Androgen Receptor Transcriptional Activity Research Articles

Abstract 2148: Exploitation of androgen receptor splice variant signaling by guanine nucleotide exchange factor Vav3 in castration resistant prostate cancer

Abstract The treatment for advanced or metastatic prostate cancer is androgen deprivation therapy. Patients inevitably relapse and the cancer is now termed androgen-independent or castration resistant (CRPC). CRPC remains dependent on androgen receptor (AR) signaling. One of the proteins implicated in the reactivation of AR transcriptional activity in CRPC is Vav3, a Rho GTPase guanine nucleotide exchange factor. Vav3 is up-regulated in human prostate cancer tumors compared to benign tissue and in preclinical models of prostate cancer progression to CRPC. Vav3 enhances AR activity at both physiological and as well as at subnanomolar androgen concentrations, similar to prostatic microenvironment levels demonstrated in CRPC. Vav3 confers castration resistant growth to a classically androgen dependent cell line in a mouse xenograft model. Thus, elevated levels of Vav3 seem to be pivotal in promoting continued AR signaling in CRPC. We examined the contributions of Vav3 to AR activity and cell proliferation in the CRPC cell lines CWR-22Rv1 and CWR-R1. Knockdown of Vav3 attenuated cell growth and reduced AR activity as compared to the shGFP control. However knockdown of full length AR had little effect, perhaps due to impaired ligand-mediated transcriptional activity of the mutated AR expressed in these cell lines. Because CWR-22Rv1 also express AR splice variants that lack the ligand binding domain and are proposed to underlie CRPC growth, we examined the possibility that Vav3 enhances AR splice variant signaling. The AR splice variant, ARV7 (also known as AR3), exhibits constitutive ligand independent activity and is linked to poor prognosis and survival. Depletion of ARV7 in 22Rv1 and CWR-R1 cell lines resulted in slowed growth and lowered AR activity. Soft agar assays revealed that knockdown of either Vav3 or ARV7 resulted in greatly decreased anchorage independent growth. Vav3 potently enhanced the activity of ARV7 in reporter gene assays. Further, knockdown of Vav3 resulted in lowered levels of nuclear AR splice variants in CWR-22Rv1. Co-immunoprecipitation revealed that ARV7 and Vav3 directly interact. These results suggest that Vav3 may enhance ARV7 activity through increased nuclear localization and that the interaction of Vav3 and ARV7 may be required for robust growth of CRPC cells. These novel data demonstrating interaction between a versatile AR coactivator and an AR splice variant provide insights into the possible mechanisms by which Vav3 exploits and enhances AR splice variant signaling in the progression of prostate cancer malignancy and castration resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2148. doi:1538-7445.AM2012-2148

DC‐SCRIPT: AR and VDR regulator lost upon transformation of prostate epithelial cells

Nuclear receptors (NR), including the Androgen Receptor (AR) and the Vitamin D Receptor (VDR), play an important role in prostate cancer etiology. We recently found that DC-SCRIPT is a prognostic marker in breast cancer and a unique NR coregulator differentially regulating different classes of NRs. Here we investigated the importance of DC-SCRIPT in prostate cancer. DC-SCRIPT mRNA expression was measured by qPCR. Immunohistochemistry was used to detect DC-SCRIPT protein expression. The functional effects of DC-SCRIPT on the transcriptional activity of AR and VDR were assessed by luciferase reporter assays and qPCR assays on well-known AR and VDR target genes. DC-SCRIPT mRNA was higher in normal than in corresponding malignant prostate tissue but could not be related to disease stage. DC-SCRIPT protein was found in morphologically normal prostate glands and in infiltrating immune cells. Strikingly, DC-SCRIPT protein expression was absent in malignant prostate epithelial tissue and prostate carcinoma cell lines. DC-SCRIPT protein expression appears to be lost prior to the basal cell marker HMW cytokeratin used in prostate carcinoma diagnostics. In addition, our data demonstrated that DC-SCRIPT repressed transcription mediated by wild-type and mutated AR while enhancing VDR mediated transcription. In addition, transient expression of DC-SCRIPT expression in prostate carcinoma cells strongly repressed cell growth. DC-SCRIPT is a key regulator of nuclear receptors AR and VDR that play an opposite role in prostate cancer etiology and loss of DC-SCRIPT may be involved in the onset of prostate cancer.

