Receptors In Prostate Cancer Research Articles

Long noncoding RNA-mediated activation of androgen receptor in prostate cancer

Remarkable progress has been made in molecular characterization of prostate cancer (PCa) with continued innovations in high throughput technologies evaluating human cancer.123 Since the completion of the Human Genome Project it has been estimated that only about 1.5%–2% of our genome codes for proteins. Various genome-wide approaches, e.g. the ENCODE project, revealed that a much larger percent of the genome is transcribed as non-protein coding (nc) RNA, including long noncoding (lnc) RNA (over 200 bps long). Although the biological roles of lncRNA (the ‘dark matter of the genome’) are not nearly as well-understood as the protein coding mRNAs, it is increasingly clear that they play important roles in almost every aspects of biology, including cancer biology.45 This is exemplified by recent genome-wide association studies revealing that over 80% of cancer-associated single nucleotide polymorphisms (SNPs) are in noncoding regions of the genome.

Acetato de abiraterona prequimioterapia. Propuesta de un algoritmo de tratamiento en el carcinoma de próstata resistente a castración

ContextProstate cancer treatment remains a challenge for the urologist. Medical control in locally advanced or metastatic prostate cancer is usually performed with LHRH analogues and/or antiandrogens. Different treatments have been proposed when there is biochemical and clinical progression of the disease and other new ones have changed the patients’ perspective and life expectancy. ObjectiveThis review has aimed to establish the current role of Abiraterone Acetate in the treatment of castration-resistant prostate cancer and facilitate decision-making by the Urologist by means of a Treatment Algorithm. Acquisition of the evidenceA search of current evidence on Abiraterone treatment in patients with castration- resistant metastatic prostate cancer was performed in PubMed, mainly analyzing those studies designed as clinical trials. In addition, we reviewed and updated the role of hormone therapy and androgen receptors in prostate cancer. Evidence synthesisThere are currently basically two clinical trials that demonstrate the effectiveness of Abiraterone in metastatic prostate cancer compared to placebo. The study COU-AA 302 shows a clear benefit with Abiraterone prior to chemotherapy in patients with castration-resistant prostate cancer, this making it possible to establish an algorithm for initial treatment that facilitates decision-making by the urologist. ConclusionAbiraterone is a pre-chemotherapy treatment option in selected patients with castration resistant metastatic prostate cancer, although it is necessary to improve the cost and to design more multicenter clinical trials to optimize the cost/benefit ratio.

TALEN-engineered AR gene rearrangements reveal endocrine uncoupling of androgen receptor in prostate cancer

Androgen receptor (AR) target genes direct development and survival of the prostate epithelial lineage, including prostate cancer (PCa). Thus, endocrine therapies that inhibit the AR ligand-binding domain (LBD) are effective in treating PCa. AR transcriptional reactivation is central to resistance, as evidenced by the efficacy of AR retargeting in castration-resistant PCa (CRPC) with next-generation endocrine therapies abiraterone and enzalutamide. However, resistance to abiraterone and enzalutamide limits this efficacy in most men, and PCa remains the second-leading cause of male cancer deaths. Here we show that AR gene rearrangements in CRPC tissues underlie a completely androgen-independent, yet AR-dependent, resistance mechanism. We discovered intragenic AR gene rearrangements in CRPC tissues, which we modeled using transcription activator-like effector nuclease (TALEN)-mediated genome engineering. This modeling revealed that these AR gene rearrangements blocked full-length AR synthesis, but promoted expression of truncated AR variant proteins lacking the AR ligand-binding domain. Furthermore, these AR variant proteins maintained the constitutive activity of the AR transcriptional program and a CRPC growth phenotype independent of full-length AR or androgens. These findings demonstrate that AR gene rearrangements are a unique resistance mechanism by which AR transcriptional activity can be uncoupled from endocrine regulation in CRPC.

