Androgen Receptor Transcriptional Activity Research Articles

TAF1 Differentially Enhances Androgen Receptor Transcriptional Activity via Its N-Terminal Kinase and Ubiquitin-Activating and -Conjugating Domains

Aberrant expression of androgen receptor (AR) coregulators has been linked to progression of prostate cancers to castration resistance. Using the repressed transactivator yeast two-hybrid system, we found that TATA binding protein-associated factor 1 (TAF1) interacted with the AR. In tissue microarrays, TAF1 was shown to steadily increase with duration of neoadjuvant androgen withdrawal and with progression to castration resistance. Glutathione S-transferase pulldown assays established that TAF1 bound through its acetylation and ubiquitin-activating/conjugating domains (E1/E2) directly to the AR N terminus. Coimmunoprecipitation and ChIP assays revealed colocalization of TAF1 and AR on the prostate-specific antigen promoter/enhancer in prostate cancer cells. With respect to modulation of AR activity, overexpression of TAF1 enhanced AR activity severalfold, whereas small interfering RNA knockdown of TAF1 significantly decreased AR transactivation. Although full-length TAF1 showed enhancement of both AR and some generic gene transcriptional activity, selective AR coactivator activity by TAF1 was demonstrated in transactivation experiments using cloned N-terminal kinase and E1/E2 functional domains. In keeping with AR coactivation by the ubiquitin-activating and -conjugating domain, TAF1 was found to greatly increase the cellular amount of polyubiquitinated AR. In conclusion, our results indicate that increased TAF1 expression is associated with progression of human prostate cancers to the lethal castration-resistant state. Because TAF1 is a coactivator of AR that binds and enhances AR transcriptional activity, its overexpression could be part of a compensatory mechanism adapted by cancer cells to overcome reduced levels of circulating androgens.

Acne vulgaris

The epidemic-like high prevalence of acne of more than 85% of adolescents exposed to westernized life style points to the role of nutritional factors. Insulinotropic foods, especially milk, dairy products and carbohydrates with high glycemic index and smoking lead to pathological exaggeration of already physiologically increased growth factor signaling of puberty. Food-derived growth factors activate the oncogenic phosphoinositide-3-kinase/Akt signaling pathway, which increases androgen receptor transcriptional activity and de-represses FoxO1-suppressed target genes of follicular keratinocyte proliferation and sebaceous lipogenesis. Thus, acne is the visible metabolic syndrome of skin exaggerated by growth factor signaling of westernized malnutrition. These insights allow a new classification of western life style acne as acne alimentaris and provide the rationale for dietary intervention. All efforts should be undertaken to eliminate the insulinotropic effector mechanism of milk in order to reduce the prevalence of acne and even more serious civilization diseases associated with malnutrition-dependent oncogenic signal transduction.

The molecular mechanism of Vav3 oncogene on upregulation of androgen receptor activity in prostate cancer cells

Our previous studies revealed that Vav3 oncogene is overexpressed in human prostate cancer, enhances androgen receptor (AR)-mediated signaling, and may play a role in prostate cancer development and progression. The purpose of this study was to determine the molecular mechanisms responsible for AR activation by Vav3. We found that interaction between N-terminus and C-terminus of AR is essential for its elevated activity stimulated by Vav3. The DH and PH domains of Vav3 are involved in direct interaction with AR. Both cytoplasmic and nuclear levels of AR and Vav3 are elevated and their nuclear localization is further stimulated by DHT in androgen-independent LNCaP-AI cells relative to their parental androgen-dependent LNCaP cells. Vav3 is colocalized with AR, phospho(P)-Akt, and HER2 with a short term stimulation by EGF and DHT. PI3K inhibitor LY294002 blocks colocalization of Vav3 with P-Akt. Consistently, EGF and DHT stimulate Vav3 and AR interaction and enhance PI3K-Akt signaling. Mutation of tyrosines to phenylalanines in the acidic domain or deletion of the SH2 and SH3 domains significantly enhances Vav3 ability for AR activation, while deletion of the DH domain abolishes this activity. Given that an elevated interaction of Vav3 with AR, P-Akt, and HER2 in both cytoplasm and nucleus upon the short-term of DHT stimulation, our data suggest that Vav3 may enhance non-genomic AR activity via PI3K-Akt signaling in addition to AR transcriptional activity and further support a role in androgen-independent growth in prostate cancer.

