Interaction Of Androgen Receptor Research Articles

Abstract 65: Rationally designed peptidomimetics target androgen receptor signaling in prostate cancer

Abstract INTRODUCTION AND OBJECTIVES: Androgen receptor (AR) signaling is essential for prostate cancer development, growth, and progression at all stages of disease. AR signaling occurs via both genomic and non-genomic pathways and is mediated by AR interaction with cofactors. In our laboratory, we have shown that the interaction between a cofactor, PELP1 (Proline, Glutamic acid, Leucine rich Protein 1) and AR is critical for AR nuclear translocation in response to ligand and subsequent AR-mediated genomic signaling. We hypothesized that disruption of the interaction between AR and PELP1 using rationally designed peptidomimetics could affect AR genomic signaling in prostate cancer. METHODS: We have developed rationally designed bis benzamide scaffold-based α helical peptidomimetics analogous to the LXXLL motif on PELP1 that is presumed to bind AR. Initially two peptidomimetics were created- the control D1 and a LXXLL analogue D2. Prostate cancer cell lines were pre-treated with peptidomimetics prior to incubation with androgen (DHT/R1881 (0.01-10nM)) and evaluated for proliferation. Immunofluorescence was used to examine endogenous AR translocation to the DAPI-stained nucleus. RESULTS: Initial toxicity assays using trypan blue exclusion revealed both D1 and D2 were not toxic to the cells up to 500nM concentrations. We have shown that D2 (but not the control D1) prevents the formation of the AR-PELP-1 complex as evidenced by the ability of D2 to prevent co-immunoprecipitation of AR and PELP-1 in LAPC4 and LNCaP cell lines. This effect was overcome by increased expression of either AR or PELP1. D2 was able to significantly decrease DHT-mediated transcription from both minimal ARE luciferase promoters and PSA gene expression, whereas the control D1 did not show any activity. In microarray analyses, D2 was able to abrogate the induced expression of more than 1500 of 1900 genes induced by DHT. Further, D2 was able to reduce DHT mediated proliferation of PCa cells in vitro in dose-dependent manner, whereas the control D1 did not show any activity. This ability of D2 to inhibit DHT-mediated proliferation was noted in all androgen-responsive PCa cells LAPC4, LNCaP, RWPE1, C4-2 and CWR22v1, but not PC-3 cells which lack AR expression. Interestingly, we noted that the peptidomimetic D2 blocked translocation of AR to the nucleus compared to the control D1. Finally, D2 blocked the proliferation of C4-2 sc xenografts in nude mice. CONCLUSIONS: We have shown this peptidomimetic is capable of blocking AR-PELP1 interaction, nuclear translocation of AR, AR mediated genomic signaling as well as DHT-mediated proliferation of prostate cancer cells in vitro. Selective targeting of PELP1-AR interaction affects AR genomic signaling. These data strongly indicate that this strategy may serve as a viable therapeutic target for disrupting AR function in patients with prostate cancer. 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 65. doi:1538-7445.AM2012-65

Abstract 2844: Inhibiting androgen receptor and JunD protein interaction to prevent prostate cancer progression

Abstract Development of an effective therapy to prevent prostate cancer (PCa) progression to castrate-resistant PCa (CRPCa) remains an unmet medical need, mainly due to a poor understanding of the mechanism of PCa progression. Reactive oxygen species (ROS) are produced in high amounts in PCa cells. ROS play a major role in the development and progression of PCa. We have published that the activated androgen receptor (AR)-JunD complex induces spermidine/spermine N1-acetyl transferase (SSAT). SSAT is the first, and a regulatory, enzyme in a major polyamine catabolism pathway that leads to an over-production of ROS, specifically in the polyamine-rich PCa cells. A focus of our current research is to identify compounds that specifically target and block steps in this newly discovered ROS production pathway downstream to AR activation. Such compounds have the potential to become new targeted therapeutic agents offering minimal side effects for preventing progression to CRPCa in patients with early stage progressing PCa. Here we present data on drug-like small molecule inhibitors of the AR-JunD interaction that initiates this ROS-generating pathway in PCa. A novel high throughput assay based on Gaussia Luciferase enzyme reconstitution via protein-protein interaction was used to screen NCI Diversity Set and Life Chemicals libraries of drug-like small molecules to identify inhibitors of the AR-JunD interaction. The 27 selected hits were categorized as antiandrogens or non-antiandrogens through the use of a published fluorescence polarization assay. Sixteen non-antiandrogens, acting downstream of AR activation, were chosen for further study. In vitro studies entailed the measurement of the ability of the compounds to inhibit androgen-independent and dependent growth as well as androgen-induced ROS in human PCa cells. The top four compounds exhibited significant inhibition of growth and androgen-induced ROS at concentrations of 5 uM or less. Immunocytochemistry (ICC) studies were performed to determine the ability of the selected compounds to specifically inhibit the AR-JunD co-translocation to the nuclei. Based on the cell culture and ICC data, lead compounds were selected for pharmacokinetics studies to determine oral bioavailability. Detailed pharmacokinetic data of selected top compounds showing their bioavailability after intra venous and oral administration will be presented. 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 2844. doi:1538-7445.AM2012-2844