Posttranslational Modification of the Androgen Receptor in Prostate Cancer

The androgen receptor (AR) is important in the development of the prostate by regulating transcription, cellular proliferation, and apoptosis. AR undergoes posttranslational modifications that alter its transcription activity, translocation to the nucleus and stability. The posttranslational modifications that regulate these events are of utmost importance to understand the functional role of AR and its activity. The majority of these modifications occur in the activation function-1 (AF1) region of the AR, which contains the transcriptional activation unit 1 (TAU1) and 5 (TAU5). Identification of the modifications that occur to these regions may increase our understanding of AR activation in prostate cancer and the role of AR in the progression from androgen-dependent to castration-resistant prostate cancer (CRPC). Most of the posttranslational modifications identified to date have been determined using the full-length AR in androgen dependent cells. Further investigations into the role of posttranslational modifications in androgen-independent activation of full-length AR and constitutively active splicing variants are warranted, findings from which may provide new therapeutic options for CRPC.

Nuclear localization of the androgen receptor in prostate cancer circulating tumor cells from men who have failed androgen deprivation therapy.

e22141 Background: Prostate cancer (PRCA) is driven by the Androgen Receptor (AR), a cytoplasmic protein that translocates to the nucleus to activate a transcriptional program critical to PRCA tumorigenesis. The AR is the major therapeutic target in PRCA, with multiple drugs that improve survival and quality of life for men with advanced PRCA. However, few biomarkers can assay for AR activity to identify those most likely to benefit from AR-targeting therapies. Methods: Utilizing a novel microfluidic technology known as the VERSA (Vertical Exclusion-based Rare Sample Analysis) platform, CTCs were captured and purified from men with advanced PRCA who had failed androgen deprivation therapy. CTCs were then stained and imaged via epiflourescent microscopy for EpCAM, AR, Hoechst and CD45. The enhanced optical imaging of the VERSA platform enabled subcellular localization of the AR and calculation of the relative amount of AR localized to the nucleus as a biomarker of resistance to AR-targeting therapies. Results: CTCs were prospectively collected and analyzed for AR nuclear localization. Significant heterogeneity in AR localization was found between patients who had failed androgen deprivation therapy with the mean nuclear AR ranging from 20-57% of total AR. Significant intra-patient heterogeneity was also identified with the percentage of AR localized to the nucleus ranging from 11-77% of total AR. DNA and mRNA have been isolated from these cells to analyze for underlying mechanisms of nuclear localization. Conclusions: The VERSA platform is able to analyze subcellular localization of proteins in CTCs followed by total nucleic acid extraction. We show that among men whose disease has progressed through androgen deprivation, a significant portion of the AR is localized to the nucleus as a possible biomarker of resistance to AR targeting therapies. We also identify significant heterogeneity in AR localization among CTCs, suggesting resistant PRCA clones that have greater proliferative potential.

Role of the androgen receptor in prostate cancer

AbstractInhibition of the androgen receptor leads to regression of normal and malignant prostate tissue. The authors discuss therapeutic strategies for directly targeting the androgen receptor to help address the problem of castrate‐resistant prostate cancer. Copyright © 2013 John Wiley & Sons

Abstract 753: Reciprocal regulation of HOXB13 and androgen receptor in prostate cancer.