Abstract B92: Combating prostate cancer by targeting androgen receptor with phytochemical resveratrol

Abstract The chemopreventive effects of resveratrol (RSV) on prostate cancer have been well established; the androgen receptor (AR) plays pivotal roles in prostatic tumorigenesis. However, the exact underlying molecular mechanisms about the effects of RSV on AR have not been fully elucidated. A model system is needed to determine whether and how RSV represses AR transcriptional activity. The AR cDNA was first cloned into the retroviral vector pOZ-N and then integrated into the genome of AR-negative HeLa cells to generate the AR(+) cells. The constitutively expressed AR was characterized by monitoring hormone-stimulated nuclear translocation, DNA binding, and transcriptional activation, with the AR(−) cells serving as controls. AR(+) cells were treated with RSV, and both AR protein levels and AR transcriptional activity were measured simultaneously. Chromatin immunoprecipitation (ChIP) assays were used to detect the effects of RSV on the recruitment of AR to its cognate element (ARE). AR in the AR (+) stable cell line functions in a manner similar to that of endogenously expressed AR. Using this model system we clearly demonstrated that RSV represses AR transcriptional activity independently of any effects on AR protein levels. However, neither the hormone-mediated nucleus translocation nor the AR/ARE interaction was affected by RSV treatment. We demonstrated unambiguously that RSV regulates AR target gene expression, at least in part, by repressing AR transcriptional activity. Repressive effects of RSV on AR activity result from mechanisms other than the affects of AR nuclear translocation or DNA binding. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B92.

PI3K-AKT-mTOR Pathway is Dominant over Androgen Receptor Signaling in Prostate Cancer Cells

Background: Androgen receptor (AR) and the phosphatidylinositol-3 kinase (PI3K) signaling are two of the most important pathways implicated in prostate cancer. Previous work has shown that there is crosstalk between these two pathways; however, there are conflicting findings and the molecular mechanisms are not clear. Here we studied the AR–PI3K pathway crosstalk in prostate cancer cells in vitro as well as in vivo.Methods: Quantitative PCR, Western analysis, reporter assays, and proliferation analyses in vitro and in vivo were used to evaluate the effect of PI3K pathway inhibition on AR signaling and cell growth.Results: Transcriptional activity of AR was increased when the PI3K pathway was inhibited at different levels. In the androgen responsive prostate cancer cell line LNCaP, androgen and the mTOR inhibitor rapamycin synergistically activated androgen target genes. Despite increased androgen signaling, rapamycin treatment reduced LNCaP cell growth; the AR antagonist bicalutamide potentiated this effect. Furthermore, the rapamycin derivative CCI-779 reduced the growth of CWR22 prostate cancer xenografts while increasing AR target gene expression.Conclusions: These findings suggest that inhibition of the PI3K pathway activates AR signaling. Despite the increase in AR signaling which has proliferative effects, the result of PI3K pathway inhibition is antiproliferative. These findings suggest that the PI3K pathway is dominant over AR signaling in prostate cancer cells which should be considered in developing novel therapeutic strategies for prostate cancer.

FKBP51 and Cyp40 are positive regulators of androgen-dependent prostate cancer cell growth and the targets of FK506 and cyclosporin A

