrogen-resistant PCa cells, and also the high-grade and hormone-refractory clinical PCa tissues. Stable overexpression of TLX in AR-positive LNCaP cells could promote both androgen-independent and antiandrogen-resistant cell growth in vitro, and enhance tumor growth capacity in castrated male SCID mice in vivo. Decreased expression of AR and AR target genes in LNCaP-TLX cells were identified, while results of AR-promoter driven luciferase reporter assay also revealed that TLX overexpression could suppress the AR transcription in non-prostatic HEK293 cells. Furthermore, we also identified the direct binding site of TLX on the AR promoter by chromatin immunoprecipitation analyses in TLX-transfected HEK293 cells. The molecular mechanism may involve the TLX-mediated recruitment of lysine-specific demethylase 1 (LSD1) as treatment with an inhibitor of LSD1 pargyline could reverse the transrepressive regulation of TLX on AR-promoter driven luciferase activity and restore AR gene expression levels in LNCaP cells. In summary, our results showed for the first time that overexpression of TLX might contribute to the hormone-resistant PCa cell growth and advanced progression of PCa. LSD1 might serve as a corepressor in TLX-mediated direct transrepression on AR gene expression. This work is partly supported by an RGC-General Research Fund CUHK461012. Citation Format: Lin Jia, Dinglan Wu, Shan Yu, Franky Leung Chan. Orphan nuclear receptor TLX recruits lysine-specific demethylase 1 to repress androgen receptor gene transcription and functions to promote hormone-resistant growth of prostate cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A21.

ARF represses androgen receptor transactivation in prostate cancer.

Androgen receptor (AR) signaling is essential for prostate cancer (PCa) development in humans. The initiation of prostate malignancy and progression to a castration-resistant stage are largely contributed by the modulation of AR activity through its coregulatory proteins. We and others previously reported that p14 alternative reading frame (ARF) expression is positively correlated with the disease progression and severity of PCa. Here, we provide evidence that p14ARF physically interacts with AR and functions as an AR corespressor in both an androgen-dependent and androgen-independent manner. Endogenous ARF (p14ARF in human and p19ARF in mouse) and AR colocalize in both human PCa cells in vitro and PCa tissues of mouse and human in vivo. Overexpression of p14ARF in PCa cells significantly attenuates the activities of androgen response region (ARR2)-probasin and prostate-specific antigen (PSA) promoters. The forced expression of p14ARF in cells resulted in a suppression of PSA and NK transcription factor locus 1 (NKX3.1) expression. Conversely, knockdown of endogenous p14ARF in human PCa cells with short hairpin RNA enhanced AR transactivation activities in a dose-dependent and p53-independent manner. Furthermore, we demonstrated that p14ARF binds to both the N-terminal domain and the ligand-binding domain of AR, and the human double minute 2 (HDM2)-binding motif of p14ARF is required for the interaction of p14ARF and AR proteins. p14ARF perturbs the androgen-induced interaction between the N terminus and C terminus of AR. Most importantly, we observed that the expression of PSA is reversely correlated with p14ARF in human prostate tissues. Taken together, our results reveal a novel function of ARF in modulation of AR transactivation in PCa.

Androgen receptor repression of gonadotropin-releasing hormone gene transcription via enhancer 1

Gonadotropin-releasing hormone (GnRH) plays a major role in the hypothalamic-pituitary–gonadal (HPG) axis, and synthesis and secretion of GnRH are regulated by gonadal steroid hormones. Disruptions in androgen levels are involved in a number of reproductive defects, including hypogonadotropic hypogonadism and polycystic ovarian syndrome. Androgens down-regulate GnRH mRNA synthesis in vivo and in vitro via an androgen receptor (AR)-dependent mechanism. Methyltrienolone (R1881), a synthetic AR agonist, represses GnRH expression through multiple sites in the proximal promoter. In this study, we show AR also represses GnRH transcription via the major enhancer (GnRH-E1). A multimer of the −1800/−1766 region was repressed by R1881 treatment. Mutation of two bases, −1792 and −1791, resulted in decreased basal activity and a loss of AR-mediated repression. AR bound to the −1796/−1791 sequence in electrophoretic mobility shift assays, indicating a direct interaction with DNA or other transcription factors in this region. We conclude that AR repression of GnRH-E1 acts via multiple AR-responsive regions, including the site at −1792/−1791.

