Repression Of Androgen Receptor Research Articles

-Tocopheryl succinate and derivatives mediate the transcriptional repression of androgen receptor in prostate cancer cells by targeting the PP2A-JNK-Sp1-signaling axis

As part of our effort to understand the mechanism underlying alpha-tocopheryl succinate [vitamin E succinate (VES)]-mediated antitumor effects, we investigated the signaling pathway by which VES suppresses androgen receptor (AR) expression in prostate cancer cells. VES and, to a greater extent, its truncated derivative TS-1 mediated transcriptional repression of AR in prostate cancer cells but not in normal prostate epithelial cells; a finding that underscores the differential susceptibility of normal versus malignant cells to the antiproliferative effect of these agents. This AR repression was attributable to the ability of VES and TS-1 to facilitate the proteasomal degradation of the transcription factor Sp1. This mechanistic link was corroborated by the finding that proteasome inhibitors or ectopic expression of Sp1 protected cells against drug-induced AR ablation. Furthermore, evidence suggests that the destabilization of Sp1 by VES and TS-1 resulted from the inactivation of Jun N-terminal kinases (JNKs) as a consequence of increased phosphatase activity of protein phosphatase 2A (PP2A). Stable transfection of LNCaP cells with the dominant-negative JNK1 plasmid mimicked drug-induced Sp1 repression, whereas constitutive activation of JNK kinase activity or inhibition of PP2A activity by okadaic acid protected Sp1 from VES- and TS-1-induced degradation. From a mechanistic perspective, the ability of VES and TS-1 to activate PP2A activity underscores their broad spectrum of effects on multiple signaling mechanisms, including those mediated by Akt, mitogen-activated protein kinases, nuclear factor kappaB, Sp1 and AR. This pleiotropic effect in conjunction with low toxicity suggests the translational potential for developing TS-1 into potent PP2A-activating agents for cancer therapy.

Secreted Frizzled-related protein-1 is a negative regulator of androgen receptor activity in prostate cancer.

Secreted Frizzled-related protein-1 (sFRP1) associates with Wnt proteins and its loss can lead to activation of Wnt/β-catenin signalling. It is frequently downregulated in cancer, including prostate cancer, but its function in prostate cancer is unclear because it can increase proliferation of prostate epithelial cells. We investigated the function of sFRP1 in androgen-dependent prostate cancer and found that sFRP1 inhibited androgen receptor (AR) transcriptional activity. In addition, sFRP1 inhibited the proliferation of androgen-dependent LNCaP cells but not of an androgen-independent subline LNCaP-r, suggesting a role in androgen-dependent growth. The inhibition of AR by sFRP1 was unaffected by co-expression of Wnt3a, stabilised β-catenin or β-catenin shRNA, suggesting it does not involve Wnt/β-catenin signalling. Wnt5a also inhibited AR and expression of Wnt5a and sFRP1 together did not further inhibit AR, suggesting that Wnt5a and sFRP1 activate the same signal(s) to inhibit AR. However, sFRP1 inhibition of AR was unaffected by inhibitors of kinases involved in Wnt/Ca2+ and Wnt/planar cell polarity non-canonical Wnt signalling. Interestingly, the cysteine-rich domain of sFRP1 interacted with Frizzled receptors expressed in prostate cancer cells, suggesting that sFRP1/Frizzled complexes activate a signal that leads to repression of AR. Taken together, these observations highlight the function of β-catenin-independent Wnt signalling in the control of AR activity and provide one explanation for sFRP1 downregulation in prostate cancer.