Prostate cancer (PCa) growth is dependent on androgens and the androgen receptor (AR), which acts by modulating gene transcription. Tetratricopeptide repeat (TPR) proteins (FKBP52, FKBP51 and Cyp40) interact with AR in PCa cells, suggesting roles in AR-mediated gene transcription and cell growth. We report here that FKBP51 and Cyp40, but not FKBP52, are significantly elevated in PCa tissues and in androgen-dependent (AD) and -independent (AI) cell lines. Overexpression of FKBP51 in AD LNCaP cells increased AR transcriptional activity in the presence and absence of androgen, whereas siRNA knockdown of FKBP51 dramatically decreased AD gene transcription and proliferation. Knockdown of Cyp40 also inhibited androgen-mediated transcription and growth in LNCaP cells. However, disruption of FKBP51 and Cyp40 in the AI C4-2 cells caused only a small reduction in proliferation, indicating that Cyp40 and FKBP51 predominantly regulate AD cell proliferation. Under knock-down conditions, the inhibitory effects of TPR ligands, CsA and FK506, on AR activity were not observed, indicating that Cyp40 and FKBP51 are the targets of CsA and FK506, respectively. Our findings demonstrate that FKBP51 and Cyp40 are positive regulators of AR that can be selectively targeted by CsA and FK506 to achieve inhibition of androgen-induced cell proliferation. These proteins and their cognate ligands thus provide new strategies in the treatment of PCa

14-3-3η Amplifies Androgen Receptor Actions in Prostate Cancer

PURPOSE: Androgen receptor abundance and androgen receptor-regulated gene expression in castration-recurrent prostate cancer are indicative of androgen receptor activation in the absence of testicular androgen. Androgen receptor transactivation of target genes in castration-recurrent prostate cancer occurs in part through mitogen signaling that amplifies the actions of androgen receptor and its coregulators. Herein we report on the role of 14-3-3eta in androgen receptor action. Experimental Design and RESULTS: Androgen receptor and 14-3-3eta colocalized in COS cell nuclei with and without androgen, and 14-3-3eta promoted androgen receptor nuclear localization in the absence of androgen. 14-3-3eta interacted with androgen receptor in cell-free binding and coimmunoprecipitation assays. In the recurrent human prostate cancer cell line, CWR-R1, native endogenous androgen receptor transcriptional activation was stimulated by 14-3-3eta at low dihydrotestosterone concentrations and was increased by epidermal growth factor. Moreover, the dihydrotestosterone- and epidermal growth factor-dependent increase in androgen receptor transactivation was inhibited by a dominant negative 14-3-3eta. In the CWR22 prostate cancer xenograft model, 14-3-3eta expression was increased by androgen, suggesting a feed-forward mechanism that potentiates both 14-3-3eta and androgen receptor actions. 14-3-3eta mRNA and protein decreased following castration of tumor-bearing mice and increased in tumors of castrate mice after treatment with testosterone. CWR22 tumors that recurred 5 months after castration contained 14-3-3eta levels similar to the androgen-stimulated tumors removed before castration. In a human prostate tissue microarray of clinical specimens, 14-3-3eta localized with androgen receptor in nuclei, and the similar amounts expressed in castration-recurrent prostate cancer, androgen-stimulated prostate cancer, and benign prostatic hyperplasia were consistent with androgen receptor activation in recurrent prostate cancer. CONCLUSION: 14-3-3eta enhances androgen- and mitogen-induced androgen receptor transcriptional activity in castration-recurrent prostate cancer. (Clin Cancer Res 2009;15(24):7571-81).

Abstract A219: Universal efficacy of novel androgen receptor pure antagonist, CH5137291, against broad spectrum prostate cancer including castration-resistant prostate cancers

