Androgen-independent Activation Of Androgen Receptor Research Articles

Poly-glutamine-dependent self-association as a potential mechanism for regulation of androgen receptor activity.

The androgen receptor (AR) plays a central role in prostate cancer. Development of castration resistant prostate cancer (CRPC) requires androgen-independent activation of AR, which involves its large N-terminal domain (NTD) and entails extensive epigenetic changes depending in part on histone lysine demethylases (KDMs) that interact with AR. The AR-NTD is rich in low-complexity sequences, including a polyQ repeat. Longer polyQ sequences were reported to decrease transcriptional activity and to protect against prostate cancer, although they can lead to muscular atrophy. However, the molecular mechanisms underlying these observations are unclear. Using NMR spectroscopy, here we identify weak interactions between the AR-NTD and the KDM4A catalytic domain, and between the AR ligand-binding domain and a central KDM4A region that also contains low-complexity sequences. We also show that the AR-NTD can undergo liquid-liquid phase separation in vitro, with longer polyQ sequences phase separating more readily. Moreover, longer polyQ sequences hinder nuclear localization in the absence of hormone and increase the propensity for formation of AR-containing puncta in the nucleus of cells treated with dihydrotestosterone. These results lead us to hypothesize that polyQ-dependent liquid-liquid phase separation may provide a mechanism to decrease the transcriptional activity of AR, potentially opening new opportunities to design effective therapies against CRPC and muscular atrophy.

Androgen Receptor Could Be a Potential Therapeutic Target in Patients with Advanced Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a male-dominant disease with poor prognosis. Sorafenib is the only approved systemic chemotherapeutic drug for patients with advanced HCC. Previous studies have shown that androgen and androgen receptor (AR) are involved in human hepatocarcinogenesis and the development of HCC. Here, we discuss the recent data on AR and HCC, and the combination of sorafenib and inhibitors of AR for advanced-HCC patients. Androgen-dependent and androgen-independent AR activation exist in human hepatocarcinogenesis. AR could directly control hepatocarcinogenesis and regulate the innate immune system to influence HCC progression. Combination of sorafenib with AR inhibitors might represent a potential treatment for patients with advanced HCC.

LncRNA HOTAIR Enhances the Androgen-Receptor-Mediated Transcriptional Program and Drives Castration-Resistant Prostate Cancer.

SUMMARYUnderstanding the mechanisms of androgen receptor (AR) activation in the milieu of low androgen is critical to effective treatment of castration-resistant prostate cancer (CRPC). Here, we report HOTAIR as an androgen-repressed lncRNA, and, as such, it is markedly upregulated following androgen deprivation therapies and in CRPC. We further demonstrate a distinct mode of lncRNA-mediated gene regulation, wherein HOTAIR binds to the AR protein to block its interaction with the E3 ubiquitin ligase MDM2, thereby preventing AR ubiquitination and protein degradation. Consequently, HOTAIR expression is sufficient to induce androgen-independent AR activation and drive the AR-mediated transcriptional program in the absence of androgen. Functionally, HOTAIR overexpression increases, whereas HOTAIR knockdown decreases, prostate cancer cell growth and invasion. Taken together, our results provide compelling evidence of lncRNAs as drivers of androgen-independent AR activity and CRPC progression, and they support the potential of lncRNAs as therapeutic targets.

Mechanisms of resistance in castration-resistant prostate cancer (CRPC).

Despite advances in prostate cancer diagnosis and management, morbidity from prostate cancer remains high. Approximately 20% of men present with advanced or metastatic disease, while 29,000 men continue to die of prostate cancer each year. Androgen deprivation therapy (ADT) has been the standard of care for initial management of advanced or metastatic prostate cancer since Huggins and Hodges first introduced the concept of androgen-dependence in 1972, but progression to castration-resistant prostate cancer (CRPC) occurs within 2-3 years of initiation of ADT. CRPC, previously defined as hormone-refractory prostate cancer, is now understood to still be androgen dependent. Multiple mechanisms of resistance help contribute to the progression to castration resistant disease, and the androgen receptor (AR) remains an important driver in this progression. These mechanisms include AR amplification and hypersensitivity, AR mutations leading to promiscuity, mutations in coactivators/corepressors, androgen-independent AR activation, and intratumoral and alternative androgen production. More recently, identification of AR variants (ARVs) has been established as another mechanism of progression to CRPC. Docetaxel chemotherapy has historically been the first-line treatment for CRPC, but in recent years, newer agents have been introduced that target some of these mechanisms of resistance, thereby providing additional survival benefit. These include AR signaling inhibitors such as enzalutamide (Xtandi, ENZA, MDV-3100) and CYP17A1 inhibitors such as abiraterone acetate (Zytiga). Ultimately, these agents will also fail to suppress CRPC. While some of the mechanisms by which these agents fail are unique, many share similarities to the mechanisms contributing to CRPC progression. Understanding these mechanisms of resistance to ADT and currently approved CRPC treatments will help guide future research into targeted therapies.