Inhibition of MAPK-signaling pathway promotes the interaction of the corepressor SMRT with the human androgen receptor and mediates repression of prostate cancer cell growth in the presence of antiandrogens

Prostate cancer is one of the most prominent malignancies of elderly males. The growth of normal prostate and prostate cancer (PCa) cells depend on functional androgen receptor (AR), a ligand controlled transcription factor and member of the nuclear receptor superfamily. Binding of agonistic ligand enhances the transactivation function of AR and hence promotes the growth of prostate epithelial cells. We have earlier shown that AR antagonistic ligands such as cyproterone acetate (CPA) promote the recruitment of transcriptional corepressors such as silencing mediator of retinoid and thyroid receptor (SMRT) leading to repression of AR transactivation in non-PCa cells. Unfortunately, however, in LNCaP PCa cells, CPA functions as an agonist and thereby increases AR transactivation function. Here, we show that activated MEK signaling cascade inhibits functional recruitment of corepressor SMRT to CPA-bound AR in PCa cells. Chemical blockade of MEK kinase using a specific inhibitor U0126 increases the interaction and hence repression of AR by the corepressor SMRT in LNCaP PCa cells. This inhibition also results in enhanced antagonistic behavior of CPA as assessed by reporter and cell-growth assays. Moreover, the growth of LNCaP cells stably overexpressing SMRT was more robustly inhibited in the presence of CPA and U1026. In line with this, the growth rate of LNCaP cells was decelerated in the presence of both CPA and U0126. This suggests that activated MEK signaling pathway attenuates the functional recruitment of corepressor SMRT to AR induced by antagonists and thus indicates the important role of corepressors in mediating repression of both AR transactivation and PCa cell growth by antagonists. Furthermore, these findings suggest that combining receptor antagonists with signaling inhibitors could be a beneficial approach for PCa treatment.

Loss of Androgen Receptor in Aging and Oxidative Stress through Myb Protooncoprotein-regulated Reciprocal Chromatin Dynamics of p53 and Poly(ADP-ribose) Polymerase PARP-1

Poly(ADP-ribosyl)ation of transcription factors and coregulators, mediated by the poly(ADP-ribose) polymerase PARP-1, has been emerging as an important epigenetic mechanism that controls transcriptional dynamics in response to diverse intra- and extracellular signals. PARP-1 activity is also implicated in the regulation of mammalian lifespan. Herein we show that transcriptional down-regulation of androgen receptor (AR) in the aging rat liver and in oxidatively stressed hepatoma cells involves exchange of a PARP-1-associated, p/CAF-containing coactivator assembly for a p53-interacting, Groucho/TLE1-, and mSin3A-included corepressor complex at an age- and oxidant-responsive DNA element (age-dependent factor (ADF) element) in the AR promoter. The coregulator switch is mediated by B-Myb and c-Myb, which bind to the ADF element and physically associate with PARP-1 and the tumor suppressor p53. Heterogeneous nuclear ribonucleoprotein K, residing at the ADF element in association with PARP-1, may serve a platform role in stabilizing the activating complex. PARP-1 coactivated B-Myb- and c-Myb-mediated transactivation of the AR promoter, and p53 antagonized the B-Myb/c-Myb-induced AR promoter activation. PARP-1, heterogeneous nuclear ribonucleoprotein K, B-Myb, and c-Myb each serves as a positive regulator of cellular AR content, whereas p53 negatively regulates AR expression. Our results identify a shared, PARP-1-regulated sensing mechanism that coordinates transcriptional repression of AR during aging and in response to oxidative stress. This study may provide insights as to how advancing age and intracellular redox balance might influence androgen-regulated physiology.

Pharmacological Exploitation of the Peroxisome Proliferator-Activated Receptor γ Agonist Ciglitazone To Develop a Novel Class of Androgen Receptor-Ablative Agents