Abstract The androgen receptor (AR), a member of the nuclear receptor superfamily of ligand-regulated transcription factors, plays a pivotal role in the initiation and progression of prostate cancer. HOXB13, a prostate-specific homeodomain-containing transcription factor, interacts with AR to regulate the cellular response to androgens (Norris et al. Mol Cell 2009). Our group recently identified a rare but recurrent germline mutation (G84E) in HOXB13 as a prostate cancer susceptibility allele by sequencing the 17q21-22 region of the genomic DNA from families with hereditary prostate cancer. (Ewing et al. NEJM 2012). Our research goal is to study how HOXB13 regulates AR function and how this mechanism might be affected by mutant HOXB13. The overexpression of wild type HOXB13 in LNCaP cells led to an increased expression of AR at the mRNA and protein levels; this induction of AR was attenuated with HOXB13 G84E over-expression. Upon the reduction of endogenous HOXB13 by siRNA in LNCaP cells, the amount of AR decreased at the mRNA and protein levels, indicating that HOXB13 promotes the expression of AR. Conversely, when AR was knocked down with siRNA, the level of HOXB13 transcripts and proteins increased, indicating that AR suppresses HOXB13 expression. Furthermore, co-immunoprecipitation experiments demonstrate that FOXA1, the pioneer factor implicated to control prostate-specific transcription of HOXB13 (McMullin et al. 2009), interacts with AR and HOXB13. These analyses implicate at least a three factor mechanism whereby the levels of critical components of prostate-specific androgen signaling network might recruit and/or titrate one another, and suggest this network may be perturbed by mutations in HOXB13. Since LNCaP cells contain a mutated copy of HOXB13 (L144P), these analyses need to be performed in additional prostate cancer cells before general conclusions can be reached. Citation Format: Shuangling Chen, Dorhyun Johng, William B. Isaacs. Reciprocal regulation of HOXB13 and androgen receptor in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 753. doi:10.1158/1538-7445.AM2013-753

Abstract 4245: Silencing of the PMEPA1 gene, a key regulator of androgen receptor in prostate cancer.

Abstract Introduction and Objectives: PMEPA1 is an androgen inducible gene, abundantly expressed in prostate epithelial cells and controls androgen receptor (AR) protein levels by recruiting NEDD4-1 E3 ubiquitin ligase to degrade AR. PMEPA1 gene expression is either reduced or lost in two-thirds of prostate cancer (CaP). Methylation of the PMEPA1 promoter may contribute to PMEPA1 gene silencing. Previously we have shown that AR binds to an Androgen Responsive Element (ARE) in the proximal promoter of the PMEPA1 gene in a hormone-dependent manner. These results prompted us to investigate the methylation status of proximal ARE and intronic SP1 enhancer sites in cell lines and in laser capture micro-dissected (LCM) human prostate tumors. Methods: Methylation status of the PMEPA1 promoter was evaluated in AR positive LNCaP, VCaP and LAPC4 cell lines and in LCM-enriched tumor cells using a previously validated method combining precipitation of methylated-DNA and methylation-sensitive restriction enzyme digestion (COMPARE-MS). Expression of PMEPA1 at protein and transcript levels was assessed by Western blot and Q-PCR, respectively in response to DNA methyl transferase inhibitor 5-aza-2’-deoxycitidine. Results: In a panel of LCM derived genomic DNA (N=50), PMEPA1 promoter sequences were found to be hypermethylated in 44% of human prostate tumors (N=22). PMEPA1 methylation was consistent with loss of PMEPA1 gene expression. PMEPA1 methylation was also detected in androgen-sensitive VCaP, LNCaP and LAPC4 CaP cell lines. Western blot and Q-PCR analysis revealed that demethylation restores PMEPA1 expression in CaP cells. Evaluation of proximal promoter sequences of PMEPA1 suggested that DNA methylation may directly inhibit AR binding to the proximal ARE and to the downstream intronic SP1 enhancer site of PMEPA1 gene. PMEPA1 methylation status showed a trend towards association with race, with a very low (12%) incidence of PMEPA1 methylation in African Americans subjects. Conclusions: PMEPA1 promoter regulatory sequences are hypermethylated in prostate cancer cell lines and in primary tumors suggesting that DNA methylation may contribute to the silencing of PMEPA1 gene in a significant subset of prostate tumors. These data, along with our earlier observation showing elevation of AR levels in response to the inhibition of PMEPA1 expression suggest a role of DNA methylation in the silencing of PMEPA1 gene and AR regulation in human CaP. Overall, reduced or eliminated PMEPA1 expression is likely to interfere with AR signaling and/ or other pathways in CaP. Source of Funding: This study was supported by the NIH grant RO CA106653 to S.S. Citation Format: Shashwat E. Sharad, Hua Li, Micheal Haffner, Srinivasan Yegnasubramanian, Lakshmi Ravindranth, Yongmei Chen, Alagarsamy Srinivasan, Gyorgy Petrovics, Shiv Srivastava, Albert Dobi. Silencing of the PMEPA1 gene, a key regulator of androgen receptor in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4245. doi:10.1158/1538-7445.AM2013-4245