Ligand-independent and tissue-selective androgen receptor inhibition by pyrvinium.

Pyrvinium pamoate (PP) is a potent noncompetitive inhibitor of the androgen receptor (AR). Using a novel method of target identification, we demonstrate that AR is a direct target of PP in prostate cancer cells. We demonstrate that PP inhibits AR activity via the highly conserved DNA binding domain (DBD), the only AR inhibitor that functions via this domain. Furthermore, computational modeling predicts that pyrvinium binds at the interface of the DBD dimer and the minor groove of the AR response element. Because PP acts through the DBD, PP is able to inhibit the constitutive activity of AR splice variants, which are thought to contribute to the growth of castration resistant prostate cancer (CRPC). PP also inhibits androgen-independent AR activation by HER2 kinase. The antiandrogen activity of pyrvinium manifests in the ability to inhibit the in vivo growth of CRPC xenografts that express AR splice variants. Interestingly, PP was most potent in cells with endogenous AR expression derived from prostate or bone. PP was able to inhibit several other hormone nuclear receptors (NRs) but not structurally unrelated transcription factors. PP inhibition of other NRs was similarly cell-type selective. Using dual-energy X-ray absorptiometry, we demonstrate that the cell-type specificity of PP manifests in tissue-selective inhibition of AR activity in mice, as PP decreases prostate weight and bone mineral density but does not affect lean body mass. Our results suggest that the noncompetitive AR inhibitor pyrvinium has significant potential to treat CRPC, including cancers driven by ligand-independent AR signaling.

Androgen Receptor in Human Health: A Potential Therapeutic Target

Androgen is a key for the activation of Androgen Receptor (AR) in most of the disease conditions, however androgen-independent activation of AR is also found in aggressive type human malignancies. An intense search for the inhibitors of AR is underway to cure AR-dependent diseases. In addition to targeting various components of AR signaling pathway, compounds which directly target AR are under preclinical and clinical investigation. Various In vitro and preclinical animal studies suggest that different natural compounds have potential to act against AR. Some natural compounds have been found to be pharmacologically effective against AR irrespective of varying routs of administration viz; oral, intra-peritoneal and intravenous. This mini-review summarizes the studies conducted with different natural agents in determining their pharmacological utility against AR signaling.

Abstract C38: Whole-genome sequencing of paired androgen-dependent and -independent prostate cancer cell lines: Potential role of novel targets in PI3K and Notch signaling pathways in resistance to hormonal therapy