475 RECIPROCAL REGULATION OF SLUG AND ANDROGEN RECEPTOR FACILITATES PROSTATE CANCER TO ACQUIRE CASTRATION RESISTANT AND STEM-LIKE PHENOTYPES

You have accessJournal of UrologyProstate Cancer: Basic Research III1 Apr 2012475 RECIPROCAL REGULATION OF SLUG AND ANDROGEN RECEPTOR FACILITATES PROSTATE CANCER TO ACQUIRE CASTRATION RESISTANT AND STEM-LIKE PHENOTYPES Kaijie Wu, Crystal Gore, Lin Yang, Gleave Martin, Rey-Chen Pong, Jer-Tsong Hsieh, and Dalin He Kaijie WuKaijie Wu Xi'an, China, People's Republic of More articles by this author , Crystal GoreCrystal Gore Dallas, TX More articles by this author , Lin YangLin Yang Xi'an, China, People's Republic of More articles by this author , Gleave MartinGleave Martin Vancouver, Canada More articles by this author , Rey-Chen PongRey-Chen Pong Dallas, TX More articles by this author , Jer-Tsong HsiehJer-Tsong Hsieh Dallas, TX More articles by this author , and Dalin HeDalin He Xi'an, China, People's Republic of More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.544AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Castration resistant prostate cancer (CRPC) is eventually incurable; however, its cell origin and characteristics are still poorly understood. Although the onset of CRPC is an inevitable outcome from hormonal therapy, androgen receptor (AR) hyperactivation is often associated with CRPC. There are several mechanisms such as gene amplification, mutation and splice variants. In this study, we report a new mechanism that the interaction between Slug and AR promote castration resistance of PCa cells with stem-like phenotypes. METHODS Slug gene regulation by AR was determined in three AR-positive PCa cells (LNCaP, C4-2 and CWR22RV1) with quantitative RT-PCR and western blot. A series of biochemical, molecular and cell biologic methods were employed to characterize the impacts of Slug on AR in PCa cells. Also, xenograft model and clinical specimens were used to validate in vitro results. RESULTS Slug appeared to be an androgen responsive gene in AR-positive PCa cells, and the presence of constitutively active AR splice variants often found in CRPC could also induce Slug expression. On the other hand, Slug formed a complex with either wild type AR or its splicing variants by stabilizing AR protein and enhancing AR transcriptional activities with or without androgen. The presence of this complex also increased in vitro growth of PCa in the absence of androgen, and provided the growth advantage of PCa cells to resist castration in xenograft animal model. We also noticed that Slug was able to increase stem cell phenotypes in PCa cells in which Slug-AR complex directly bound to the promoter region of two stem cell markers (i.e., CD117 and CD44) and increased their gene expression. Immunostaining of Slug and AR in three tissue microarrays unveiled a significant correlation of the expression of these two proteins during PCa progression. In addition, data from several cDNA microarrays indicated a positive correlation of mRNA expression between Slug and CD44 or CD117 in human clinical specimens. CONCLUSIONS A reciprocal regulation between Slug and AR plays a critical role in promoting the onset of CRPC in which both AR activation and stem-like phenotypes are highly elevated. Overall, this study unveils a new regulatory mechanism of AR with Slug as a novel co-activator, in which Slug appears to be a potent prognostic marker and therapeutic target for CRPC. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e194-e195 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kaijie Wu Xi'an, China, People's Republic of More articles by this author Crystal Gore Dallas, TX More articles by this author Lin Yang Xi'an, China, People's Republic of More articles by this author Gleave Martin Vancouver, Canada More articles by this author Rey-Chen Pong Dallas, TX More articles by this author Jer-Tsong Hsieh Dallas, TX More articles by this author Dalin He Xi'an, China, People's Republic of More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Anti‐androgenic effects of S‐40542, a novel non‐steroidal selective androgen receptor modulator (SARM) for the treatment of benign prostatic hyperplasia