Ericifolin: a novel antitumor compound from allspice that silences androgen receptor in prostate cancer

Silencing of androgen receptor (AR) signaling is a specific and effective mechanism to cure cancer of the prostate (CaP). In this study, the isolation and characterization of a compound from the aromatic berries of Pimenta dioica (allspice) that silences AR is presented. Potential antitumor activities of an aqueous allspice extract (AAE) and a compound purified from the extract were tested on CaP cells. AAE inhibited tumor cell proliferation and colony formation (50% growth inhibition ∼40-85 µg/ml) but not the viability of quiescent normal fibroblasts or non-tumorigenic prostate cells. In tumor cells, AAE inhibited cell cycle progression at G1/S, induced apoptosis or autophagy. Apoptosis was by caspase-dependent poly (ADP ribose) polymerase cleavage. A caspase-independent, apoptosis-inducing factor-mediated mechanism of apoptosis caused cell death in castration-resistant AR-positive or AR-negative CaP cells, such as CWR22RV1, PC-3 or DU145 cells. Treatment with AAE decreased the levels of AR messenger RNA (mRNA), protein and silenced AR activity in AR-positive cells. AR depletion was due to inhibition of AR promoter activity and mRNA stability. Delayed tumor growth (~55%) without measurable systemic toxicity was observed in LNCaP tumor-bearing mice treated with AAE by oral or intraperitoneal routes. LNCaP tumor tissues from AAE-treated mice revealed increased apoptosis as a potential mechanism of antitumor activity of AAE. The chemical identity of bioactive compound in AAE was established through multistep high-performance liquid chromatography fractionation, mass and Nuclear Magnetic Resonance spectroscopies. The compound, eugenol 5-O-β-(6'-galloylglucopyranoside) or ericifolin (EF), showed antiproliferative, pro-apoptosis and anti-AR transcription activities. These results demonstrate a potential use of AAE and EF against prostate cancer.

Sequencing the transcriptional network of androgen receptor in prostate cancer

The progression of prostate cancer is largely dependent on the activity of the androgen receptor (AR), which in turn, correlates with the net output of the AR transcriptional regulatory network. A detailed and thorough understanding of the AR transcriptional regulatory network is therefore critical in the strategic manipulation of AR activity for the targeted eradication of prostate cancer cells. In this mini-review, we highlight some of the novel and unexpected mechanistic and functional insights of the AR transcriptional network derived from recent targeted sequencing (ChIP–Seq) studies of AR and its coregulatory factors in prostate cancer cells.

Tumor-associated Stromal Cells Expressing E-prostanoid 2 or 3 Receptors in Prostate Cancer: Correlation With Tumor Aggressiveness and Outcome by Angiogenesis and Lymphangiogenesis

To clarify the detailed pathologic roles of prostaglandin E(2) in prostate cancer tissues, the present study investigated the clinical significance and prognostic roles of the density of tumor-associated stromal cells expressing specific receptors for prostaglandin E2, termed "E-prostanoid (EP)1-4 receptors (EP1R-4Rs)." The expression of each receptor was immunohistochemically examined in 114 formalin-fixed biopsy specimens. Correlations with clinicopathologic features were investigated in these specimens. Angiogenesis and lymphangiogenesis were measured by the percentage of CD34-stained vessels (microvessel density) and D2-40-stained vessels (lymph vessel density). The relationships between the density of each EPR-stained cells and the microvessel density or lymph vessel density were evaluated in 62 prostate cancer tissues obtained by radical surgery for more detailed analysis in a wider area of prostate cancer tissue. The density of tumor-associated cells with EP2R expression was positively associated with the N (P<.001) and M (P=.002) stages. Similarly, EP3R-positive stromal cell density was significantly associated with the N (P=.033) and M (P=.026) stages. The density of EP2R- and EP3R-stained cells correlated with the microvessel density (r=0.42, P<.001) and lymph vessel density (r=0.36, P=.012), respectively. A greater density of EP2R-stained cells was recognized as an independent predictor of progression (hazard ratio 7.26, P=.002) on multivariate analysis. EP2R- and EP3R-stained cells might play important roles in tumor progression, angiogenesis, and lymphangiogenesis in prostate cancer. The density of EP2R-stained stromal cells could offer a useful predictor of biochemical recurrence after radical surgery.