Abstract The growth of the majority of prostate tumors is driven by androgens signaling through the androgen receptor. Most patients initially respond to androgen deprivation therapies such as LHRH analogues and anti-androgens like bicalutamide, but after a varying duration of treatment, patients generally develop resistance to these therapies and their tumor progresses to an androgen independent state. Molecular mechanisms that drive androgen independent disease include mutation and amplification of androgen receptor (AR), androgen-independent activation of AR, and AR-independent mechanisms. The aim of this project was to sequence the entire genome of pairs of cell lines, modeling the progression to androgen independent disease, to increase our understanding of these mechanisms and identify new therapeutic targets. Four ‘parental’ prostate cancer cell lines, all of which are sensitive to hormone deprivation, and an androgen independent derivative of each line, derived either by culturing in bicalutamide or growing in the absence of androgens, were subjected to whole genome sequencing. Genomic variants present in the androgen independent but not the parental lines, presumed to be acquired during the progression to androgen independence, were then identified. To find any commonality between the four different pairings, the derived variants were then mapped to functional biological pathways and their likely impact on protein function predicted. Interestingly, derived mutations in the PI3K and Notch signaling pathways were identified in multiple cell lines. In addition to the analysis of derived mutations, baseline characterization of genomic rearrangements and copy number changes in cell lines highlighted variability in the patterns and prevalence of these events as well as uncovering aberrations in genes known to impact prostate cancer development (PTEN, AR, TMPRSS2 rearrangement). This extensive genome sequencing effort has yielded a deeper understanding of potential pathways and molecular events that occur during the acquisition of resistance to anti-androgen therapy, using in vitro models of prostate cancer. These candidates now require validation in tissue samples from prostate cancer patients and functional assays. Citation Format: Gillian L. Dalgliesh, Joanne L. Hartley, Cary H. O'Donnell, James R. Bradford, Iain McKendrick, Barry R. Davies. Whole-genome sequencing of paired androgen-dependent and -independent prostate cancer cell lines: Potential role of novel targets in PI3K and Notch signaling pathways in resistance to hormonal therapy [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 C38.

Abstract B92: Differences in gene expression between primary chemotherapy-naive prostate carcinomas and hormone-refractory prostate carcinomas.

Abstract Introduction: Despite the widespread use of prostate specific antigen screening for early detection, prostate cancer remains the second leading cause of cancer related death among men in the US. Metastatic, hormone refractory prostate cancer (HRPCa) is the end stage, lethal form of the disease. Defining the molecular mechanisms underlying the transition of an androgen responsive prostate cancer represents an important clinical problem. Currently, no effective therapies exist for end stage, hormone refractory disease. In this study, we investigated the differentially expressed genes in primary prostate tumor vs. hormone refractory prostate tumor in order to identify potentially important therapeutic targets. Methods: Formalin fixed paraffin embedded tumor samples from 14 cases (8 primary, chemotherapy-naïve and 6 locally recurrent, hormonal therapy-refractory carcinomas) were analyzed for the whole genome RNA (29,285 transcripts) microarray analysis using Illumina cDNA-mediated annealing, selection, extension and ligation (DASL) process with the HumanHT-12 v4 beadChip (Illumina Inc., San Diego, CA). Additionally, a select number of chemotherapy-predictive (theranostic) biomarkers were analyzed using immunohistochemical methods. Results: Transcriptome analysis identified 299 genes highly differentially expressed between the primary and recurrent groups (p-value < 0.01, Student's t-test). Using these 299 genes, we performed two dimensional hierarchical clustering and were able to discover 8 clusters corresponding to 5 general patterns: Pattern 1-underexpressed in both groups but more so in recurrent than primary (2 clusters with 28 and 22 genes respectively); Pattern 2-slightly overexpressed in primary and underexpressed or no change in recurrent (1 cluster with 65 genes); Pattern 3-slightly under expressed/ no change in primary and overexpressed in recurrent (1 cluster, 12 genes); Pattern 4-overexpressed in primary and no change in recurrent (1 cluster, 25 genes); Pattern 5 - overexpressed in both groups but more so in recurrent than primary (3 clusters with 58, 32, and 57 genes in each cluster). Individual gene analysis revealed upregulation of androgen receptor (AR) mRNA in recurrent HRPCa. This was, however, accompanied by a down regulation of regulator of G-protein signaling 2 (RGS2) and ras responsive element binding protein 1 (RREB1). Consistent down regulation of excision repair cross-complementing group 1 (ERCC1) and thymidylate synthase (TS) proteins were also observed in HRPCa. Conclusion: Up-regulation of AR in HRPCa is associated with down regulation of androgen receptor signaling co-regulators (RGS2 and RREB1), providing a mechanism of androgen-independent activation of AR in HRPCa. Platinum based drugs may have potential benefit in treating of HRPCa due to the down regulation of nucleotide excision repair protein ERCC1, while fluoropyrimidines are potentially beneficial due to the lack of TS protein expression in HRPCa. The heat map derived signature readily distinguishes between prostate cancer specimens from men who were treatment naïve vs. hormone refractory cancers with 5 different expression patterns. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B92.