Selective androgen receptor modulators (SARMs) would provide alternative therapeutic agent for androgen-related diseases. We identified a tetrahydroquinoline (THQ) derivative, 1-(8-nitro-3a, 4, 5, 9b-tetrahydro-3H-cyclopenta[c]quinolin-4-yl) ethane-1, 2-diol (S-40542) as a novel SARM antagonist. Affinity for nuclear receptors of S-40542 was evaluated in receptor-binding studies. Androgen receptor (AR) transcriptional activity of S-40542 was investigated by luciferase reporter assay in DU145AR cells. Normal and benign prostatic hyperplasia (BPH) model rats were repeatedly treated with S-40542 and flutamide. The tissue weights of prostate and levator ani muscle as well as blood levels of testosterone and luteinizing hormone were measured. S-40542 bound to the AR with high affinity. S-40542 at relatively high concentrations increased the transcriptional activity. This agent also showed a concentration-dependent AR antagonistic action in the presence of 1 nM 5α-dihydrotestosterone. Repeated treatment with S-40542 and flutamide decreased dose-dependently the weights of the prostate to a similar extent. In contrast, the tissue weight-reducing effect by S-40542 treatment on the levator ani muscle was much weaker than that of flutamide. S-40542 had little effect on the blood level of testosterone and luteinizing hormone, whereas flutamide increased the level of both hormones. Furthermore, S-40542 decreased dose-dependently prostate weight of BPH rats. The current results indicate that S-40542 possesses the prostate-selective SARM activity, suggestive of clinical benefit against benign prostate hyperplasia. THQ compounds may be useful for the research of mode of action of SARMs and for the development of safe SARM antagonists.

ARN-509: A Novel Antiandrogen for Prostate Cancer Treatment

Continued reliance on the androgen receptor (AR) is now understood as a core mechanism in castration-resistant prostate cancer (CRPC), the most advanced form of this disease. While established and novel AR pathway-targeting agents display clinical efficacy in metastatic CRPC, dose-limiting side effects remain problematic for all current agents. In this study, we report the discovery and development of ARN-509, a competitive AR inhibitor that is fully antagonistic to AR overexpression, a common and important feature of CRPC. ARN-509 was optimized for inhibition of AR transcriptional activity and prostate cancer cell proliferation, pharmacokinetics, and in vivo efficacy. In contrast to bicalutamide, ARN-509 lacked significant agonist activity in preclinical models of CRPC. Moreover, ARN-509 lacked inducing activity for AR nuclear localization or DNA binding. In a clinically valid murine xenograft model of human CRPC, ARN-509 showed greater efficacy than MDV3100. Maximal therapeutic response in this model was achieved at 30 mg/kg/d of ARN-509, whereas the same response required 100 mg/kg/d of MDV3100 and higher steady-state plasma concentrations. Thus, ARN-509 exhibits characteristics predicting a higher therapeutic index with a greater potential to reach maximally efficacious doses in man than current AR antagonists. Our findings offer preclinical proof of principle for ARN-509 as a promising therapeutic in both castration-sensitive and castration-resistant forms of prostate cancer.

Androgen Receptor Serine 81 Phosphorylation Mediates Chromatin Binding and Transcriptional Activation

Our previous findings indicated that androgen receptor (AR) phosphorylation at serine 81 is stimulated by the mitotic cyclin-dependent kinase 1 (CDK1). In this report, we extended our previous study and confirmed that Ser-81 phosphorylation increases during mitosis, coincident with CDK1 activation. We further showed blocking cell cycle at G(1) or S phase did not disrupt androgen-induced Ser-81 phosphorylation and AR-dependent transcription, consistent with a recent report that AR was phosphorylated at Ser-81 and activated by the transcriptional CDK9. To assess the function of Ser-81 phosphorylation in prostate cancer (PCa) cells expressing endogenous AR, we developed a ligand switch strategy using a ligand-binding domain mutation (W741C) that renders AR responsive to the antagonist bicalutamide. An S81A/W741C double mutant AR stably expressed in PCa cells failed to transactivate the endogenous AR-regulated PSA or TMPRSS2 genes. ChIP showed that the S81A mutation prevented ligand-induced AR recruitment to these genes, and cellular fractionation revealed that the S81A mutation globally abrogated chromatin binding. Conversely, the AR fraction rapidly recruited to chromatin after androgen stimulation was highly enriched for Ser-81 phosphorylation. Finally, inhibition of CDK1 and CDK9 decreased AR Ser-81 phosphorylation, chromatin binding, and transcriptional activity. These findings indicate that Ser-81 phosphorylation by CDK9 stabilizes AR chromatin binding for transcription and suggest that CDK1-mediated Ser-81 phosphorylation during mitosis provides a pool of Ser-81 phosphorylation AR that can be readily recruited to chromatin for gene reactivation and may enhance AR activity in PCa.