Tendencies for higher co-expression of EGFR and HER2 and downregulation of HER3 in prostate cancer lymph node metastases compared with corresponding primary tumors

The epidermal growth factor receptor (EGFR) family members are potential targets for therapy using extra-cellular domain receptor binding agents, such as the antibodies trastuzumab and cetuximab, or antibodies labeled with therapeutically useful radionuclides or toxins. This is especially the case when the tumor cells are resistant to chemotherapy and tyrosine kinase inhibitors. Studies concerning the expression of these receptors in prostate cancer vary in the literature, possibly due to differences in patient inclusion, sample preparations and scoring criteria. In our study, EGFR, HER2 and HER3 expression was analyzed in prostate cancer samples from primary tumors and corresponding lymph node metastases from 12 patients. The expression of HER2 and EGFR was scored from immunohistochemical preparations and the HercepTest criteria (0, 1+, 2+ or 3+), while HER3 expression was scored as no, weak or strong staining. There were 5 EGFR-positive (2+ or 3+) primary tumors and 6 EGFR-positive lymph node metastases, and there was EGFR upregulation in one metastasis. Only 4 of the 12 patients had marked HER2 expression (2+ or 3+) in their primary tumors and there was one downregulation and 5 cases of upregulation in the metastases. Thus, a total of 8 out of 12 analyzed metastases were HER2-positive. Of the 12 primary tumors, 9 expressed HER3 while only 2 of the lymph node metastases expressed recognizable HER3 staining, so 7 metastases appeared to have downregulated HER3 expression. In one of the primary tumors there was positive co-expression of EGFR and HER2, while this co-expression was observed in 4 of the metastases. Thus, there were tendencies for upregulation of HER2, increased co-expression of EGFR and HER2 and downregulation of HER3 in the prostate cancer lymph node metastases in comparison to the primary tumors. The results are encouraging for studies involving more patients. Possible strategies for EGFR- and HER2-targeted therapy are briefly discussed in the present study, especially with regard to the expression and co-expression of EGFR and HER2 in metastases.

Differential expression of VEGF ligands and receptors in prostate cancer

Prostate cancer disseminates to regional lymph nodes, however the molecular mechanisms responsible for lymph node metastasis are poorly understood. The vascular endothelial growth factor (VEGF) ligand and receptor family have been implicated in the growth and spread of prostate cancer via activation of the blood vasculature and lymphatic systems. The purpose of this study was to comprehensively examine the expression pattern of VEGF ligands and receptors in the glandular epithelium, stroma, lymphatic vasculature and blood vessels in prostate cancer. The localization of VEGF-A, VEGF-C, VEGF-D, VEGF receptor (VEGFR)-1, VEGFR-2, and VEGFR-3 was examined in cancerous and adjacent benign prostate tissue from 52 subjects representing various grades of prostate cancer. Except for VEGFR-2, extensive staining was observed for all ligands and receptors in the prostate specimens. In epithelial cells, VEGF-A and VEGFR-1 expression was higher in tumor tissue compared to benign tissue. VEGF-D and VEGFR-3 expression was significantly higher in benign tissue compared to tumor in the stroma and the endothelium of lymphatic and blood vessels. In addition, the frequency of lymphatic vessels, but not blood vessels, was lower in tumor tissue compared with benign tissue. These results suggest that activation of VEGFR-1 by VEGF-A within the carcinoma, and activation of lymphatic endothelial cell VEGFR-3 by VEGF-D within the adjacent benign stroma may be important signaling mechanisms involved in the progression and subsequent metastatic spread of prostate cancer. Thus inhibition of these pathways may contribute to therapeutic strategies for the management of prostate cancer.