On the basis of our finding that the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist ciglitazone at high doses was able to mediate PPARgamma-independent transcriptional repression of androgen receptor (AR) in a tumor cell-specific manner, we used Delta2CG, a PPARgamma-inactive analogue of ciglitazone, to conduct lead optimization to develop a novel class of AR-ablative agents. Structure-activity analysis indicates a high degree of flexibility in realigning Delta2CG's structural moieties without compromising potency in AR repression, as evidenced by the higher AR-ablative activity of the permuted isomer 9 [( Z)-5-(4-hydroxybenzylidene)-3-(1-methylcyclohexylmethyl)thiazolidine-2,4-dione]. Further modifications of 9 gave rise to 12 [( Z)-5-(4-hydroxy-3-trifluoromethylbenzylidene)-3-(1-methylcyclohexylmethyl)thiazolidine-2,4-dione], which completely inhibited AR expression in LNCaP cells at low micromolar concentrations. This AR down-regulation led to growth inhibition in LNCaP cells through apoptosis induction. Moreover, the role of AR repression in the antiproliferative effect of compound 12 was validated by the differential inhibition of cell viability between androgen-responsive and androgen-nonresponsive cells.

Cyclin D3/CDK11p58 Complex Is Involved in the Repression of Androgen Receptor

Androgen receptor (AR) is essential for the maintenance of the male reproductive systems and is critical for the carcinogenesis of human prostate cancers (PCas). D-type cyclins are closely related to the repression of AR function. It has been well documented that cyclin D1 inhibits AR function through multiple mechanisms, but the mechanism of how cyclin D3 exerts its repressive role in the AR signaling pathway remains to be identified. In the present investigation, we demonstrate that cyclin D3 and the 58-kDa isoform of cyclin-dependent kinase 11 (CDK11p58) repressed AR transcriptional activity as measured by reporter assays of transformed cells and prostate-specific antigen expression in PCa cells. AR, cyclin D3, and CDK11p58 formed a ternary complex in cells and were colocalized in the luminal epithelial layer of the prostate. AR activity is controlled by phosphorylation at specific sites. We found that AR was phosphorylated at Ser-308 by cyclin D3/CDK11p58 in vitro and in vivo, leading to the repressed activity of AR transcriptional activation unit 1 (TAU1). Furthermore, androgen-dependent proliferation of PCa cells was inhibited by cyclin D3/CDK11p58 through AR repression. These data suggest that cyclin D3/CDK11p58 signaling is involved in the negative regulation of AR function.

Estrogen Sensitivity of Target Genes and Expression of Nuclear Receptor Co-Regulators in Rat Prostate after Pre- and Postnatal Exposure to the Ultraviolet Filter 4-Methylbenzylidene Camphor

Background and objectivesIn previous studies, we found that the ultraviolet filter 4-methyl-benzylidene camphor (4-MBC) exhibits estrogenic activity, is a preferential estrogen receptor (ER)-β ligand, and interferes with development of female reproductive organs and brain of both sexes in rats. Here, we report effects on male development.Methods4-MBC (0.7, 7, 24, 47 mg/kg/day) was administered in chow to the parent generation before mating, during gestation and lactation, and to offspring until adulthood. mRNA was determined in prostate lobes by real-time reverse transcription–polymerase chain reaction and protein was determined by Western blot analysis.Results4-MBC delayed male puberty, decreased adult prostate weight, and slightly increased testis weight. Androgen receptor (AR), insulin-like growth factor-1 (IGF-1), ER-α, and ER-β expression in prostate were altered at mRNA and protein levels, with stronger effects in dorsolateral than ventral prostate. To assess sensitivity of target genes to estrogens, offspring were castrated on postnatal day 70, injected with 17β-estradiol (E2; 10 or 50 μg/kg, sc) or vehicle on postnatal day 84, and sacrificed 6 hr later. Acute repression of AR and IGF-1 mRNAs by E2, studied in ventral prostate, was reduced by 4-MBC exposure. This was accompanied by reduced co-repressor N-CoR (nuclear receptor co-repressor) protein in ventral and dorsolateral prostate, whereas steroid receptor coactivator-1 (SRC-1) protein levels were unaffected.ConclusionsOur data indicate that 4-MBC affects development of male reproductive functions and organs, with a lowest observed adverse effect level of 0.7 mg/kg. Nuclear receptor coregulators were revealed as targets for endocrine disruptors, as shown for N-CoR in prostate and SRC-1 in uterus. This may have widespread effects on gene regulation.