Abstract PR-06: PKC∈-activated Stat3 and subsequent AR interaction are potential molecular targets for prevention of late-stage prostate cancer

Abstract Prostate cancer (PCa) is the second most common malignancy in men and the second leading cause of cancer-related deaths in the Western World. Androgen ablation therapy remains the gold standard for the treatment of PCa. Despite the initial success of androgen ablation therapy, resistance to anti-androgen therapy manifests by progression to castration-resistant prostate cancer (CRPC). CRPC is the end stage that accounts for the majority of PCa patient deaths. An understanding of the mechanisms linked to the emergence of CRPC is the key to the prevention of this disease. Overwhelming evidence supports the concept that development of CRPC is usually related to continued activation of androgen receptor (AR). Several mechanisms are proposed for androgen-dependent (AD) and androgen-independent (AI) activation of AR in CRPC. Gene amplification and mutations in AR are frequently observed in recurrent PCa, which may account for the hypersensitivity of the AR to low castrate level of androgens and altered ligand specificity. In the AR-dependent pathway, the functions of AR are modified significantly and involve AR interaction with many co-activators and co-repressors. Increased AR activity in CRPC is perhaps caused by cross talk of AR with multiple intracellular signaling cascades. We present here that PKC∈ and Stat3, which correlate with the aggressiveness of PCa, may play roles in both AI AR activation and promotion of PCa cell survival for emergence and progression of CRPC. Stat3 has two conserved amino acid residues (Tyr705 and Ser727), which have to be phosphorylated for maximum transcriptional activity. PKC∈, which is constitutively activated in PCa, mediates phosphorylation of both Stat3Tyr705 and Stat3Ser727. Specific observations are our finding that PKC∈ deletion in TRAMP mice, which prevents PCa development and metastasis, inhibits the expression levels of IL-6, pStat3Ser727 and pStat3Tyr705. PKC∈ overexpression is sufficient to transform AD LNCaP cells into an AI variant. We further explored the possibility that PKC∈− activated Stat3 is a non-androgen activator of AR. In this experiment, human PCa LNCaP (AD) cells were infected with adenoviral vector containing PKC∈ or empty vector; 48 h later, cells were harvested and whole cell lysates were prepared. The effect of PKC∈ overexpression on the interaction of Stat3 with AR was determined by performing reciprocal immunoprecipitation/blotting experiments. Overexpression of PKC∈ increased the interaction of Stat3 with AR. We also found similar interaction of Stat3 with AR in human PCa C4–2 (AI) cells treated with IL-6. To determine the link of PKC∈ in Stat3 interaction with AR and the growth of C4–2 cells, we used plumbagin (PL). PL is a unique plant-derived chemopreventive agent, which selectively inhibits expression of PKC∈. PL administration delayed the growth of C4–2 cells xenograft tumors as determined by both tumor weight and volume. In a reciprocal immunoprecipation/blotting experiments, PL inhibited Stat3-AR interaction. We conclude that: 1) PKC∈-mediated Stat3Ser727 phosphorylation and subsequent Stat3-AR interaction are essential components of the emergence and progression of CRPC, 2) Progression of CRPC is the result of cross-talk of PKC∈-Stat3 signaling with other signaling pathways (AKT and MAPK) which activate AR as well as promote cell survival and 3) PKC∈-activated Stat3 and subsequent AR interaction are potential molecular targets for prevention of late stage PCa. Support: NIH Grant CA138761. Citation Information: Cancer Prev Res 2011;4(10 Suppl):PR-06.

Epigenetic repression of regulator of G-protein signaling 2 promotes androgen-independent prostate cancer cell growth.