2225 INFLUENCE OF METABOLIC SYNDROME ON PROSTATE VOLUME AND PROSTATE SPECIFIC ANTIGEN

N 162, 43.8% ERG , p 0.001). In the younger age group ERG patients had a significant lower median serum PSA of 4.0 ng/ml compared to 6.0 ng/ml in ERG patients (p 0.011). The older patient cohort showed no significant differences in PSA values (ERG 5.6 ng/ml, ERG 6.3 ng/ml, p 0.079). No associations of ERG status with other clinical and histopathological parameters were found. CONCLUSIONS: In our PSA screening PCa cohort ERG overexpression is significantly more frequent in tumors detected at a lower age than in tumors diagnosed in older patients. This indicates an acceleration of tumor development by ERG overexpression. Furthermore, ERG overexpression seems to lower PSA sequestration into the circulation. This could reflect a lower production of androgen regulated genes like the PSA gene caused by interactions of ERG and androgen receptor.

Androgen receptor and HIF-1α interaction in prostate cancer.

197 Background: Prostate cancer is the most common noncutaneous cancer among men in the United States, and its progression is largely controlled by the androgen receptor (AR). Androgen deprivation therapy (ADT) is an initially effective treatment for prostate cancer, but most tumors eventually become castrate resistant. Tumor hypoxia also appears to be associated with a poor prognosis in prostate cancer. HIF-1a regulates the transcription of genes that allow tumor survival and growth in low oxygen conditions. Our laboratory has data showing that in response to castration and anti-androgen therapy in mice, there was a strong transcriptional relationship between HIF-1a and AR, as measured by quantitative RT-PCR, suggesting an interaction between the two proteins. Thus, the purpose of this study was to determine if there is a molecular interaction between HIF-1a and AR in prostate cancer cells. Methods: We used Western blot analysis to examine the expression levels of HIF-1a and AR in LNCaP prostate cancer cells to determine if they are upregulated together at the protein level. Four experimental conditions were tested: control (no treatment), DHT for induction of AR expression, CoCl2 for induction of HIF-1a, and a combination treatment of DHT and CoCl2. Immunoprecipitation experiments were carried out to determine if there is an association between HIF-1a and AR. In addition, cells were fractionated into nuclear and cellular cells extracts, followed by Western blot analysis to determine where in the cells this interaction occurs. Results: Western blot analysis of cell lysates showed synergistic upregulation of both HIF-1a and AR expression only under combined CoCl2 and DHT treatment conditions. In addition, immunoprecipitation experiments showed that HIF-1a and AR exist in a complex with one another, and fractionation experiments indicated this complex occurs in the nucleus. Conclusions: Our results demonstrate that HIF-1a and AR associate with one another in cells. Binding assays are in progress to determine the nature of this interaction. In addition, we are examining the cellular consequences of this protein interaction, as it is possible that upregulation of HIF-1a in response to low androgen contributes to the development of tumor resistance to ADT.

“True” Antiandrogens—Selective Non-Ligand-Binding Pocket Disruptors of Androgen Receptor–Coactivator Interactions: Novel Tools for Prostate Cancer

Prostate cancer (PCa) therapy typically involves administration of “classical” antiandrogens, competitive inhibitors of androgen receptor (AR) ligands, dihydrotestosterone (DHT) and testosterone (tes), for the ligand-binding pocket (LBP) in the ligand-binding domain (LBD) of AR. Prolonged LBP-targeting leads to resistance, and alternative therapies are urgently required. We report the identification and characterization of a novel series of diarylhydrazides as selective disruptors of AR interaction with coactivators through application of structure and ligand-based virtual screening. Compounds demonstrate full (“true”) antagonism in AR with low micromolar potency, selectivity over estrogen receptors α and β and glucocorticoid receptor, and partial antagonism of the progesterone receptor. MDG506 (5) demonstrates low cellular toxicity in PCa models and dose responsive reduction of classical antiandrogen-induced prostate specific antigen expression. These data provide compelling evidence for such non-LBP intervention as an alternative approach or in combination with classical PCa therapy.