A comparison of the effects of Angiotensin IV on androgen-dependent and androgen-independent prostate cancer cell lines

Angiotensin IV is one of the biologically active peptides of the renin-angiotensin system. Limited data suggests that this hexapeptide could contribute to cancer development and/or progression. Using the MTT reduction assay as an indicator of cell viability, and the bromodeoxyuridine incorporation assay as an indicator of cell proliferation, the influence of Angiotensin IV was evaluated on two human prostate cancer lines: androgen-dependent (LNCaP) and androgen-independent (DU-145). The potential effect of Angiotensin IV classic angiotensin receptors was examined by using the selective antagonists losartan and PD123319. Finally, the changes in expression levels of AT1 and AT2 receptors were compared, before and after angiotensin treatment. Angiotensin IV caused significant changes in cell viability and proliferation in LNCaP cells but not in DU-145. It was found that AT2 receptor blocker (PD123319) was able to diminish the suppressor effect of Angiotensin IV on bromodeoxyuridine incorporation into the DNA of androgen-dependent prostate cancer cells. Simultaneously, it was reported that Angiotensin IV is the factor that modulates the density of AT1 and AT2 receptors in prostate cancer cells. These findings suggested that Angiotensin IV can modulate tumour cell proliferation in the early stage of androgen-dependent prostate cancer. The effect might be promoted by the change of the angiotensin receptor level.

Abstract 1940: Anti-tumor action of adenosine A3 receptor in prostate cancer involves suppression of micro RNA-21

Abstract Introduction: Adenosine-based drugs have shown promising results against various solid tumors including prostate cancer. These drugs exert their effects via adenosine receptors (ARs), and in prostate cancer, the A3AR subtype appears to play a major role in its anti-tumor action. MicroRNAs (miRs) are small non-coding RNAs that regulate the coding RNAs at post-transcriptional level. One of these, miR-21, has been shown to promote prostate cancer growth and invasiveness. In the present study, we hypothesized that A3AR agonists suppressed prostate tumor growth by reducing miR-21 expression. Methods: In vivo studies were performed in xenograft model of prostate cancer in severe combined immunodeficient (SCID) mice. Mice were injected subcutaneously with PC3-MM human prostate cancer cells, treated with vehicle, the agonist N’-(3-iodobenzyl) adenosine-5′-N-methyluronamide (IB-MECA), the antagonist 9-Chloro-2-(2-furanyl)-5-((phenylacetyl)amino)-[1,2,4]triazolo[1,5-c]quinazoline (MRS1220) or MRS1220 + IB-MECA, on alternate day for 2-3 weeks, and tumor growth was measured. Primary tumors were then excised and used to determine miR-21 (by TaqMan assay) and maspin, PDCD4 and TPM1 (by Western blotting and RT-PCR). In vitro studies were done in PC3-MM cells grown in cell culture plates. The levels of miR-21 were also detected in RNA samples obtained from human prostate cancer tissues (OriGene Technologies), while the levels of maspin and PDCD4 were determined in the formalin-fixed paraffin-embedded (FFPE) prostate tumor sections by immunohistochemistry. Results: IB-MECA suppressed tumor growth in SCID mice. This effect was blunted in presence of the antagonist, MRS1220. Reductions in tumor volume were associated with reduced expression of miR-21 in the primary tumors. Furthermore, we observed up-regulation of the tumor suppressor proteins maspin, PDCD4 and TPM1, which are known to be negatively regulated by miR-21. Similar findings were obtained in in vitro cell culture studies. In human prostate cancer RNA samples from patients with different stages of prostate cancer there was high expression of miR-21. This finding co-related with down-regulation of its target proteins, maspin and PDCD4, in human prostate tumor sections. Conclusions: Based on the data, we conclude that activation of the A3AR reduces the prostate cancer growth by suppressing miR-21 expression and up-regulating tumor suppressor proteins such as maspin, PDCD4 and TPM1. These data support the use of A3AR agonists as a viable therapeutic option against the advanced forms of 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 1940. doi:1538-7445.AM2012-1940