G-protein-coupled receptor (GPCR)-stimulated androgen-independent activation of androgen receptor (AR) contributes to acquisition of a hormone-refractory phenotype by prostate cancer. We previously reported that regulator of G-protein signaling (RGS) 2, an inhibitor of GPCRs, inhibits androgen-independent AR activation (Cao et al., Oncogene 2006;25:3719-34). Here, we show reduced RGS2 protein expression in human prostate cancer specimens compared to adjacent normal or hyperplastic tissue. Methylation-specific PCR analysis and bisulfite sequencing indicated that methylation of the CpG island in the RGS2 gene promoter correlated with RGS2 downregulation in prostate cancer. In vitro methylation of this promoter suppressed reporter gene expression in transient transfection studies, whereas reversal of this promoter methylation with 5-aza-2'-deoxycytidine (5-Aza-dC) induced RGS2 reexpression in androgen-independent prostate cancer cells and inhibited their growth under androgen-deficient conditions. Interestingly, the inhibitory effect of 5-Aza-dC was significantly reduced by an RGS2-targeted short hairpin RNA, indicating that reexpressed RGS2 contributed to this growth inhibition. Restoration of RGS2 levels by ectopic expression in androgen-independent prostate cancer cells suppressed growth of xenografts in castrated mice. Thus, RGS2 promoter hypermethylation represses its expression and unmasks a latent pathway for AR transactivation in prostate cancer cells. Targeting this reversible process may provide a new strategy for suppressing prostate cancer progression by reestablishing its androgen sensitivity.

1449 P38 MAP KINASE ASSOCIATES WITH ANDROGEN RECEPTOR (AR) AND MODULATES AR SIGNALING IN PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 20111449 P38 MAP KINASE ASSOCIATES WITH ANDROGEN RECEPTOR (AR) AND MODULATES AR SIGNALING IN PROSTATE CANCER CELLS Sweaty Koul, Douglas Lim, Fernando J. Kim, Randall B. Meacham, and Hari Koul Sweaty KoulSweaty Koul Aurora, CO More articles by this author , Douglas LimDouglas Lim Aurora, CO More articles by this author , Fernando J. KimFernando J. Kim Aurora, CO More articles by this author , Randall B. MeachamRandall B. Meacham Aurora, CO More articles by this author , and Hari KoulHari Koul Aurora, CO More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1362AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES p38 MAP kinase signal transduction pathway is activated by various forms of cellular stress. While, in many tissues activation of p38 MAP kinase is associated with apoptosis, in some tissues p38 activation is critical for survival. Indeed activation of p38 MAP kinase pathway has been shown in several tumor types, but the role of p38 MAP kinase to prostate cancer biology and castrate resistant PCa has not been clearly delineated. Present studies aimed at understanding the role of p38 MAP kinase pathway in prostate cancer with special emphasis on Androgen Receptor signaling. METHODS LNCaP cells, were grown at high densities in RPMI medium supplemented with 10% Fetal Calf Serum, 1mM Sodium Pyruvate and antibiotics and maintained at 37 °C in 5% CO2 incubator. Western blot analysis was used to measure amount and activity of p38 MAP kinase, and Luciferase reporter assays were used to determine the activity of Androgen Receptor proteins. Immuno precipitation and Western blot combination was used to probe interaction of AR with p38 MAP kinase. Chemical inhibitor, dominant negative expression and shRNA to p38 MAP kinase was used to regulate p38 MAP kinase activity and levels. Well established protocols in the lab probed the effects of these treatments on cell viability, invasion, clonogenic activity and invasive behavior of the cells. RESULTS We observed that suppression of p38 MAP kinase activity by a variety of approaches decreased viability and growth of LNCaP cells in culture. We also observed that androgen depletion was associated with increased activation of p38 MAP kinase activity and inhibition of p38 MAP kinase sensitized these cells to androgen depletion. Our results show that activation of p38 MAP kinase during androgen depletion was associated with androgen independent AR activation. Results of IP experiments show for the first time that AR co-localized withp38 MAP kinase and finally and most surprisingly, stable suppression of p38 MAP kinase using Lentiviral shRNA approach resulted in complete loss of AR activity and AR protein levels. CONCLUSIONS These data show, for the first time, critical requirement for p38 MAP kinase in androgen receptor signaling in prostate cancer cells; and show for the first time, in any cell type, the regulation of AR expression by p38 MAP kinase signaling pathway. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e581 Peer Review Report Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sweaty Koul Aurora, CO More articles by this author Douglas Lim Aurora, CO More articles by this author Fernando J. Kim Aurora, CO More articles by this author Randall B. Meacham Aurora, CO More articles by this author Hari Koul Aurora, CO More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract 1710: Epigenetic repression of regulator of G-protein signaling 2 promotes prostate cancer progression