Chromatin accessibility reveals insights into androgen receptor activation and transcriptional specificity

BackgroundEpigenetic mechanisms such as chromatin accessibility impact transcription factor binding to DNA and transcriptional specificity. The androgen receptor (AR), a master regulator of the male phenotype and prostate cancer pathogenesis, acts primarily through ligand-activated transcription of target genes. Although several determinants of AR transcriptional specificity have been elucidated, our understanding of the interplay between chromatin accessibility and AR function remains incomplete.ResultsWe used deep sequencing to assess chromatin structure via DNase I hypersensitivity and mRNA abundance, and paired these datasets with three independent AR ChIP-seq datasets. Our analysis revealed qualitative and quantitative differences in chromatin accessibility that corresponded to both AR binding and an enrichment of motifs for potential collaborating factors, one of which was identified as SP1. These quantitative differences were significantly associated with AR-regulated mRNA transcription across the genome. Base-pair resolution of the DNase I cleavage profile revealed three distinct footprinting patterns associated with the AR-DNA interaction, suggesting multiple modes of AR interaction with the genome.ConclusionsIn contrast with other DNA-binding factors, AR binding to the genome does not only target regions that are accessible to DNase I cleavage prior to hormone induction. AR binding is invariably associated with an increase in chromatin accessibility and, consequently, changes in gene expression. Furthermore, we present the first in vivo evidence that a significant fraction of AR binds only to half of the full AR DNA motif. These findings indicate a dynamic quantitative relationship between chromatin structure and AR-DNA binding that impacts AR transcriptional specificity.

Molecular Pathophysiology and Disease-Modifying Therapies for Spinal and Bulbar Muscular Atrophy

Spinal and bulbar muscular atrophy (SBMA), or Kennedy disease, is an adult-onset lower motor neuron disease characterized by slowly progressive muscle weakness and atrophy. The disease is caused by the expansion of a trinucleotide CAG repeat encoding a polyglutamine tract within the first exon of the androgen receptor (AR) gene. During the 2 decades since the discovery of the AR gene mutation in SBMA, basic and clinical research have deepened our understanding of the disease phenotype and pathophysiology. Spinal and bulbar muscular atrophy exclusively affects men, whereas women homozygous for the AR mutation do not fully develop the disease. The ligand-dependent nuclear accumulation of pathogenic AR protein is central to the pathogenesis, although additional steps, eg, DNA binding and interdomain interactions of AR, are required for toxicity. Downstream molecular events, eg, transcriptional dysregulation, axonal transport disruption, and mitochondrial dysfunction, are implicated in the neurodegeneration in SBMA. Pathogenic AR-induced myopathy also contributes to the degeneration of motor neurons. Several potential therapies, including hormonal manipulation, have emerged from animal studies, some of which have been tested in clinical trials.

Anti-androgen effects of cypermethrin on the amino- and carboxyl-terminal interaction of the androgen receptor

The pyrethroid insecticide, cypermethrin has been demonstrated to be an environmental anti-androgen in the androgen receptor (AR) reporter gene assay. The amino- and carboxyl-terminal (N/C) interaction is required for transcription potential of the AR. In order to characterize the anti-androgen effects of cypermethrin involved in the N/C interaction of AR, the mammalian two-hybrid assay has been developed in the study. The fusion vectors pVP16-ARNTD, pM-ARLBD and the pG5CAT Reporter Vector were cotransfected into the CV-1 cells. The assay displayed appropriate response to the potent, classical AR agonist 5α-dihydrotestosterone (DHT) and known AR antagonist nilutamide. The N/C interaction was induced by DHT from 10−11M to 10−5M in a dose-dependent manner. Nilutamide did not activate N/C interaction, while inhibited DHT-induced AR N/C interaction at the concentrations from 10−7M to 10−5M. Treatment of CV-1 cells with cypermethrin alone did not activate the reporter CAT. Cypermethrin significantly decreased the DHT-induced reporter CAT expression at the higher concentration of 10−5M. The mammalian two-hybrid assay provides a promising tool both for defining mechanism involved in AR N/C interaction of EDCs and for screening of chemicals with androgen agonistic and antagonistic activities. Cypermethrin exhibits inhibitory effects on the DHT-induced AR N/C interaction, while the potency is weaker than that of nilutamide.