MiR‐141 modulates androgen receptor transcriptional activity in human prostate cancer cells through targeting the small heterodimer partner protein

Aberrant expressions of microRNAs, including upregulation of miR-141, are closely associated with the tumorigenesis of prostate cancer (PCa). The orphan receptor small heterodimer partner (Shp) is a co-repressor to androgen receptor (AR) and represses AR-regulated transcriptional activity. Here, we investigated the correlation of Shp expression with the cellular level of miR-141 and its effects on AR transcriptional activity in non-malignant and malignant human prostate epithelial cell lines. We found that Shp was downregulated in multiple PCa cell lines. The mature form of miR-141 was upregulated in PCa cells. miR-141 could target 3'-untranslated region of Shp mRNA resulting in translational suppression and RNA degradation. Moreover, enforced expression of Shp or inhibition of miR-141 function by anti-miR-141 attenuated AR-regulated transcriptional activity in AR-responsive LNCaP cells. Phenethyl isothiocyanate, a natural constituent of many edible cruciferous vegetables, increased Shp expression, downregulated miR-141, and inhibited AR transcriptional activity in LNCaP cells. Shp is a target for miR-141 and it is downregulated in cultured human PCa cells with the involvement of upregulation of miR-141, which promotes AR transcriptional activity. Moreover, Shp and miR-141 could be targets for chemoprevention for PCa.

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.

Abstract A55: ETS1 transcriptional activity promotes the castrate-resistant phenotype in prostate cancer.

Abstract Androgen deprivation therapy (ADT) is the treatment of choice for advanced prostate cancer. ADT targets the transcriptional activity of the androgen receptor (AR) to inhibit prostate cancer tumor growth. Unfortunately, the success of this treatment is not sustained as tumors circumvent the effects of ADT by restoring AR transcriptional function to become castrate resistant. Despite recent improvements in the treatment of castrate resistant prostate cancer (CRPC), therapies remain palliative. Castrate resistance accounts for practically all prostate cancer mortalities, therefore preventing its occurrence by keeping tumors static, i.e., castrate sensitive, would significantly impact patient survival. Aberrant ETS1 (v-ets erythroblastosis virus E26 oncogene 1) activity is associated with poor prognosis in several tumor types through the transcriptional regulation of genes associated with aggressive cancer phenotypes. Our studies indicate that inhibition of ETS1 can restore sensitivity to ADT in castrate resistant cells and stall the invasive processes that promote its deadly effects. In human prostate cancer tissue ETS1 expression is increased in tumors Gleason 7 and above. In vitro, ETS1 expression and nuclear phosphorylation correlated with both aggression and the castrate resistant phenotype in the LNCaP model of prostate cancer progression. Targeted AR activity altered ETS1 expression levels and loss of ETS1 expression restored sensitivity to AR antagonist treatment in castrate resistant cells. AKT activity is increased in CRPC. Elevated AKT activity was demonstrated to increase ETS1 levels specifically in castrate resistant cells and exogenous ETS1 expression was sufficient to restore the invasive potential decreased by AKT inhibition. MMP1 (matrix metalloproteinase 1) is an ETS1 transcriptional target. Increased expression of MMP1 reflects the malignant phenotype and more aggressive tumor characteristics. ELISA assays show that MMP1 levels are increased in castrate resistant compared to castrate sensitive cells and are further increased by exogenous ETS1 expression. Luciferase reporter assays show that ETS1 significantly enhances MMP1 promoter activation in castrate resistant compared to castrate sensitive cells when exogenously expressed at comparable levels. In mouse xenograft models, increased ETS1 expression in castrate sensitive LNCaP cells promotes tumor growth to levels observed for castrate resistant cells. Significantly, exogenous ETS1 expression in castrate sensitive cells promotes tumor growth in androgen deprived (castrated) mice. Restoration of androgen receptor (AR) transcriptional activity is believed to be critical for progression to the castrate resistant phenotype. The mechanism by which AR activity is restored in CRPC remains unclear but increased co-recruitment of AR co-regulatory factors is critical. In combination these data suggest a role for ETS1 transcriptional function in promoting the restoration of AR transcriptional activity in castrate resistant cells. Citation Format: Ashley Marie Smith, Victoria Jane Findlay, Savannah Bandurraga, Emily Kistner-Griffin, Laura Spruill, Angen Liu, Ali Golshayan, David Paul Turner. ETS1 transcriptional activity promotes the castrate-resistant phenotype in prostate cancer [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 A55.