Abstract Prostate cancer is the most common cancer in American men and acquisition of androgen independence by prostate cancer is the key problem causing prostate cancer progression to highly aggressive form of cancer, leaving limited modalities in its treatment. Recent studies suggest that G protein-coupled receptor (GPCR)-stimulated androgen-independent activation of androgen receptor (AR) contributes to development of androgen independence. We previously reported that RGS2, an inhibitor of GPCR signaling, inhibits androgen-independent AR activation in prostate cancer cells (Cao et al., (2006) Oncogene, 25: 3719-3734). Here, we report that RGS2 protein expression was significantly reduced in human prostate cancer specimens compared to adjacent normal prostate tissues. Interestingly, RGS2 repression in androgen-independent prostate cancer cells and human prostate cancer specimens is not simply due to the loss of the RGS2 gene. Instead, methylation-specific PCR analysis and bisulfite sequencing indicated that methylation of the RGS2 gene promoter correlates with RGS2 down-regulation in prostate cancer cells and human prostate cancer specimens. Hypermethylation of the RGS2 gene promoter abolished its promoter activity and suppressed RGS2 gene expression. Conversely, the DNA methyltransferase inhibitor, 5-aza-2’-deoxycytidine induced re-expression of RGS2 in androgen-independent prostate cancer cells via demethylation of the RGS2 promoter to inhibit androgen-independent prostate cancer growth. Furthermore, ectopic re-expression of RGS2 in androgen-independent prostate cancer cells by RGS2-adenovirus infection suppressed tumorigenicity and growth of xenografts in castrated nude mice. Taken together, our results suggest that RGS2 repression due to hypermethylation of its promoter could unmask a latent pathway for AR activation by various GPCRs, thus contributing to prostate cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1710.

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.

Hypoxia-associated p38 mitogen-activated protein kinase-mediated androgen receptor activation and increased HIF-1alpha levels contribute to emergence of an aggressive phenotype in prostate cancer.

Androgen Receptor (AR) -signaling plays a critical role in the development and progression of prostate cancer. Tumor microvasculature contributes to continual exposure of prostate cancer cells to hypoxia-reoxygenation, however, the role of hypoxia-reoxygenation in prostate cancer progression and modulation of AR signaling is not understood. In the present study, we evaluated the effects of hypoxia-reoxygenation in LNCaP cells, a line of hormone responsive human prostate cancer cells. Our results demonstrate that hypoxia-reoxygenation resulted in increased survival, higher clonogenicity, and enhanced invasiveness of these cells. Moreover, hypoxia-reoxygenation was associated with an increased AR activity independent of androgens as well as increased Hypoxia Inducible Factor (HIF-1α) levels and activity. We also observed that the activation of p38 MAP kinase pathway was an early response to hypoxia, and inhibition of p38 MAP kinase pathway by variety of approaches abolished hypoxia-reoxygenation induced increased AR activity as well as increased survival, clonogenicity, and invasiveness. These results demonstrate a critical role for hypoxia induced p38 MAP kinase pathway in androgen independent AR activation in prostate cancer cells, and suggest that hypoxia re-oxygenation may select for aggressive androgen-independent prostate cancer phenotype.

Mitogenic action of the androgen receptor sensitizes prostate cancer cells to taxane-based cytotoxic insult: Hess-Wilson JK, Daly HK, Zagorski WA, Montville CP, Knudsen KE, Department of Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH

Prostate cancer cells are dependent on androgen for growth and survival; as such, inhibition of androgen receptor (AR) activity is the first line of intervention for disseminated disease. Recently, specific cytotoxic agents have been shown to extend survival times in patients with advanced disease. Given the established ability of androgen to modify cell survival in prostate cancer cells, it is imperative to determine the effect of the hormonal environment on cytotoxic response. Here, we show that the response of prostate cancer cells to taxane-induced cell death is significantly enhanced by androgen stimulation in AR-positive, androgen-dependent prostate cancer cells. Similar results were observed on androgen-independent AR activation. By contrast, AR-positive yet androgen-independent or AR-negative cells were refractory to androgen influence on taxane function. The ability of androgen to potentiate taxane activity was dependent on its mitogenic capacity and was separable from overall AR activity, as co-administration of AR antagonists, G(1) cyclin-dependent kinase inhibitors, or high-dose (growth inhibitory) androgen nullified the proapoptotic function of androgen. Observed induction of cell death was attributed to caspase-dependent apoptosis and correlated with p53 activation. Combined, these data indicate that the cytotoxic effects of taxanes are substantially influenced by the hormonal environment and/or status of AR activity in prostate cancer cells and provide the foundation for refinement and optimization of cytotoxic intervention in prostate cancer.