Peroxisome Proliferator-Activated Receptor γ–Independent Suppression of Androgen Receptor Expression by Troglitazone Mechanism and Pharmacologic Exploitation

Previously, we showed that the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist troglitazone at high doses was able to suppress androgen receptor (AR) expression in LNCaP prostate cancer cells independently of PPARgamma. Pharmacologic exploitation of this finding led to STG28, a PPARgamma-inactive analogue of troglitazone with substantially higher potency in AR repression. Considering the pivotal role of AR in prostate tumorigenesis, this study investigates the mechanism by which troglitazone and derivatives suppress AR expression in LNCaP cells. Reverse transcription-PCR and reporter gene assays indicate that this drug-induced AR repression occurs at both mRNA and protein levels. Evidence suggests that troglitazone and derivatives mediate the transcriptional repression of AR by facilitating the ubiquitin-dependent proteasomal degradation of the transcriptional factor Sp1. These agents also cause the proteolysis of two proteins that regulate Sp1-mediated transcription (i.e., the TATA-binding protein-associated factor TAF(II)250 and cyclin D1). However, their involvement in the transcriptional repression of AR is refuted by the finding that small interfering RNA knockdown of these two regulatory proteins does not cause AR down-regulation. STG28 does not cause significant reduction in Sp1 or AR expression in normal prostate epithelial cells. This discriminatory effect underscores the differential susceptibility of malignant versus normal cells to the inhibitory effect of STG28 on cell viability. From a translational perspective, STG28 provides a proof of principle that potent AR-ablative agents could be developed through structural modifications of troglitazone. Moreover, as the control of Sp1 degradation remains unclear, STG28 represents a unique pharmacologic probe to investigate the ubiquitin-proteasome system that regulates Sp1 proteolysis.

Clioquinol, a Therapeutic Agent for Alzheimer's Disease, Has Proteasome-Inhibitory, Androgen Receptor–Suppressing, Apoptosis-Inducing, and Antitumor Activities in Human Prostate Cancer Cells and Xenografts

Tumor growth and metastasis depend on angiogenesis that requires the cofactor copper. Consistently, high levels of copper have been found in many types of human cancers, including prostate, breast, colon, and lung. Recent studies suggest that copper could be used as a novel selective target for cancer therapies. Clioquinol is capable of forming stable complexes with copper and currently used in clinics for treatment of Alzheimer's disease. Most recently, it has been reported that clioquinol possesses antitumor effects. However, the underlying molecular mechanism is unclear. We report here that after binding to copper, clioquinol can inhibit the proteasomal chymotrypsin-like activity, repress androgen receptor (AR) protein expression, and induce apoptotic cell death in human prostate cancer LNCaP and C4-2B cells. In addition, clioquinol alone exhibits similar effects in prostate cancer cell lines with elevated copper at concentrations similar to those found in patients. Addition of dihydrotestosterone did not affect clioquinol-mediated proteasome inhibition in both prostate cancer cell lines. However, dihydrotestosterone partially inhibited clioquinol-induced AR suppression and apoptosis only in androgen-dependent LNCaP cells. Animal studies show that clioquinol treatment significantly inhibits the growth of human prostate tumor C4-2B xenografts (by 66%), associated with in vivo proteasome inhibition, AR protein repression, angiogenesis suppression, and apoptosis induction. Our study provides strong evidence that clioquinol is able to target tumor proteasome in vivo in a copper-dependent manner, resulting in formation of an active AR inhibitor and apoptosis inducer that is responsible for its observed antiprostate tumor effect.