Abstract C7: Targeting the androgen receptor with novel combination therapeutic strategies in metastatic/castrate-resistant prostate cancer

Abstract While recent advances have resulted in the FDA approval of novel therapies for metastatic/castrate resistant prostate cancer, responses are not often durable and novel therapeutic interventions are still required. Recent work within our laboratory has resulted in the development of a novel orthotopic syngeneic transplant mouse model of prostate cancer. Further, treatment with a HDAC and mTORC1 inhibitor in combination resulted in greater anti-tumor and increased overall survival in tumor bearing animals. HDAC/mTORC1 combination therapy also resulted in decreased androgen receptor (AR) transcriptional activity without loss of AR protein expression. Current investigations are underway to delineate molecular mechanisms underlying sustained AR expression. Through this understanding, novel therapeutic interventions which will augment combined HDAC/mTORC1 inhibitor therapy are under investigation. Citation Format: Leigh Ellis, Swathi Ramakrishnan, Shengyu Ku, Roberto Pili. Targeting the androgen receptor with novel combination therapeutic strategies in metastatic/castrate-resistant prostate cancer [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 C7.

Abstract B5: Epigenetic modification of AR in PC3 cells negatively regulates mTORC1/2 activity

Abstract Wild type androgen receptor (AR) acts as a tumor suppressor and mediates the differentiation of non-malignant prostate epithelial cells. However, in the development of prostate cancer AR activity becomes critical for the development and progression of disease. Previous studies demonstrate that re-expressing wild type AR within an AR negative cell line (PC3) possesses tumor suppressor quality by reducing cell proliferation and tumorgenicity in mice. Further, it is documented that acetylation of AR in non-malignant cells is a post-translational modification associated with increased transcriptional activity of AR, whereas acetylation of AR in tumor cells results in negative regulation of AR transcriptional activity. We therefor are seeking to investigate the regulation of re-expressed AR within PC3 cells by induced acetylation post HDAC inhibition. Our preliminary data reveals that sensitivity to HDAC inhibition is increased with the re-expression of AR in PC3 cells. Further, epigenetic modification of AR in this model negatively regulates mTORC1 and mTORC2 expression and activity, independent of AR transcriptional activity. Ongoing experiments are being conducted to investigate further the role of epigenetic post-translational modification of AR and its negative regulation of mTORC1/2 signaling. Citation Format: Leigh Ellis, Remi Adelaiye, Shengyu Ku, Alejandro Godoy, Roberto Pili. Epigenetic modification of AR in PC3 cells negatively regulates mTORC1/2 activity [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 B5.

Abstract B14: Developing small-molecule inhibitors to the androgen receptor N-terminus domain for the treatment of advanced prostate cancer

Abstract Androgen ablation therapy remains the gold standard for the treatment of advanced prostate cancer, but unfortunately, it is not curative and eventually the disease will return as lethal castration-resistant prostate cancer (CRPC). There is evidence supporting the concept that development of CRPC is causally related to continued transactivation of androgen receptor (AR). Suspected mechanisms for continued AR activity in spite of castrate levels of androgen include: amplification or overexpression of AR; gain-of-function mutations allowing AR to be activated by steroids or antiandrogens; ligand-independent activation by growth factors, cytokines, or kinases; overexpression of AR coactivators; intracrine signaling by increased intratumoral androgens; and/or expression of constitutively active splice variants of AR that lack the C-terminal ligandbinding domain (LBD). All current therapies that target the AR are dependent on the presence of its C-terminal LBD. However, it is the N-terminal domain (NTD) of the AR that is the “Achilles Heel” of AR activity, with activation function-1 (AF-1) being essential for AR activity regardless of androgen. Our efforts have been focused upon developing drugs to the AR NTD and have yielded EPI-001 a small molecule, sintokamide peptides, and decoys to the AR NTD. Of these, EPI-001 is the best characterized as previously shown to inhibit essential protein-protein interactions that are required for AR transcriptional activity. EPI-001 and its analogues (generally referred to as “EPI”) have great promise for clinical development based upon its unique mechanism of action, specificity, low toxicity, and cytoreductive antitumor activity. EPI blocked transcriptional activity of full-length and AR variants as well as specifically inhibited AR-dependent cell proliferation. EPI directly and specifically interacted with AF1 and did not interact with denatured AF1 as shown using in vitro binding assays. Specific and direct interaction of EPI with the endogenous AR occurred in living cells as shown using click chemistry. EPI-001 had 86% oral bioavailability, a half-life of 3.4 hours, and plasma levels at the effective concentration of 10 ug/ml were achieved with oral dosing. Consistent with excellent oral bioavailability, oral dosing of EPI inhibited VCaP tumor growth in castrated animals. VCaP human prostate cancer cells express an abundance of full-length AR as well as constitutively active AR splice variant lacking LBD. Evidence for EPI targeting the AR transcriptional program in vivo, was provided by reduced transcripts of UBE2C, CDC20, cyclinA2, and AKT1 in harvested VCaP xenografts from animals treated orally with EPI. In conclusion, EPI is an antagonist of AR NTD that blocks the activity of AR, including constitutively active AR splice variants, by a mechanism that involves direct interaction with the NTD. Oral dosing of EPI has antitumor activity in prostate cancer xenografts that express AR variant. Together these data support the clinical development of EPI for the treatment of CRPC. Funding: NIH (2R01 CA105304) and US Army Medical Research and Materiel Command Prostate Cancer Research Program (PC100761). Note: This abstract was not presented at the conference because the presenter was unable to attend. Citation Format: Marianne D. Sadar, Jae-Kyung Myung, Iain McEwan, Stephen Plymate, Raymond J. Andersen, Carmen A. Banuelos, Nasrin R. Mawji, Jun Wang, Javier Garcia Fernandez, Amy Tien, Iran Tavakoli, Yu Chi Yang, Simon Haile. Developing small-molecule inhibitors to the androgen receptor N-terminus domain for the treatment of advanced prostate cancer [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 B14.