Abstract Background: Prostate cancer resistant to total androgen depletion therapies is termed castration-resistant prostate cancer and novel therapeutic approaches are in demand. The mechanisms of castration resistance in prostate cancer are reported to be: 1) hypersensitivity to androgen derived from overexpression of androgen receptor (AR), 2) androgen-independent activation of AR, termed “outlaw”, and 3) loss of ligand specificity derived from mutation of AR. To address this demand, we screened and developed an AR antagonist without agonist activity, a so-called AR pure antagonist, CH5137291. Methods: We compared the inhibitory effects of CH5137291 with bicalutamide on the growth of hormone-sensitive and castration-resistant prostate cancer cells. To confirm the in vivo efficacy of CH5137291, we used xenograft models. The antagonist/agonist effect of CH5137291 on AR transcriptional activity was analyzed using reporter gene assays. To elucidate the mechanisms of CH5137291, we examined the effect of CH5137291 on the protein level of nuclear AR in LNCaP-CS10 and the effect of CH5137291 on the subcellular localization of AR in LNCaP cells. Finally, the exposure of CH5137291 and its effect on serum prostate-specific antigen (PSA) level were investigated in cynomolgus monkey. Results: CH5137291 showed in vitro inhibitory effects superior to bicalutamide in all models tested including hormone-sensitive VCaP, LNCaP and castration-resistant, LNCaP-BC2 (overexpression model) and LNCaP-CS10 (outlaw model). CH5137291 treatment (10, 100 mg/kg) inhibited the tumor growth to initial levels and PSA production to lower than initial levels in castration-resistant LuCaP35V, LNCaP-BC2 and LNCaP-CS10 xenograft models. In contrast, bicalutamide (10, 100 mg/kg) did not inhibit tumor growth or PSA production in any of the xenograft models. In addition, CH5137291 exhibited prolongation of the time to progression compared with bicalutamide in the LNCaP xenograft model. In vitro functional assays clarified that CH5137291 showed pure antagonist activity against wild type, bicalutamide-resistant type (W741C), and flutamide-resistant type (T877A) AR in transcription and inhibited the nuclear translocation of all types of AR tested. CH5137291 also exhibited a dose-dependent increase in serum concentration and inhibited PSA production in cynomolgus monkey. Conclusion: Our novel AR pure antagonist CH5137291 inhibited AR nuclear translocation and showed not only superior efficacy in prostate cancers with castration-resistance acquired from AR overexpression, mutation and outlaw mechanisms but also prolonged time to progression in hormone-sensitive prostate cancer compared with bicalutamide. CH5137291 is expected to contribute to novel therapeutic approaches against a broad spectrum of prostate cancers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A219.

Abstract A220: Design and synthesis of an androgen receptor pure antagonist (CH5137291) for treatment of castration-resistant prostate cancer

Abstract Background: We hypothesized that the androgen receptor (AR) pure antagonists, which exhibit no agonist activities, would inhibit AR signaling completely and would be efficacious against castration-resistant prostate cancer (CRPC). Based on our understanding of estrogen receptor pure antagonists, we designed dihydrotestosterone (DHT) derivatives and nonsteroidal RU56187 derivatives that inhibit the folding of helix 12 of AR and screened AR pure antagonists. Although a non-steroidal derivative (CH4933468) showed improved metabolic stability compared with DHT derivatives, it metabolized into an extremely small amount of a dealkylated metabolite with strong agonist effect. Therefore, CH4933468 did not show antitumor activity against the CRPC xenograft model. Methods: For metabolic stability, we designed and synthesized sulfonamide-substituted aryl compounds without dealkylated agonist metabolites. The production of agonist metabolites was examined in rat, mouse, monkey and dog. The efficacy of our compounds was measured using an in vitro reporter assay, a cell growth assay and in vivo prostate cancer xenograft models. Results: As a result of our optimization, CH5137291 was discovered and found to be an orally-active androgen receptor pure antagonist without agonistic activities derived from dealkylated agonist metabolites in vivo in rat, mouse, monkey and dog. CH5137291 exhibited AR antagonistic activities of IC50 of 300 nM and no agonist activity even at 30,000 nM in the reporter gene assay. In LNCaP-BC2 cells (AR overexpression CRPC model), CH5137291 was more efficient than bicalutamide. CH5137291 completely inhibited cell growth, in contrast to the partial inhibition of bicalutamide. Furthermore, CH5137291 inhibited the tumor growth and PSA production in the LNCaP-BC2 xenograft model. CH5137291 also inhibited AR nuclear translocation in the presence of R1881. According to the binding models of CH5137291 and bicalutamide to wild type AR, the sulfonamide of CH5137291 directly collided with M895 and thus prevented the folding of helix 12 completely. On the other hand, bicalutamide did not collide with M895 from helix 12 and inhibited helix 12 folding indirectly through W741. Hence, CH5137291 might act as an AR pure antagonist, but bicalutamide apparently does not. Conclusions: CH5137291, a novel AR pure antagonist without agonistic activities derived from dealkylated agonist metabolites, has shown antitumor activities in both in vitro and in vivo CRPC models. Our experimental results for clinical candidate CH5137291 corroborate our hypothesis that pure antagonists inhibit the folding of helix 12 completely, leading to the complete inhibition of the transcription activity of AR. Based on the strong efficacy against CRPC xenograft model and the inhibition of AR nuclear translocation, CH5137291 may offer more benefit than bicalutamide. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A220.