Androgen receptor repression of GnRH gene transcription.

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

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.

Corepressor effect on androgen receptor activity varies with the length of the CAG encoded polyglutamine repeat and is dependent on receptor/corepressor ratio in prostate cancer cells

The response of prostate cells to androgens reflects a combination of androgen receptor (AR) transactivation and transrepression, but how these two processes differ mechanistically and influence prostate cancer risk and disease outcome remain elusive. Given recent interest in targeting AR transrepressive processes, a better understanding of AR/corepressor interaction and responses is warranted. Here, we used transactivation and interaction assays with wild-type and mutant ARs, and deletion AR fragments, to dissect the relationship between AR and the corepressor, silencing mediator for retinoic acid and thyroid hormone receptors (SMRT). We additionally tested how these processes are influenced by AR agonist and antagonist ligands, as well as by variation in the polyglutamine tract in the AR amino terminal domain (NTD), which is encoded by a polymorphic CAG repeat in the gene. SMRT was recruited to the AR ligand binding domain by agonist ligand, and as determined by the effect of strategic mutations in activation function 2 (AF-2), requires a precise conformation of that domain. A distinct region of SMRT also mediated interaction with the AR-NTD via the transactivation unit 5 (TAU5; residues 315–538) region. The degree to which SMRT was able to repress AR increased from 17% to 56% as the AR polyglutamine repeat length was increased from 9 to 42 residues, but critically this effect could be abolished by increasing the SMRT:AR molar ratio. These data suggest that the extent to which the CAG encoded polyglutamine repeat influences AR activity represents a balance between corepressor and coactivator occupancy of the same ligand-dependent and independent AR interaction surfaces. Changes in the homeostatic relationship of AR to these molecules, including SMRT, may explain the variable penetrance of the CAG repeat and the loss of AR signaling flexibility in prostate cancer progression.

IL-6 causes multiple effects in androgen-sensitive and -insensitive prostate cancer cell lines

The androgen receptor (AR) is expressed in most human prostate cancers. It can be activated in a hypersensitive manner by androgens, nonandrogenic steroids, nonsteroidal anti-androgens and in a ligand-independent manner. IL-6 is a proinflammatory cytokine that activates several signaling pathways. It can either stimulate or inhibit growth of prostate cancer cells. IL-6 is an important positive regulator of AR activity and can stimulate the expression of prostate-specific antigen in a ligand-independent manner. IL-6/AR interaction may either result in growth stimulation (MDA PCa 2b cells) or inhibition (LAPC-4 cells). IL-8 and IL-4 have also been observed to activate AR. Cells generated by prolonged treatment with IL-6 acquire a growth advantage. There are several therapeutic options to target IL-6 in prostate cancer and most laboratory studies have been performed with the monoclonal antibody siltuximab. Endogenous suppressors of cytokine signaling (SOCS)-3 and -1 are expressed in prostate cancer tissue. SOCS-3 inhibits apoptosis in AR-negative cells. However, in androgen-sensitive prostate cancer cell lines, SOCS-3 is induced by androgen and blocks its effects on proliferation and secretion. It is currently understood that there are numerous interactions between androgen and IL-6 signaling in human prostate cancer.

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 ...

Hormone Depletion-Insensitivity of Prostate Cancer Cells Is Supported by the AR Without Binding to Classical Response Elements

A need for androgen response elements (AREs) for androgen receptor (AR)-dependent growth of hormone depletion-insensitive prostate cancer is generally presumed. In such cells, androgen-independent activation by AR of certain genes has been attributed to selective increases in basal associations of AR with putative enhancers. We examined the importance of AR binding to DNA in prostate cancer cells in which proliferation in the absence of hormone was profoundly (∼ 90%) dependent on endogenous AR and where the receptor was not up-regulated or mutated but was predominantly nuclear. Here, ARE-mediated promoter activation and the binding of AR to a known ARE in the chromatin remained entirely androgen dependent, and the cells showed an androgen-responsive gene expression profile with an unaltered sensitivity to androgen dose. In the same cells, a different set of genes primarily enriched for cell division functions was activated by AR independently of hormone and significantly overlapped the signature gene overexpression profile of hormone ablation-insensitive clinical tumors. After knockdown of endogenous AR, hormone depletion-insensitive cell proliferation and AR apoprotein-dependent gene expression were rescued by an AR mutant that was unable to bind to ARE but that could transactivate through a well-established AR tethering protein. Hormone depletion-insensitive AR binding sites in the chromatin were functional, binding, and responding to both the wild-type and the mutant AR and lacked enrichment for canonical or noncanonical ARE half-sites. Therefore, a potentially diverse set of ARE-independent mechanisms of AR interactions with target genes must underlie truly hormone depletion-insensitive gene regulation and proliferation in prostate cancer.