Polychlorinated Biphenyls Affect Histone Modification Pattern in Early Development of Rats: A Role for Androgen Receptor-Dependent Modulation?

The epigenome represents an important target of environmental pollution. Early-life exposure to polychlorinated biphenyls (PCBs) modifies sex steroid enzymes and receptor transcription patterns. Steroid receptors, such as androgen receptor (AR), function as coregulators of histone modification enzymes. To clarify if a PCB early-life exposure might affect the epigenome in rat liver, we analyzed some histone post-translational modifications (H3K4me3 and H4K16Ac) and the corresponding histone remodeling enzymes, and the AR as a histone enzyme coregulator. We observed a decrease of H4K16Ac and H3K4me3 levels, possibly linked to the induction of chromatin-modifying enzymes SirtT1 and Jarid1b, and a decrease of AR. PCBs also seem to induce AR transcriptional activity. Some of the observed effects are sex dimorphic. Our data suggest that an early-life exposure to PCB sometimes modifies the epigenome in the offspring liver in a dimorphic way. AR might be involved in modulating PCB effects on the epigenome.

AR intragenic deletions linked to androgen receptor splice variant expression and activity in models of prostate cancer progression

Reactivation of the androgen receptor (AR) during androgen depletion therapy (ADT) underlies castration-resistant prostate cancer (CRPCa). Alternative splicing of the AR gene and synthesis of constitutively-active COOH-terminally truncated AR variants lacking the AR ligand binding domain has emerged as an important mechanism of ADT-resistance in CRPCa. In a previous study, we demonstrated that altered AR splicing in CRPCa 22Rv1 cells was linked to a 35 kb intragenic tandem duplication of AR exon 3 and flanking sequences. In this study, we demonstrate that complex patterns of AR gene copy number imbalances occur in PCa cell lines, xenografts, and clinical specimens. To investigate whether these copy number imbalances reflect AR gene rearrangements that could be linked to splicing disruptions, we carried out a detailed analysis of AR gene structure in the LuCaP 86.2 and CWR-R1 models of CRPCa. By deletion-spanning PCR, we discovered a 8,579 bp deletion of AR exons 5, 6, and 7 in the LuCaP 86.2 xenograft, which provides a rational explanation for synthesis of the truncated AR v567es AR variant in this model. Similarly, targeted re-sequencing of the AR gene in CWR-R1 cells led to the discovery of a 48 kb deletion in AR intron 1. This intragenic deletion marked a specific CWR-R1 cell population with enhanced expression of the truncated AR-V7/AR3 variant, a high level of androgen-independent AR transcriptional activity, and rapid androgen independent growth. Together, these data demonstrate that structural alterations in the AR gene are linked to stable gain-of-function splicing alterations in CRPCa.