Abstract B12: LSD1 mediates global AR transcription suppression in prostate cancer cells

Abstract This project is to study the molecular basis of LSD1-dependent AR suppression in prostate cancer cells. Androgen receptor (AR) is highly expressed in prostate cancer (PCa) and plays a pivotal role in tumor growth. Patients generally respond to androgen deprivation therapy (ADT). However, the tumors invariably relapse, and these relapsed tumors (called castration-resistant prostate cancer, CRPC) are generally more aggressive and relatively resistant to current AR antagonist treatments. Significantly, these relapsed tumors express increased levels of AR mRNA and express multiple AR regulated genes, indicating that AR transcriptional activity has been restored. Despite the critical role AR plays in PCa development and progression to CRPC, the mechanisms that contribute to the consistent substantial increases in AR mRNA in CRPC are poorly understood. Using a VCaP xenograft model, we initially detected a rapid and substantial upregulation of AR mRNA upon castration, suggesting a negative feedback loop regulating AR mRNA levels, which may make a significant contribution to increasing AR mRNA in CRPC (Cancer Research 2009, 69:6027). In a recent published study (Cancer Cell 2011, 20:457), we reported that the agonist liganded AR binds to an enhancer in the second intron of the AR gene, and that increased AR gene expression and progression to CRPC is associated with increased H3K4 methylation and increased recruitment of other transcription factors to this site. Significantly, in contrast to AR function as a transcriptional activator on previously studied androgen responsive elements (AREs), we found that the agonist liganded AR functions as a direct transcriptional repressor at this site. The repression is due to AR recruitment of an H3K4 demethylase, LSD1. Interestingly, AR binding to this site and repression of AR gene expression occurs at slightly higher androgen levels than those required for classical androgen regulated genes, consistent with a negative feedback loop to regulate AR signaling. Pathway analyses of additional AR repressed genes showed marked enrichment for genes mediating DNA synthesis and cell cycle progression, while AR stimulated genes were associated with lipid/protein synthesis and cellular metabolism. Significantly, a set of these androgen repressed genes that are associated with proliferation are overexpressed in CRPC. Taken together, these results indicate that the agonist liganded AR functions as a transcriptional repressor on a subset of genes that enhance proliferation. We propose that ADT is effective because it initially suppresses all AR functions, but that the partial restoration of androgen levels and AR activity in CRPC cells may provide a strong growth advantage by stimulating cellular metabolism without downregulation of AR repressed genes that enhance cellular proliferation. Combining the expression array data and ChIP-seq analysis on AR in VCaP and VCaP-derived castration-resistant VCS2 cells, we have identified gene subsets with AR direct binding on the gene locus. Interestingly, while AR binding is highly enriched (∼2fold) in AR-activated gene subset, the binding is less enriched (1.4fold) in AR-suppressed gene subset. The lower enrichment could mean that fewer genes in the AR –repressed group are directly regulated by AR, but could also be in part technical and reflect somewhat weaker binding of AR to AR-repressed genes. To further elucidate the molecular basis for AR suppression function, we focused on the role of LSD1 in the global AR transcriptional repression as previous results showed that expressions of a number of AR-suppressed genes are dependent on LSD1 activity. LSD1 was initially identified in corepressor complexes and shown to function by demethylating mono- and dimethylated H3K4. However, it was subsequently shown to also function as a coactivator through demethylation of repressive mono- and dimethylated H3K9 when associated with AR. Our data indicate that the association with AR does not determine the coactivator versus corepressor function of LSD1, and that it is instead determined by properties of the element to which it is being recruited. Through genome wide ChIP-seq analysis of LSD1 in VCaP and LNCaP cells, we found that LSD1 binding is significantly enriched for both AR-activated and –repressed (both ∼1.5fold) genes, suggesting that both AR activation and repression are dependent on LSD1 activity. Furthermore, we searched the transcription factor-binding motif on LSD1 binding sites. While E2F1 is highly enriched for LSD1 binding in both activation and repression loci, the ZBTB (zinc finger and BTB domain containing) transcription repressor binding motif was only significantly enriched in repression loci. Among this family, PLZF and LRF are highly expressed in prostate cancer cells. Therefore, the current work has focused on these two factors and we propose that LSD1 mediates AR gene suppression through interaction with ZBTB transcription repressors. Citation Format: Changmeng Cai, Housheng He, Sen Chen, X. Shirley Liu, Myles Brown, Steven P. Balk. LSD1 mediates global AR transcription suppression in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B12.

Androgen receptor repression of GnRH gene transcription.