G‐protein alpha‐s and ‐12 subunits are involved in androgen‐stimulated PI3K activation and androgen receptor transactivation in prostate cancer cells

The androgen receptor (AR) is a ligand-dependent transcription factor that mediates androgenic hormone action in cells. We recently demonstrated the involvement of phosphoinositide 3-OH kinase (PI3K) p110beta in AR transactivation and gene expression. In this study, we determined the upstream signals that lead to PI3K/p110beta activation and AR transactivation after androgen stimulation. Human prostate cancer LAPC-4 and 22Rv1 cell lines were used for the experiments. AR transactivation was assessed using an androgen responsive element-driven luciferase (ARE-LUC) assay. Cell proliferation was examined using BrdU incorporation and MTT assays. Target genes were silenced using small interfering RNA (siRNA) approach. Gene expression was evaluated at the mRNA level (real-time RT-PCR) and protein level (Western blot). PI3K kinase activities were measured using immunoprecipitation-based in vitro kinase assay. The AR-DNA-binding activity was determined using chromatin-immunoprecipitation (ChIP) assay. First, at the cellular plasma membrane, disrupting the integrity of caveolae microdomain with methyl-β-cyclodextrin (M-β-CD) abolished androgen-induced AR transactivation and gene expression. Then, knocking down caveolae structural proteins caveolin-1 or -2 with the gene-specific siRNAs significantly reduced androgen-induced AR transactivation. Next, silencing Gα(s) and Gα(12) genes but not other G-proteins blocked androgen-induced AR transactivation and cell proliferation. Consistently, overexpression of Gα(s) or Gα(12) active mutants enhanced androgen-induced AR transactivation, of which Gα(s) active mutant sensitized the AR to castration-level of androgen (R1881). Most interestingly, knocking down Gα(s) but not Gα(12) subunit significantly suppressed androgen-stimulated PI3K p110beta activation. However, ChIP analysis revealed that both Gα(s) or Gα(12) subunits are involved in androgen-induced AR interaction with the AR target gene PSA promoter region. These data suggest that caveolae-associated G-protein alpha subunits are involved in AR transactivation by modulating the activities of different PI3K isoforms.

Green tea polyphenol EGCG blunts androgen receptor function in prostate cancer

Androgen deprivation therapy is the major treatment for advanced prostate cancer (PCa). However, it is a temporary remission, and the patients almost inevitably develop hormone refractory prostate cancer (HRPC). HRPC is almost incurable, although most HRPC cells still express androgen receptor (AR) and depend on the AR for growth, making AR a prime drug target. Here, we provide evidence that epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, is a direct antagonist of androgen action. In silico modeling and FRET-based competition assay showed that EGCG physically interacts with the ligand-binding domain of AR by replacing a high-affinity labeled ligand (IC(50) 0.4 μM). The functional consequence of this interaction was a decrease in AR-mediated transcriptional activation, which was due to EGCG mediated inhibition of interdomain N-C termini interaction of AR. Treatment with EGCG also repressed the transcriptional activation by a hotspot mutant AR (T877A) expressed ectopically as well as the endogenous AR mutant. As the physiological consequence of AR antagonism, EGCG repressed R1881-induced PCa cell growth. In a xenograft model, EGCG was found to inhibit AR nuclear translocation and protein expression. We also observed a significant down-regulation of androgen-regulated miRNA-21 and up-regulation of a tumor suppressor, miRNA-330, in tumors of mice treated with EGCG. Taken together, we provide evidence that EGCG functionally antagonizes androgen action at multiple levels, resulting in inhibition of PCa growth.