Hypoxia enhances ligand-occupied androgen receptor activity

Hypoxia and the androgen receptor (AR) play important roles in the development and progression of prostate cancer. In this study, the combined effects of dihydrotestosterone (DHT) and hypoxia on AR-mediated transactivation were investigated. Hypoxia alone did not induce a detectable ARE-mediated response in the absence of DHT. DHT-induced AR transcriptional activity was dramatically increased by hypoxia or ectopic expression of HIF-1α, as determined by introducing ARE-responsive reporter plasmids into LNCaP prostate cancer cells. The secretion of VEGF was enhanced by the combination of hypoxia and DHT as compared to each treatment alone. These effects were not due to increased expression of the AR or HIF-1α as a result of hypoxia and DHT treatment. These results provide evidence that hypoxia may stimulate as yet unknown factors, which further stimulate AR signal transduction pathways.

Inhibitory mechanisms of the transcriptional activity of androgen receptor by resveratrol : Implification of DNA binding and acetylation of the receptor

Inhibitory mechanisms of the transcriptional activity of androgen receptor by resveratrol : Implification of DNA binding and acetylation of the receptor

ASC-J9 Suppresses Castration-Resistant Prostate Cancer Growth through Degradation of Full-length and Splice Variant Androgen Receptors

Early studies suggested androgen receptor (AR) splice variants might contribute to the progression of prostate cancer (PCa) into castration resistance. However, the therapeutic strategy to target these AR splice variants still remains unresolved. Through tissue survey of tumors from the same patients before and after castration resistance, we found that the expression of AR3, a major AR splice variant that lacks the AR ligand-binding domain, was substantially increased after castration resistance development. The currently used antiandrogen, Casodex, showed little growth suppression in CWR22Rv1 cells. Importantly, we found that AR degradation enhancer ASC-J9 could degrade both full-length (fAR) and AR3 in CWR22Rv1 cells as well as in C4-2 and C81 cells with addition of AR3. The consequences of such degradation of both fAR and AR3 might then result in the inhibition of AR transcriptional activity and cell growth in vitro. More importantly, suppression of AR3 specifically by short-hairpin AR3 or degradation of AR3 by ASC-J9 resulted in suppression of AR transcriptional activity and cell growth in CWR22Rv1-fARKD (fAR knockdown) cells in which DHT failed to induce, suggesting the importance of targeting AR3. Finally, we demonstrated the in vivo therapeutic effects of ASC-J9 by showing the inhibition of PCa growth using the xenografted model of CWR22Rv1 cells orthotopically implanted into castrated nude mice with undetectable serum testosterone. These results suggested that targeting both fAR- and AR3-mediated PCa growth by ASC-J9 may represent the novel therapeutic approach to suppress castration-resistant PCa. Successful clinical trials targeting both fAR and AR3 may help us to battle castration-resistant PCa in the future.

SOD Mimetics: A Novel Class of Androgen Receptor Inhibitors That Suppresses Castration-Resistant Growth of Prostate Cancer

Advanced prostate cancer is the second leading cause of cancer-related deaths among American men. The androgen receptor (AR) is vital for prostate cancer progression, even in the face of castrate levels of serum testosterone following androgen ablation therapy, a mainstay therapy for advanced prostate cancer. Downregulation of superoxide dismutase 2 (SOD2), a major intracellular antioxidant enzyme, occurs progressively during prostate cancer progression to advanced states and is known to promote AR activity in prostate cancer. Therefore, this study investigated the effects of SOD mimetics on AR expression and function in AR-dependent LNCaP, CWR22Rv1, and LAPC-4AD prostate cancer cells. Treatment with Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), a SOD mimetic, not only lowered cellular superoxide levels but also concomitantly attenuated AR transcriptional activity and AR target gene expression in a dose- and time-dependent manner, in the presence and absence of dihydrotestosterone, the major endogenous AR agonist. Inhibition of AR by Tempol was mediated, in large part, by its ability to decrease AR protein via increased degradation, in the absence of any inhibitory effects on other nuclear receptors. Inhibitory effects of Tempol on AR were also reproducible with other SOD mimetics, MnTBAP and MnTMPyP. Importantly, effects of Tempol on AR function were accompanied by significant in vitro and in vivo reduction in castration-resistant prostate cancer (CRPC) survival and growth. Collectively, this study has shown for the first time that SOD mimetics, by virtue of their ability to suppress AR function, may be beneficial in treating the currently incurable CRPC, in which SOD2 expression is highly suppressed.

PD01-07: AR Overexpression and Aromatase Inhibitor Resistance in Breast Cancer.