Suberoylanilide hydroxamic acid (vorinostat) represses androgen receptor expression and acts synergistically with an androgen receptor antagonist to inhibit prostate cancer cell proliferation

Growth of prostate cancer cells is initially dependent on androgens, and androgen ablation therapy is used to control tumor growth. Unfortunately, resistance to androgen ablation therapy inevitably occurs, and there is an urgent need for better treatments for advanced prostate cancer. Histone deacetylase inhibitors, such as suberoylanilide hydroxamic acid (SAHA; vorinostat), are promising agents for the treatment of a range of malignancies, including prostate cancer. SAHA inhibited growth of the androgen-responsive LNCaP prostate cancer cell line at low micromolar concentrations and induced caspase-dependent apoptosis associated with chromatin condensation, DNA fragmentation, and mitochondrial membrane depolarization at higher concentrations (>/=5 mumol/L). Gene profiling and immunoblot analyses showed a decrease in androgen receptor (AR) mRNA and protein in LNCaP cells cultured with SAHA compared with control cells, with a corresponding decrease in levels of the AR-regulated gene, prostate-specific antigen. Culture of LNCaP cells in steroid-free medium markedly sensitized the cells to SAHA. Moreover, a combination of low, subeffective doses of SAHA and the AR antagonist bicalutamide resulted in a synergistic reduction in cell proliferation and increase in caspase-dependent cell death. Addition of exogenous androgen prevented the induction of cell death, indicating that suppression of androgen signaling was required for synergy. At the subeffective concentrations, these agents had no effect, alone or in combination, on proliferation or death of AR-negative PC-3 prostate cancer cells. Our findings indicate that SAHA is effective in targeting the AR signaling axis and that androgen deprivation sensitizes prostate cancer cells to SAHA. Consequently, combinatorial treatments that target different components of the AR pathway may afford a more effective strategy to control the growth of prostate cancer cells.

Mechanisms of androgen receptor repression in prostate cancer

Anti-androgens used in prostate cancer therapy inhibit AR (androgen receptor) activity via largely unknown mechanisms. Although initially successful in most cases, they eventually fail and the disease progresses. We need to elucidate how anti-androgens work to understand why they fail, and prolong their effects or design further therapies. Using a cellular model, we found different anti-androgens have diverse effects on subcellular localization of AR, revealing that they work via different mechanisms and suggesting that an informed sequential treatment regime may benefit patients. In the presence of the anti-androgens bicalutamide and hydroxyflutamide, a significant proportion of the AR is translocated to the nucleus but remains inactive. Receptor inhibition under these conditions is likely to involve recruitment of co-repressor proteins, which interact with antagonist-occupied receptor but inhibit receptor-dependent transcription. Which co-repressors are required in vivo for AR repression by anti-androgens is not clear, but one candidate is the Notch effector Hey1. This inhibits ligand-dependent activity of the AR but not other steroid receptors. Further, it is excluded from the nucleus in most human prostate cancers, suggesting that abnormal subcellular distribution of co-repressors may contribute to the aberrant hormonal responses observed in prostate cancer. A decrease in co-repressor function is one possible explanation for the development of anti-androgen-resistant prostate cancer, and this suggests that it may not occur at the gross level of protein expression.

Prohibitin, a protein downregulated by androgens, represses androgen receptor activity

Prohibitin (PHB) is a cell cycle regulatory protein, known to repress E2F1-mediated gene activation via recruitment of transcriptional regulatory factors such as retinoblastoma and histone deacetylase 1 (HDAC1). We previously identified PHB as a target protein of androgen signaling in prostate cancer cells and showed that downregulation of PHB is required for androgen-induced cell cycle entry in these cells. We now present evidence that PHB, which has 54% homology at the protein level to the oestrogen receptor corepressor REA (repressor of oestrogen receptor activity), can repress androgen receptor (AR)-mediated transcription and androgen-dependent cell growth. Depletion of endogenous PHB resulted in an increase in expression of the androgen-regulated prostate-specific antigen gene. The repression appears to be specific to androgen and closely related receptors, as it is also evident for the glucocorticoid and progesterone, but not oestrogen, receptors. In spite of interaction of PHB with HDAC1, HDAC activity is not required for this repression. Although AR and PHB could be co-immunoprecipitated, no direct interaction was detectable, suggesting that PHB forms part of a repressive complex with the AR. Competition with the co-activator SRC1 further suggests that formation of a complex with AR, PHB and other cofactors is the mechanism by which repression is achieved. It appears then that repression of AR activity is one mechanism by which PHB inhibits androgen-dependent growth of prostate cells. Further, this study implies that the AR itself could, by mediating downregulation of a corepressor, be involved in the progression of prostate tumours to the hormone refractory stage.