Melanoma Antigen Gene Protein-A11 (MAGE-11) F-box Links the Androgen Receptor NH2-terminal Transactivation Domain to p160 Coactivators

Androgen-dependent transcriptional activity by the androgen receptor (AR) and its coregulators is required for male reproductive development and function. In humans and other primates, melanoma antigen gene protein-A11 (MAGE-11) is an AR selective coregulator that increases AR transcriptional activity. Here we show that the interaction between AR and MAGE-11 is mediated by AR NH(2)-terminal FXXLF motif binding to a highly conserved MAGE-11 F-box in the MAGE homology domain, and is modulated by serum stimulation of mitogen-activated protein kinase phosphorylation of MAGE-11 Ser-174. The MAGE-11-dependent increase in AR transcriptional activity is mediated by a direct interaction between MAGE-11 and transcriptional intermediary factor 2 (TIF2) through the NH(2)-terminal region of TIF2, and by a MAGE-11 FXXIF motif interaction with an F-box-like region in activation domain 1 of TIF2. The results suggest that MAGE-11 functions as a bridging factor to recruit AR coactivators through a novel FXX(L/I)F motif-F-box interaction paradigm.

Hormonal Therapy for Prostate Cancer: Toward Further Unraveling of Androgen Receptor Function

Prostate cancer is a major cause of cancer-related death in men. Prostate cancer is an androgen-responsive tumor and the treatment of advanced prostate cancer involves hormonal therapy. First-line treatment for advanced prostate cancer is androgen deprivation therapy (ADT), usually with agents that suppress gonadotropins through a pituitary mechanism. Gonadotropin-releasing hormone agonists and antagonists both suppress gonadal release of testosterone, although their activity profiles vary. ADT down-regulates androgen receptor (AR) transcriptional activity in the tumor but the response in metastatic disease is transient and tumors progress as castration-resistant prostate cancer (CRPC). Although serum testosterone concentrations decline dramatically with ADT, CRPC growth remains largely dependent on AR activity. Secondary hormonal therapies are then often employed to further dampen AR-driven transcription. These secondary hormonal therapies either further deplete adrenal or intratumoral androgen synthesis, or directly and competitively antagonize AR. New hormonal agents with both of these mechanisms are in clinical trials and show promising activity in patients with CRPC. Abiraterone acetate is an inhibitor of CYP17, which is an enzyme required for the synthesis of all androgens and estrogens. MDV3100 is an AR antagonist that has a higher affinity for AR than any other AR antagonist in clinic use. In phase I and phase II clinical trials, both agents have significant activity. These agents and the promise of the development of others provide hope that more effective hormonal therapies may soon be offered to patients, which will improve clinical outcomes.

Androgen via p21 Inhibits Tumor Necrosis Factor α-induced JNK Activation and Apoptosis

The male hormone androgen is a growth/survival factor for its target tissues or organs. Yet, the underlying mechanism is incompletely understood. Here, we report that androgen via p21 inhibits tumor necrosis factor alpha-induced JNK activation and apoptosis. Inhibition by androgen requires the transcription activity of androgen receptor (AR) and de novo protein synthesis. Androgen.AR induces expression of p21 that in turn inhibits tumor necrosis factor alpha-induced JNK and apoptosis. Furthermore, genetic interruption of p21 alleles abolishes the inhibition by androgen. Our results reveal a novel cross-talk between androgen x AR and JNK, thereby providing a molecular mechanism underlying the survival function of androgen.

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.