Abstract Background: Aromatase inhibitors (AIs) have emerged as the therapy of choice for the treatment of estrogen receptor alpha (ERα)-positive breast cancer. However, many patients develop resistance to AI treatment. Although the involvement of the ERα in AI resistance is well established, the role of the androgen receptor (AR) is not known. It has been estimated that about 60%-70% of ERα-positive breast cancer co-express the AR, and that AR agonists can either inhibit or stimulate breast cancer cell proliferation. Thus it is important to determine if there are biomarkers predicting AR's effects in breast tumors. We have previously shown a role for AR-overexpression in tamoxifen resistance in ERα-positive MCF-7 breast cancer cells; here we hypothesized that AR overexpression might similarly be involved in resistance to the AI anastrazole (Anas). Materials and Methods: Stable transfection of MCF-7 cells was performed to generate cell lines that express the aromatase gene (MCF-7 BK Arom) and then co-transfected with an AR expression vector (MCF-7 AR Arom). Aromatase and AR expression levels were evaluated by western blot analysis, and the enzyme activity was evaluated using aromatase activity assays. Proliferation was tested using anchorage independent soft agar assays and MTT in the presence of the androgen substrate androstenedione (AD), or AD plus Anas. ERα and AR transcriptional activities were tested with ERE-luciferase reporter assays. Localization of ERα and AR within the cells was visualized using immunofluorescence microscopy. Results: ERα-positive MCF-7 cells were stably transfected with either aromatase, or aromatase plus AR. MCF-7 aromatase clones overexpressing AR were resistant to the growth inhibitory effects of Anas when stimulated with the androgen AD. Resistance was not mediated through changes in aromatase expression or activity. The growth of several of the AR Arom-overexpressing cells was stimulated with treatment of Anas alone, suggesting that Anas was acting as an agonist. As expected, AD treatment stimulated ERα transcriptional activity, but Anas was unable to block AD-stimulated activity in AR Arom-overexpressing cells using ERE-Luciferase reporter assay. Anas was able to enhance AR and ERα colocalization in AR-overxpressing cells. Resistance was not associated with activation of known mechanisms of resistance, such as HER2, IGF-1R, or MAPK. However AR-overexpressing cells had higher constitutive phosphorylation of Akt. Accordingly, resistance to Anas was blocked using an Akt1/2 inhibitor. Conclusion: Using a model of ERα-positive breast cancer cells expressing exogenous aromatase and AR, we have demonstrated that AR overexpression confers resistance to the AI Anas. These results suggest that in patients recurring on hormonal therapy whose tumors express elevated levels of AR, targeted therapy to Akt might restore hormone sensitivity. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD01-07.

Identification of Novel Androgen-Regulated Pathways and mRNA Isoforms through Genome-Wide Exon-Specific Profiling of the LNCaP Transcriptome

Androgens drive the onset and progression of prostate cancer (PCa) by modulating androgen receptor (AR) transcriptional activity. Although several microarray-based studies have identified androgen-regulated genes, here we identify in-parallel global androgen-dependent changes in both gene and alternative mRNA isoform expression by exon-level analyses of the LNCaP transcriptome. While genome-wide gene expression changes correlated well with previously-published studies, we additionally uncovered a subset of 226 novel androgen-regulated genes. Gene expression pathway analysis of this subset revealed gene clusters associated with, and including the tyrosine kinase LYN, as well as components of the mTOR (mammalian target of rapamycin) pathway, which is commonly dysregulated in cancer. We also identified 1279 putative androgen-regulated alternative events, of which 325 (∼25%) mapped to known alternative splicing events or alternative first/last exons. We selected 30 androgen-dependent alternative events for RT-PCR validation, including mRNAs derived from genes encoding tumour suppressors and cell cycle regulators. Of seven positively-validating events (∼23%), five events involved transcripts derived from alternative promoters of known AR gene targets. In particular, we found a novel androgen-dependent mRNA isoform derived from an alternative internal promoter within the TSC2 tumour suppressor gene, which is predicted to encode a protein lacking an interaction domain required for mTOR inhibition. We confirmed that expression of this alternative TSC2 mRNA isoform was directly regulated by androgens, and chromatin immunoprecipitation indicated recruitment of AR to the alternative promoter region at early timepoints following androgen stimulation, which correlated with expression of alternative transcripts. Together, our data suggest that alternative mRNA isoform expression might mediate the cellular response to androgens, and may have roles in clinical PCa.