Alterations in androgen levels lead to reproductive defects in both males and females, including hypogonadotropic hypogonadism, anovulation, and infertility. Androgens have been shown to down-regulate GnRH mRNA levels through an androgen receptor (AR)-dependent mechanism. Here, we investigate how androgen regulates expression from the GnRH regulatory region in the GT1-7 cell line, a model of GnRH neurons. A synthetic androgen, R1881, repressed transcription from the GnRH promoter (GnRH-P) in an AR-dependent manner, and liganded AR associated with the chromatin at the GnRH-P in live GT1-7 cells. The three known octamer-binding transcription factor-1 (Oct-1) binding sites in GnRH-P were required for AR-mediated repression, although other sequences were also involved. Although a multimer of the consensus Oct-1 binding site was not repressed, a multimer of the cluster of Oct-1, Pre-B cell leukemia transcription factor (Pbx)/Prep, and NK2 homeobox 1 (Nkx2.1) binding sites, found at -106/-91 in GnRH-P, was sufficient for repression. In fact, overexpression of any of these factors disrupted the androgen response, indicating that a balance of factors in this tripartite complex is required for AR repression. AR bound to this region in EMSA, indicating a direct interaction of AR with DNA or with other transcription factors bound to GnRH-P at this sequence. Collectively, our data demonstrate that GnRH transcription is repressed by AR via multiple sequences in GnRH-P, including three Oct-1 binding sites, and that this repression requires the complex interaction of several transcription factors.

Inability of NCoR/SMRT to repress androgen receptor transcriptional activity in prostate cancer cell lines

The molecular mechanisms leading to castration-resistant prostate cancer (CRPC) are poorly understood. Among several mechanisms leading to CRPC growth a dysregulation of androgen receptor (AR) co-regulators (i.e. up-regulation of co-activators or down-regulation of co-repressors) is discussed. There are numerous reports demonstrating an increased expression of co-activators during prostate cancer progression. On the contrary, the impact of co-repressors on tumor growth and development is less clear. In this study we compared the effects of two known co-repressors, NCoR and SMRT, on AR transcriptional activity in prostate cancer (PCa) cell lines and compared them to that in COS-1 cells. Interestingly, we found that NCoR/SMRT overexpression did not repress AR-dependent gene expression in the PCa cell lines, but rather activated it. This finding is probably due to an impaired AR-co-repressor interaction in the prostate cancer cell lines. In conclusion, we provide evidence that up-regulation of NCoR or SMRT may increase transcriptional activity of the AR in a cell type-specific context.

Repression of Androgen Receptor Activity by HEYL, a Third Member of the Hairy/Enhancer-of-split-related Family of Notch Effectors

The Hairy/Enhancer-of-split-related with YRPW-like motif (HEY) family of proteins are transcriptional repressors and downstream effectors of Notch signaling. We previously reported that HEY1 and HEY2 selectively repress androgen receptor (AR) signaling in mammalian cell lines and have shown that in human tissue HEY1 is excluded from the nuclei in prostate cancer but not benign prostatic hyperplasia. We have now characterized a third member of this family, HEYL, which is a more potent repressor of AR activity. HEYL interacted with and repressed AR activation function-1 domain and competitively inhibited SRC1e activation of AR transcriptional activity. Using a cell line inducibly expressing exogenous HEYL, we showed that HEYL represses endogenous AR-regulated genes and reduces androgen-dependent prostate cancer cell growth. Using a trans-repression assay, we identified both trichostatin-sensitive and -insensitive domains within HEYL; however, analysis of endogenous AR target genes suggested that HEYL represses AR activity through histone deacetylase I/II-independent mechanisms. Immunohistochemical analyses of tissue indicated that, in a fashion similar to that previously reported for HEY1, HEYL is excluded from the nuclei in prostate cancer but not adjacent benign tissue. This suggests that nuclear exclusion of HEY proteins may be an important step in the progression of prostate cancer.

FGFR1 abrogates inhibitory effect of androgen receptor concurrent with induction of androgen‐receptor variants in androgen receptor‐negative prostate tumor epithelial cells