Repressive Effects of Resveratrol on Androgen Receptor Transcriptional Activity

BackgroundThe chemopreventive effects of resveratrol (RSV) on prostate cancer have been well established; the androgen receptor (AR) plays pivotal roles in prostatic tumorigenesis. However, the exact underlying molecular mechanisms about the effects of RSV on AR have not been fully elucidated. A model system is needed to determine whether and how RSV represses AR transcriptional activity.MethodologyThe AR cDNA was first cloned into the retroviral vector pOZ-N and then integrated into the genome of AR-negative HeLa cells to generate the AR(+) cells. The constitutively expressed AR was characterized by monitoring hormone-stimulated nuclear translocation, DNA binding, and transcriptional activation, with the AR(-) cells serving as controls. AR(+) cells were treated with RSV, and both AR protein levels and AR transcriptional activity were measured simultaneously. Chromatin immunoprecipitation (ChIP) assays were used to detect the effects of RSV on the recruitment of AR to its cognate element (ARE).ResultsAR in the AR (+) stable cell line functions in a manner similar to that of endogenously expressed AR. Using this model system we clearly demonstrated that RSV represses AR transcriptional activity independently of any effects on AR protein levels. However, neither the hormone-mediated nucleus translocation nor the AR/ARE interaction was affected by RSV treatment.ConclusionWe demonstrated unambiguously that RSV regulates AR target gene expression, at least in part, by repressing AR transcriptional activity. Repressive effects of RSV on AR activity result from mechanisms other than the affects of AR nuclear translocation or DNA binding.

Four and a half LIM domain 2 alters the impact of aryl hydrocarbon receptor on androgen receptor transcriptional activity

Aryl hydrocarbon receptor (AhR) ligands modulate androgen receptor (AR) signaling in prostate cancer cells through partially defined mechanisms. Furthermore, these facilitatory and inhibitory effects of AhR on AR signaling appear to be cell or context specific. In the present study we demonstrate that both AhR and AhR-nuclear translocator (ARNT) interact with AR. AhR but not ARNT enhanced the AR-transcriptional activity which was independent of exogenous AhR ligand treatment (2,3,7,8-tetrachlorodibenzo- p-dioxin, TCDD). We then tested if coactivators common to both receptors alter the facilitatory effect of AhR on AR activity. NcoA4 overexpression did not alter the AhR facilitatory effect on AR, whereas SRC1 overexpression further enhanced the effect. In contrast, FHL2 overexpression blocked the facilitatory effect of AhR. In the presence of exogenous FHL2 expression, AhR repressed AR activity, whereas at low endogenous levels of FHL2 expression, AhR overexpression enhanced AR activity. At high FHL2 expression levels, TCDD treatment decreased AR activity and this effect was reversed by AhR overexpression. These findings demonstrate that AhR modulation of AR activity is differentially altered by the level of FHL2 and AhR present in the cell.

Heat shock protein 27 mediates repression of androgen receptor function by protein kinase D1 in prostate cancer cells

We have previously shown that protein kinase D1 (PKD1), charter member of PKD protein family, is downregulated in advanced prostate cancer (PC) and influences androgen receptor (AR) function in PC cells. Other independent studies showed that serine 82 residue in heat shock protein 27 (Hsp27) undergoes substrate phosphorylation by PKD1 and is associated with nuclear transport of AR resulting in increased AR transcriptional activity. In this study, we show that PKD1 interacts and phosphorylates Hsp27 at Ser82 in PC cells, which is mediated by p38-dependent mitogen-activated protein kinase pathway and is necessary for PKD1 repression of AR transcriptional activity and androgen-dependent proliferation of PC cells. The study provides first in vivo evidence that Hsp27 is a mediator of repression of AR function by PKD1 in PC cells, thereby linking the data in the published literature.

Androgen Receptor Phosphorylation and Activity Are Regulated by an Association with Protein Phosphatase 1