Repression of androgen receptor in prostate cancer cells by phenethyl isothiocyanate

Repression of androgen receptor in prostate cancer cells by phenethyl isothiocyanate

Repression of androgen receptor in prostate cancer cells by phenethyl isothiocyanate

Prostate cancer usually progresses to androgen refractory after an initial anti-androgen treatment. The androgen receptor (AR) is a pivotal factor for the androgen-mediated growth and maintenance of the prostate. Abnormality of the AR, such as overexpression has been postulated to be related to the hormone independent growth of the cancer. Although we previously demonstrated that the AR expression could be modulated by isothiocyanates, which are natural constituents of cruciferous vegetables, the mechanism, however, remained to be clarified. We have since investigated the mechanism of phenethyl isothiocyanate (PEITC) in AR regulation. A human androgen dependent prostate cancer cell line LNCaP (AD) and its sub-line LNCaP (AI), i.e. androgen independent but overexpressing AR, were exposed to PEITC. The effects of PEITC on cell growth and AR expression/transcription were analyzed with MTT assay, real-time PCR and western blotting. The AR promoter activity was analyzed with the reporter activity after transfection with pAR-luc. The effects on Sp1, the major transcription factor of the AR, were tested with Sp1-luc activity, western blotting and electrophoretic mobility shift assay. PEITC induced a significant growth inhibition, with equal IC(50), in both AD and AI cells. The AR present in both cells was repressed as demonstrated with real-time PCR and western blot. PEITC mediates dual effects at transcriptional and post-translational levels to regulate the AR. At transcriptional level the AR level was reduced via inhibition of the transcription factor Sp1, and at post-translational level by accelerating protein degradation. PEITC represses AR transcription and expression, and mediates growth arrest in androgen dependent and independent prostate cancer cells. With the AR modulation and growth attenuation, PEITC and possibly other isothiocyanates, may prevent and inhibit hormone sensitive and refractory prostate cancer.

Sumoylation of CCAAT/Enhancer-binding Protein α and Its Functional Roles in Hepatocyte Differentiation

The sumoylation of CCAAT/enhancer-binding proteins (C/EBPs) by small ubiquitin-related modifier-1 (SUMO-1) has been reported recently. In this study, we investigated the functional role of the sumoylation of C/EBPalpha in the differentiation of hepatocytes. The amount of sumoylated C/EBPalpha gradually decreased during the differentiation, which suggests that the sumoylation is important for the control of growth/differentiation especially in the fetal liver. To analyze the function of the sumoylation of C/EBPalpha in liver-specific gene expression, we studied its effects on the expression of the albumin gene. The C/EBPalpha-mediated transactivation of the albumin gene was reduced by sumoylation of C/EBPalpha in primary fetal hepatocytes. The enhancement of C/EBPalpha-mediated transactivation by BRG1, a core subunit of the SWI/SNF chromatin remodeling complex, was hampered by sumoylation in a luciferase reporter assay. In addition, we discovered that sumoylation of C/EBPalpha blocked its inhibitory effect on cell proliferation by leading to the disruption of a proliferation-inhibitory complex because of a failure of the sumoylated C/EBPalpha to interact with BRG1. BRG1 was recruited to the dihydrofolate reductase promoter in nonproliferating C33a cells but was not detected in proliferating cells where C/EBPalpha, BRG1, and SUMO-1 were overexpressed. This result suggests that BRG1 down-regulates the expression of the dihydrofolate reductase gene. These findings provide the insight that SUMO acts as a space regulator, which affects protein-protein interactions.