Despite dramatic positive effects, there is evidence that the androgen receptor (AR) may negatively influence prostate tumor progression. Understanding the AR repressor function and how it is subverted is of particular importance in anti-androgen and AR intervention strategies. AR, resident FGFR2IIIb, and ectopic FGFR1 were expressed by transfection in the AR-negative epithelial cell line DTE that predominates in cell culture of AR-positive androgen-responsive model Dunning R3327 rat prostate tumors. Androgen-responsiveness at transcription was measured by a luciferase reporter. Cell population growth rates were assessed by cell counts, DNA synthesis, and expression of cell cycle genes. AR variants (ARVs) were assessed by immunochemistry and nuclease protection of mRNA. Expression of AR inhibited cell population growth of AR-negative DTE cells at the G1-S phase of the cell cycle. Ectopic FGFR1, but not resident FGFR2IIIb abrogated the growth inhibitory effects of AR. Appearance of ARVs was coincident with co-expression of FGFR1 and AR and abrogation of the AR-dependent inhibition of cell growth. DTE cells may represent non-malignant AR-negative progenitors whose population is restricted by activation of AR in vivo. Ectopic expression of epithelial FGFR1, a common observation in tumors, overrides the inhibition of AR and thus may contribute to evolution of androgen and AR independent tumors. These results are consistent with the notion that some tumor cells are negatively restricted by AR and are unleased by androgen-deprivation or ectopic expression of FGFR1. ARV's may play a role in the bypass of the negative restrictions of AR.

184 Histone deacetylase inhibitor Belinostat (PXD-101) represses androgen receptor expression and acts synergistically with castration and bicalutamide treatment to inhibit prostate cancer growth hormone refractory models

184 Histone deacetylase inhibitor Belinostat (PXD-101) represses androgen receptor expression and acts synergistically with castration and bicalutamide treatment to inhibit prostate cancer growth hormone refractory models

Abstract 3804: Transcriptional repression of androgen receptor in human prostate cancer cells by plant flavone apigenin

Abstract Accumulating evidence suggest that androgen receptor (AR)-mediated signaling plays an important role in the development and progression of prostate cancer. Although Prostate Cancer Prevention Trial (PCPT) showed controversial results however manipulation of AR signaling pathway reduced the incidence of prostate cancer. Safer agents are needed that can modulate AR signaling pathways with effective suppression of prostate cancer. Plant flavonoids have been shown to possess weak oestrogenic and anti-androgen activity. Apigenin, a naturally occurring plant flavone, has shown promising anticancer activity by modulating several signaling pathways that have relevance to prostate cancer. However, the effect of apigenin on AR signaling has not been fully investigated. Using androgen-responsive human prostate cancer LNCaP and 22Rv1cells, we determine the effect of apigenin on androgen receptor signaling. Exposure of these cells with 5-40μM apigenin suppressed cell proliferation and arrested cells in sub G0-G1 phase of the cell cycle after 5nM dihydrotestosterone (DHT) stimulation. Apigenin inhibited endogenous prostate-specific antigen (PSA) transcription and reduced intracellular and secreted PSA protein levels induced by DHT in these cells. In LNCaP cells apigenin inhibited AR expression in a concentration-dependent manner by restricting its translocation into the nucleus. Co-transfection of PC-3 cells with wild-type AR-GFP-tag plasmid demonstrated that apigenin has ability to inhibit DHT-induced AR translocation into the nucleus. Additionally, receptor binding assays indicated that apigenin is a strong competitive inhibitor of DHT binding to the AR. These observations matched with the results of structural modeling studies exhibiting that apigenin is remarkably similar in conformational geometry and surface charge distribution to synthetic AR antagonist, flutamide, although the atomic compositions of the two substances were quite different. Taken together, our results indicate that apigenin exhibits potent anti-proliferative and anti-androgenic properties and may be developed as useful and safer agent for prevention/therapy of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3804.

HOXB13 is co-localized with androgen receptor to suppress androgen-stimulated prostate-specific antigen expression

During the prostate cancer (PCa) development and its progression into hormone independency, androgen receptor (AR) signals play a central role by triggering the regulation of target genes, including prostate-specific antigen. However, the regulation of these AR-mediated target genes is not fully understood. We have previously demonstrated a unique role of HOXB13 homeodomain protein as an AR repressor. Expression of HOXB13 was highly restricted to the prostate and its suppression dramatically increased hormone-activated AR transactivation, suggesting that prostate-specific HOXB13 was a highly potent transcriptional regulator. In this report, we demonstrated the action mechanism of HOXB13 as an AR repressor. HOXB13 suppressed androgen-stimulated AR activity by interacting with AR. HOXB13 did neither bind to AR responsive elements nor disturb nuclear translocation of AR in response to androgen. In PCa specimen, we also observed mutual expression pattern of HOXB13 and AR. These results suggest that HOXB13 not only serve as a DNA-bound transcription factor but play an important role as an AR-interacting repressor to modulate hormone-activated androgen receptor signals. Further extensive studies will uncover a novel mechanism for regulating AR-signaling pathway to lead to expose new role of HOXB13 as a non-DNA-binding transcriptional repressor.