Vasoactive Intestinal Peptide Transactivates the Androgen Receptor through a Protein Kinase A-Dependent Extracellular Signal-Regulated Kinase Pathway in Prostate Cancer LNCaP Cells

Acquisition of androgen independence by prostate cancer is the key problem of prostate cancer progression. Vasoactive intestinal peptide (VIP), a neuropeptide, may act as a survival factor for prostate cancer cells under androgen deprivation. However, the molecular mechanisms by which VIP promotes the androgen-independent growth of androgen-sensitive prostate cancer cells have not been addressed. We therefore investigated the biological effect and signal pathway of VIP in LNCaP cells, a prostate cancer cell line that requires androgens for growth. We showed that low nanomolar concentrations of VIP, acting through G(s)-protein-coupled VIP receptors, can induce LNCaP cell growth in the absence of androgen. Blockade of androgen-receptor (AR) in these cells by AR antagonist bicalutamide or by anti-AR small interfering RNA, inhibited the proliferative effect of VIP. In addition, VIP stimulated androgen-independent activation of AR with an EC(50) of 3.0 +/- 0.8 nM. We then investigated VIP-stimulated signaling events that may interact with the AR pathway in prostate cancer cells. VIP regulation of AR activation, mediated by VIP receptors, was protein kinase A (PKA)-dependent, and extracellular signal-regulated kinase 1/2 (ERK1/2) activation contributes to VIP-mediated AR activation. Furthermore, PKA-dependent Rap1 activation is required for both ERK1/2 activation and androgen-independent AR activation in LNCaP cells upon VIP stimulation. Finally, we showed that VIP-induced AR activation was also present in prostate cancer CWR22Rv1 and PC3 cells transfected with the wild-type AR. Altogether, we demonstrate that VIP acting through its G(s)-protein-coupled receptors can cause androgen-independent transactivation of AR through a PKA/Rap1/ERK1/2 pathway, thus promoting androgen-independent proliferation of androgen-sensitive prostate cancer cells.

Mitogenic Action of the Androgen Receptor Sensitizes Prostate Cancer Cells to Taxane-Based Cytotoxic Insult

Prostate cancer cells are dependent on androgen for growth and survival; as such, inhibition of androgen receptor (AR) activity is the first line of intervention for disseminated disease. Recently, specific cytotoxic agents have been shown to extend survival times in patients with advanced disease. Given the established ability of androgen to modify cell survival in prostate cancer cells, it is imperative to determine the effect of the hormonal environment on cytotoxic response. Here, we show that the response of prostate cancer cells to taxane-induced cell death is significantly enhanced by androgen stimulation in AR-positive, androgen-dependent prostate cancer cells. Similar results were observed on androgen-independent AR activation. By contrast, AR-positive yet androgen-independent or AR-negative cells were refractory to androgen influence on taxane function. The ability of androgen to potentiate taxane activity was dependent on its mitogenic capacity and was separable from overall AR activity, as coadministration of AR antagonists, G(1) cyclin-dependent kinase inhibitors, or high-dose (growth inhibitory) androgen nullified the proapoptotic function of androgen. Observed induction of cell death was attributed to caspase-dependent apoptosis and correlated with p53 activation. Combined, these data indicate that the cytotoxic effects of taxanes are substantially influenced by the hormonal environment and/or status of AR activity in prostate cancer cells and provide the foundation for refinement and optimization of cytotoxic intervention in prostate cancer.