289. Highly Inducible and Prostate Specific Oncolytic Adenovirus with E1A-AR Chimera

Top of pageAbstract Previously, we demonstrated prostate-specific replication and passive cell lysis using E1A driven by a prostate-specific antigen promoter (PSA) and enhancer (PSE). However, the expression of E1A relies on promoters that utilize the androgen receptor (AR) for optimal function. Recently we have discovered that E1A and AR are mutually antagonistic in their transcriptional regulation. We demonstrate that a chimeric fusion of AR with the E1A converts an androgen receptor repressor to a co-activator. Here, we describe the features of conditionally replication-competent adenoviruses (CRADs) with E1A-AR chimeric expression in prostate cancer cells and non-prostate cancer cells. We constructed a series of recombinant chimeric E1A-AR viruses including wild type AR (E1A-ARwt), transactivation domain (E1A-TAD), and a full-length AR with a point mutation in the ligand binding domain (E1A-ARC685Y), under the control of the prostate-specific enhancer (PSE) and rat probasin promoter (PBN). The E1A function of these viruses was evaluated in prostate cancer cells and non-prostate cancer cells using an adenovirus with Fiber-IRES-GFP lacking E1A as a reporter. The wild type Ad5 with an E3-deletion replicates in both prostate cancer cells and non-prostate cancer cells. Replication of this virus is decreased by 50% by activated androgen receptors in the LNCaP cell line. The CRAD Ad5PSE-PBN-E1A displayed a rapidly inducible replication in prostate cancer cells in the presence of R1881, however, it had a delayed and relatively high expression in the absence of R1881. The chimeric CRADs Ad5PSE-PBN-E1A-AR and Ad5PSE-PBN-E1A-C685Y displayed marked induction of E1A in LNCaP (AR+), no induction in PC3 (AR-), and non-prostate cancer cells Hela in the presence of hormone, and no expression in any cell line in the absence of hormone. The cytopathic effect (CPE) of these chimeric viruses was also confirmed using crystal violet staining. Our highly engineered E1A-AR chimeras not only convert a negative regulatory process into a positive regulatory process, but also greatly enhance the tissue specificity and the regulation of viral replication in the prostate. This strategy dramatically reduces toxicity in non-prostate tissue, while maintaining prostate specificity.

SHP Represses Transcriptional Activity via Recruitment of Histone Deacetylases

The orphan receptor short heterodimer partner (SHP) is a common partner for a great number of nuclear receptors, and it plays an important role in many diverse physiological events. In a previous study, we described SHP as a strong repressor of the androgen receptor (AR). Herein, we addressed the mechanism of action of its negative activity on transcription. We first investigated the intrinsic repressive potential of SHP and mapped two core repressive domains to the amino acids 170-210 and 210-240. From GST pull-down assays, we demonstrated a direct interaction between SHP and diverse histone deacetylases (HDACs) as well as a strong interaction between HDAC1 and SHP inhibitory domains. We further supported the evidence for an interaction between SHP and HDAC1 by showing their co-immunoprecipitation and provided evidence for the existence of a ternary complex comprising AR, SHP, and HDAC1. The use of trichostatin A (TSA), a specific inhibitor of HDAC activity, confirmed that HDACs significantly contribute to the intrinsic transrepressive activity of SHP. Finally, we showed that TSA reversed SHP-induced repression of AR, further emphasizing the relevance of the interaction between SHP and HDACs. This latter action affected in a very similar manner SHP-mediated repression of estrogen receptor alpha (ERalpha) transactivation. Altogether, our results indicate that SHP mediates most of its repressive effect through recruitment of HDACs and suggest that the physiological actions of SHP could be affected by HDAC inhibitors.

Inhibition of glycogen synthase kinase-3 represses androgen receptor activity and prostate cancer cell growth.