Reduced Tumor Necrosis Factor Receptor–Associated Death Domain Expression Is Associated with Prostate Cancer Progression

By using LNCaP and its derivative cell lines, we first observed an association between tumor necrosis factor-alpha (TNF-alpha) resistance and hormone independence. Moreover, we found that the expression of tumor necrosis factor receptor-associated death domain (TRADD) was reduced in androgen deprivation-independent cells compared with that in androgen deprivation-dependent cells. TRADD is a crucial transducer for TNF-alpha-induced nuclear factor-kappaB (NF-kappaB) activation. Knocking down TRADD expression in LNCaP cells impaired TNF-alpha-induced NF-kappaB activation and androgen receptor repression, whereas overexpression of TRADD in C4-2B cells restored their sensitivity to TNF-alpha. Finally, we found that androgen deprivation reduces TRADD expression in vitro and in vivo, suggesting that androgen deprivation therapy may promote the development of TNF-alpha resistance by reducing TRADD expression during prostate cancer progression.

Inhibition of the Androgen Receptor as a Novel Mechanism of Taxol Chemotherapy in Prostate Cancer

Taxol chemotherapy is one of the few therapeutic options for men with castration-resistant prostate cancer (CRPC). However, the working mechanisms for Taxol are not fully understood. Here, we showed that treatment of 22Rv1, a PTEN-positive CRPC cell line, with paclitaxel and its semisynthetic analogue docetaxel decreases expression of the androgen receptor (AR)-activated genes prostate-specific antigen (PSA) and Nkx3.1 but increases expression of the AR repression gene maspin, suggesting that Taxol treatment inhibits AR activity. This was further supported by the observation that the activity of AR luciferase reporter genes was inhibited by paclitaxel. In contrast, paclitaxel treatment failed to inhibit AR activity in the PTEN-null CRPC cell line C4-2. However, pretreatment of C4-2 cells with the phosphoinositide 3-kinase inhibitor LY294002 restored paclitaxel inhibition of the AR. Treatment of 22Rv1 xenografts in mice with docetaxel induced mitotic arrest and a decrease in PSA expression in tumor cells adjacent to vascular vessels. We further showed that paclitaxel induces nuclear accumulation of FOXO1, a known AR suppressive nuclear factor, and increases the association of FOXO1 with AR proteins in the nucleus. FOXO1 knockdown with small interfering RNA attenuated the inhibitory effect of paclitaxel on AR transcriptional activity, expression of PSA and Nkx3.1, and cell survival. These data reveal a previously uncharacterized, FOXO1-mediated, AR-inhibitory effect of Taxol in CRPC cells that may play an important role in Taxol-mediated inhibition of CRPC growth.

D,l-Sulforaphane causes transcriptional repression of androgen receptor in human prostate cancer cells

D,L-Sulforaphane (SFN), a synthetic analogue of cruciferous vegetable-derived L-isomer, inhibits the growth of human prostate cancer cells in culture and in vivo and retards cancer development in a transgenic mouse model of prostate cancer. We now show that SFN treatment causes transcriptional repression of androgen receptor (AR) in LNCaP and C4-2 human prostate cancer cells at pharmacologic concentrations. Exposure of LNCaP and C4-2 cells to SFN resulted in a concentration-dependent and time-dependent decrease in protein levels of total AR as well as Ser(210/213)-phosphorylated AR. The SFN-mediated decline in AR protein level was accompanied by a decrease in intracellular as well as secreted levels of prostate-specific antigen, an AR-regulated gene product. The decrease in AR protein level resulting from SFN exposure was not reversed in the presence of the protein synthesis inhibitor cycloheximide. Reverse transcription-PCR analysis revealed a dose-dependent decrease in AR mRNA levels, indicating transcriptional repression of this ligand-activated transcription factor. The SFN treatment inhibited AR promoter activity as revealed by luciferase reporter assay. Synthetic androgen (R1881)-stimulated nuclear translocation of AR was markedly suppressed in the presence of SFN in both cell lines. The SFN treatment also inhibited R1881-stimulated proliferation of LNCaP cells. Naturally occurring thio analogues (iberverin, erucin, and berteroin), but not the sulfonyl analogues (cheirolin, erysolin, and alyssin sulfone), of SFN were also effective in reducing protein levels of AR in LNCaP cells. In conclusion, the present study shows for the first time that SFN treatment causes transcriptional repression of AR and inhibition of its nuclear localization in human prostate cancer cells.