Androgen receptor (AR) is phosphorylated at multiple sites in response to ligand binding, but the functional consequences and mechanisms regulating AR phosphorylation remain to be established. We observed initially that okadaic acid, an inhibitor of the major PPP family serine/threonine phosphatases PP2A and protein phosphatase 1 (PP1), had cell type-dependent effects on AR expression. More specific inhibitors of PP2A (fostriecin) and PP1 (tautomycin and siRNA against the PP1alpha catalytic subunit) demonstrated that PP1 and protein phosphatase 2A had opposite effects on AR protein and transcriptional activity. PP1 inhibition enhanced proteasome-mediated AR degradation, while PP1alpha overexpression increased AR expression and markedly enhanced AR transcriptional activity. Coprecipitation experiments demonstrated an AR-PP1 interaction, while immunofluorescence and nuclear-cytoplasmic fractionation showed androgen-stimulated nuclear translocation of both AR and PP1 in prostate cancer cells. Studies with phosphospecific AR antibodies showed that PP1 inhibition dramatically increased phosphorylation of Ser-650, a site in the AR hinge region shown to mediate nuclear export. Significantly, PP1 inhibition caused a marked decrease in nuclear localization of the wild-type AR, but did not alter total or nuclear levels of a S650A mutant AR. These findings reveal a critical role of PP1 in regulating AR protein stability and nuclear localization through dephosphorylation of Ser-650. Moreover, AR may function as a PP1 regulatory subunit and mediate PP1 recruitment to chromatin, where it can modulate transcription and splicing.

Cell cycle regulator cdk2ap1 inhibits prostate cancer cell growth and modifies androgen‐responsive pathway function

We evaluated the effect of expressing the cell cycle regulator cdk2ap1, downregulated in prostate cancer cell lines, in inhibiting prostate cancer cell growth. Expression of cdk2ap1 using a tet-inducible lentiviral system modified growth rate, induced cell cycle arrest and apoptosis and reduced the invasive ability of prostate cancer cell lines, as assayed by cell viability, cell cycle profiling, Caspase 3/7 detection, and matrigel invasion assays. We examined the effect of expressing cdk2ap1 on gene expression profiles of cytokine, invasion, apoptotic, and androgen response pathways using quantitative real-time PCR, and used androgen-responsive reporter gene assays, and methylation-sensitive PCR to examine the mechanism of cdk2ap1 interaction with androgen-responsive pathways. The expression of cdk2ap1 correlated with a reduction in cellular growth, irrespective of inhibition or stimulation of androgen receptor (AR) signaling pathways. Cell cycle arrest, increased apoptosis, and a reduction in invasiveness phenotypes were observed upon cdk2ap1 expression. Enhanced demethylation at the AR promoter, AR expression increases, and enhanced AR transcriptional activity correlated with cdk2ap1 expression. Our findings support a novel concept by which cell cycle inhibitor genes can impact prostate cancer phenotypes by restoring a tumor suppressive function to androgen-responsive pathways and this function may involve modulation of a subset of functions of the AR.

Novel flufenamic acid analogues as inhibitors of androgen receptor mediated transcription.

The androgen receptor (AR), which mediates the signals of androgens, plays a crucial role in prostate-related diseases. Although widely used, currently marketed anti-androgenic drugs have significant side effects. Several studies have revealed that non-steroidal anti-inflammatory drugs, such as flufenamic acid, block AR transcriptional activity. Herein we describe the development of small molecule analogues of flufenamic acid that antagonize AR. This novel class of AR inhibitors binds to the hormone binding site, blocks AR transcription activity, and acts on AR target genes.

Yin Yang 1 regulates the transcriptional activity of androgen receptor

The multifunctional protein Yin Yang 1 (YY1) plays an important role in epigenetic regulation of gene expression. YY1 is highly expressed in various types of cancers, including prostate cancer. Currently, the mechanism underlying the functional role of YY1 in prostate tumorigenesis remains unclear. In this report, we investigated the functional interplay between YY1 and androgen receptor (AR), and the effect of YY1 on AR-mediated transcription. We found that YY1 physically interacts with AR both in a cell-free system and in cultured cells. YY1 is required for the optimal transcriptional activity of AR in promoting the transcription of the prostate specific antigen (PSA) promoter. However, ectopic YY1 expression in LNCaP cells did not further enhance the reporter driven by the PSA promoter, suggesting an optimal level of YY1 is already established in prostate tumor cells. Consistently, YY1 depletion in LNCaP cells reduced endogenous PSA levels, but overexpressed YY1 did not significantly increase PSA expression. We also observed that YY1-AR interaction is essential to YY1-mediated transcription activity of AR and YY1 is a necessary component in the complex binding to the androgen response element (ARE). Thus, our study demonstrates that YY1 interacts with AR and regulates its transcriptional activity.