The transcriptional activity of the androgen receptor (AR) is regulated by interaction with various coregulators, one of which is beta-catenin. Interest in the role of beta-catenin in prostate cancer has been stimulated by reports showing that it is aberrantly expressed in the cytoplasm and/or nucleus in up to 38% of hormone-refractory tumours and that overexpression of beta-catenin results in activation of AR transcriptional activity. We have examined the effect of depleting endogenous beta-catenin on AR activity using Axin and RNA interference. Axin, which promotes beta-catenin degradation, inhibited AR transcriptional activity. However, this did not require the beta-catenin-binding domain of Axin. Depletion of beta-catenin using RNA interference increased, rather than decreased, AR activity, suggesting that endogenous beta-catenin is not a transcriptional coactivator for the AR. The glycogen synthase kinase-3 (GSK-3)-binding domain of Axin prevented formation of a GSK-3-AR complex and was both necessary and sufficient for inhibition of AR-dependent transcription. A second GSK-3-binding protein, FRAT, also inhibited AR transcriptional activity, as did the GSK-3 inhibitors SB216763 and SB415286. Finally, inhibition of GSK-3 reduced the growth of AR-expressing prostate cancer cell lines. Our observations suggest a potential new therapeutic application for GSK-3 inhibitors in prostate cancer.

Cloning and functional characterization of a rat Daxx that functions as a corepressor for the androgen receptor.

The androgen receptor (AR), a ligand-activated nuclear transcription factor, plays an important role in endocrine system development and homeostasis. AR-mediated gene transcription is modulated by a wide variety of coregulator proteins, but corepressors of AR have not been well characterized. We have isolated a rat homologue of Daxx, a possible AR repressor. The clone obtained comprised a 2196 bp open reading frame, corresponding to a sequence of 731 amino acids with 80.5% homology to mouse. Basal transcription of the isolated clone was repressed in transient transcription experiments using a Gal4 reporter gene assay. Furthermore, a mammalian two-hybrid assay showed a strong interaction between Daxx and promyelocytic leukemia (PML) protein. These results indicate that the isolated clone is a rat homologue of Daxx. The rat Daxx (rDaxx) interacted with the androgen receptor (AR): in co-immunoprecipitation experiments, AR formed complexes with anti-Daxx antibody. Furthermore, overexpression of Daxx suppressed AR-mediated transcriptional activity in HeLa and CV1 cells in transient transfection experiments. Taken together, our results suggest that Daxx may function as a corepressor for the androgen receptor.

Human checkpoint protein hRad9 functions as a negative coregulator to repress androgen receptor transactivation in prostate cancer cells.

Positive responses to combined androgen elimination therapy and radiation therapy have been well documented in the treatment of prostate cancer patients. The detailed mechanisms how androgen-androgen receptor (AR) cross talks to the radiation-related signal pathways, however, remain largely unknown. Here we report the identification of hRad9, a key member of the checkpoint Rad protein family, as a coregulator to suppress androgen-AR transactivation in prostate cancer cells. In vivo and in vitro interaction assays using Saccharomyces cerevisiae two-hybrid, mammalian two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation methods prove that AR can interact with the C terminus of hRad9 via its ligand binding domain. The FXXLF motif within the C terminus of hRad9 interrupts the androgen-induced interaction between the N terminus and C terminus of AR. This interaction between AR and hRad9 may result in the suppression of AR transactivation, demonstrated by the repressed AR transactivation in androgen-induced luciferase reporter assay and the reduced endogenous prostate-specific antigen expression in Western blot assay. Addition of small interfering RNA of hRad9 can reverse hRad9 suppression effects, which suggests that hRad9 functions as a repressor of AR transactivation in vivo. Together, our data provide the first linkage between androgen-AR signals and radiation-induced responses. Further studies of the influence of hRad9 on prostate cancer growth may provide potential new therapeutic approaches.