Regulator of G‐protein Signaling 2 (RGS2) inhibits androgen‐independent activation of androgen receptor in prostate cancer cells

Hormones acting through G protein-coupled receptors (GPCRs) can cause androgen-independent activation of androgen receptor (AR) in prostate cancer cells. Regulators of G-protein Signaling (RGS) proteins, through their GTPase activating protein (GAP) activities, inhibit GPCR-mediated signaling by inactivating G proteins. Here, we identified RGS2 as a gene specifically down-regulated in androgen-independent prostate cancer cells. Expression of RGS2, but not other RGS proteins, abolished androgen-independent AR activity in androgen-independent LNCaP cells and CWR22Rv1 cells. In LNCaP cells, RGS2 inhibited Gq-coupled GPCR signaling. Expression of exogenous wild-type RGS2, but not its GAP-deficient mutant, significantly reduced AR activation by constitutively activated GqQ209L mutant whereas silencing endogenous RGS2 by siRNA enhanced GqQ209L-stimulated AR activity. RGS2 had no effect on RGS-insensitive GqQ209L/G188S-induced AR activation. Furthermore, extracellular signal-regulated kinase 1/2 (ERK1/2) were found to be involved in RGS2-mediated regulation of androgen-independent AR activity. In addition, RGS2 functioned as a growth suppressor for androgen-independent LNCaP cells whereas androgen-sensitive LNCaP cells with RGS2 silencing had a growth advantage under steroid-reducedconditions. Finally, RGS2 expression level was significantly decreased in human prostate tumor specimens. Takentogether, our results suggest RGS2 as a novel regulator of AR signaling and its repression may be an important step during prostate tumorigenesis and progression.

Detection and Quantification of Protein Biomarkers from Fewer than 10 Cells

The use of antibody microarrays continues to grow rapidly due to the recent advances in proteomics and automation and the opportunity this combination creates for high throughput multiplexed analysis of protein biomarkers. However, a primary limitation of this technology is the lack of PCR-like amplification methods for proteins. Therefore, to realize the full potential of array-based protein biomarker screening it is necessary to construct assays that can detect and quantify protein biomarkers with very high sensitivity, in the femtomolar range, and from limited sample quantities. We describe here the construction of ultramicroarrays, combining the advantages of microarraying including multiplexing capabilities, higher throughput, and cost savings with the ability to screen very small sample volumes. Antibody ultramicroarrays for the detection of interleukin-6 and prostate-specific antigen (PSA), a widely used biomarker for prostate cancer screening, were constructed. These ultramicroarrays were found to have a high specificity and sensitivity with detection levels using purified proteins in the attomole range. Using these ultramicroarrays, we were able to detect PSA secreted from 100 LNCaP cells in 3 h and from just four LNCaP cells in 24 h. Cellular PSA could also be detected from the lysate of an average of just six cells. This strategy should enable proteomic analysis of materials that are available in very limited quantities such as those collected by laser capture microdissection, neonatal biopsy microspecimens, and forensic samples.

Regulator of G-protein signaling 2 (RGS2) inhibits androgen-independent activation of androgen receptor in prostate cancer cells

Hormones acting through G protein-coupled receptors (GPCRs) can cause androgen-independent activation of androgen receptor (AR) in prostate cancer cells. Regulators of G-protein signaling (RGS) proteins, through their GTPase activating protein (GAP) activities, inhibit GPCR-mediated signaling by inactivating G proteins. Here, we identified RGS2 as a gene specifically downregulated in androgen-independent prostate cancer cells. Expression of RGS2, but not other RGS proteins, abolished androgen-independent AR activity in androgen-independent LNCaP cells and CWR22Rv1 cells. In LNCaP cells, RGS2 inhibited G(q)-coupled GPCR signaling. Expression of exogenous wild-type RGS2, but not its GAP-deficient mutant, significantly reduced AR activation by constitutively activated G(q)Q209L mutant whereas silencing endogenous RGS2 by siRNA enhanced G(q)Q209L-stimulated AR activity. RGS2 had no effect on RGS-insensitive G(q)Q209L/G188S-induced AR activation. Furthermore, extracellular signal-regulated kinase 1/2 (ERK1/2) was found to be involved in RGS2-mediated regulation of androgen-independent AR activity. In addition, RGS2 functioned as a growth suppressor for androgen-independent LNCaP cells whereas androgen-sensitive LNCaP cells with RGS2 silencing had a growth advantage under steroid-reduced conditions. Finally, RGS2 expression level was significantly decreased in human prostate tumor specimens. Taken together, our results suggest RGS2 as a novel regulator of AR signaling and its repression may be an important step during prostate tumorigenesis and progression.