HOXC8 Inhibits Androgen Receptor Signaling in Human Prostate Cancer Cells by Inhibiting SRC-3 Recruitment to Direct Androgen Target Genes

HOX (homeobox) genes encode homeodomain-containing transcription factors critical to development, differentiation, and homeostasis. Their dysregulation has been implicated in a variety of cancers. Previously, we showed that a subset of genes of the HOXC cluster is upregulated in primary prostate tumors, lymph node metastases, and malignant prostate cell lines. In the present study, we show that HOXC8 inhibits androgen receptor (AR)-mediated gene induction in LNCaP prostate cancer cells and HPr-1 AR, a nontumorigenic prostate epithelial cell line. Mechanistically, HOXC8 blocks the AR-dependent recruitment of the steroid receptor coactivators steroid receptor coactivator-3 (SRC-3), and CREB binding protein to the androgen-regulated prostate-specific antigen gene enhancer and inhibits histone acetylation of androgen-regulated genes. Inhibition of androgen induction by HOXC8 is reversed upon expression of SRC-3, a member of the SRC/p160 steroid receptor cofactor family. Coimmunoprecipitation studies show that HOXC8 expression inhibits the hormone-dependent interaction of AR and SRC-3. Finally, HOXC8 expression increases invasion in HPr-1 AR nontumorigenic cells. These data suggest a complex role for HOXC8 in prostate cancer, promoting invasiveness while inhibiting AR-mediated gene induction at androgen response element-regulated genes associated with differentiated function of the prostate. A greater understanding of HOXC8 actions in the prostate and its interactions with androgen signaling pathways may elucidate mechanisms driving the onset and progression of prostate cancer.

A Yeast Bioassay for Androgenic and Anti-Androgenic Compounds Based on the NH2- and COOH-Terminal Interaction of Androgen Receptor

Androgenic compounds induce an interaction between the NH(2)- and COOH-terminal regions (N-C interaction) of androgen receptor (AR). We describe a rapid yeast bioassay for androgenic and anti-androgenic compounds based on androgen-dependent β-catenin-enhanced N-C interaction. The bioassay was also effective at detecting compounds that inhibit the N-C interaction in ways that do not involve binding to the ligand-binding domain.

Regression of Castrate-Recurrent Prostate Cancer by a Small-Molecule Inhibitor of the Amino-Terminus Domain of the Androgen Receptor

Castration-recurrent prostate cancer (CRPC) is suspected to depend on androgen receptor (AR). The AF-1 region in the amino-terminal domain (NTD) of AR contains most, if not all, of the transcriptional activity. Here we identify EPI-001, a small molecule that blocked transactivation of the NTD and was specific for inhibition of AR without attenuating transcriptional activities of related steroid receptors. EPI-001 interacted with the AF-1 region, inhibited protein-protein interactions with AR, and reduced AR interaction with androgen-response elements on target genes. Importantly, EPI-001 blocked androgen-induced proliferation and caused cytoreduction of CRPC in xenografts dependent on AR for growth and survival without causing toxicity.

Abstract 4984: Androgen response element of the glycine N-methyltransferase gene is located in first intron

Abstract BACKGROUND. The prostrate cancer (PCa) susceptibility gene glycine N-methyltransferase (GNMT) is associated with the metastasis potential of PCa cells. Androgens, which participate in transcriptional regulation via an androgen receptor (AR), play an important role in PCa development. Our two goals were to determine whether GNMT is regulated by androgen, and to map androgen response elements (AREs). METHODS. Real-time PCR was used to evaluate GNMT mRNA expression levels after LNCaP and DU145 cells were treated with R1881 (a synthetic AR agonist). Putative AREs were identified and tested using the MatInspector program and a luciferase reporter assay respectively. Electrophoretic mobility shift assay and chromatin immunoprecipitation were used to verify the binding properties of the GNMT-ARE motif. RESULTS. Results from real-time PCR analyses indicate that R1881 is capable of inducing significant GNMT mRNA levels in AR-positive LNCaP cells, but not in AR-negative DU145 cells. According to in-silico screening prediction data, putative ARE1, ARE2 and ARE3 are located within GNMT intron 1, and ARE4 is located within intron 2. According to results from a luciferase reporter assay, only ARE3 mediated the transcriptional activation effects of R1881. Results from an electrophoretic mobility shift assay show that AR binds to ARE3 in vitro. Chromatin immunoprecipitation assay results indicate AR interaction with the ARE3-containing region in vivo. CONCLUSION. Our data show that GNMT is an AR target gene, with its functional ARE located at +1114/+1128 in GNMT intron 1. We believe this information is valuable for clarifying the relationship between androgen and GNMT gene expression. 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 4984.