Abstract LB-273: Transcriptional regulation by androgen receptor in prostate cancer

Abstract Transcriptional regulation by the androgen receptor (AR) is critical to prostate cancer development and progression. Through integrative analysis of genome-wide AR occupancy and androgen-regulated gene expression, we have recently reported AR as a globally acting transcriptional repressor. This repression is mediated by androgen responsive elements (ARE) and dictated by Polycomb group protein EZH2 and repressive chromatin remodeling. In addition, AR-repressed genes are enriched for developmental regulators controlling cell differentiation. They tend to get down-regulated in advanced prostate cancer probably accountable for stem cell-like de-differentiation and thus tumor progression. However, critical AR-repressed genes of functional relevance are yet to be identified and their roles to be carefully characterized. Through meta-analysis of several expression microarray data, we nominate one top candidate AR-repressed gene. Using this gene as a model, we present further evidence that AR directly inhibits gene expression through physically binding to the enhancer, establishing promoter-enhancer DNA looping, and inducing repressive chromatin remodeling. Further, overexpression of this gene inhibits prostate cancer cell growth, migration, and invasion in vitro and xenograft tumor growth in mice, thus exhibiting a tumor suppressive role. In addition, the loss of this gene drives castration-resistant prostate cancer. Therefore, our findings support that AR-mediated inhibition of tumor suppressors plays important roles during prostate cancer progression. Our study provides an innovative mechanism for AR in castration resistance, which may eventually lead to novel therapeutic targets for treatment of advanced 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 LB-273. doi:1538-7445.AM2012-LB-273

Abstract 2206: Molecular interaction of HIF-1α and the androgen receptor in prostate cancer

Abstract 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-1α 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-1α 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-1α and AR in prostate cancer cells. Methods: We used Western blot analysis to examine the expression levels of HIF-1α 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-1α, and a combination treatment of DHT and CoCl2. Immunoprecipitation experiments were carried out to determine if there is an association between HIF-1α 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-1α and AR expression only under combined CoCl2 and DHT treatment conditions. In addition, immunoprecipitation experiments showed that HIF-1α 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-1α 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-1α in response to low androgen contributes to the development of tumor resistance to ADT. 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 2206. doi:1538-7445.AM2012-2206

Therapeutic targeting of angiotensin II receptor type 1 to regulate androgen receptor in prostate cancer

With the limited strategies for curative treatment of castration-resistant prostate cancer (CRPC), public interest has focused on the potential prevention of prostate cancer. Recent studies have demonstrated that an angiotensin II receptor blocker (ARB) has the potential to decrease serum prostate-specific antigen (PSA) level and improve performance status in CRPC patients. These facts prompted us to investigate the direct effects of ARBs on prostate cancer growth and progression. Transgenic rat for adenocarcinoma of prostate (TRAP) model established in our laboratory was used. TRAP rats of 3 weeks of age received ARB (telmisartan or candesartan) at the concentration of 2 or 10 mg/kg/day in drinking water for 12 weeks. In vitro analyses for cell growth, ubiquitylation or reporter gene assay were performed using LNCaP cells. We found that both telmisartan and candesartan attenuated prostate carcinogenesis in TRAP rats by augmentation of apoptosis resulting from activation of caspases, inactivation of p38 MAPK and down-regulation of the androgen receptor (AR). Further, microarray analysis demonstrated up-regulation of estrogen receptor β (ERβ) by ARB treatment. In both parental and androgen-independent LNCaP cells, ARB inhibited both cell growth and AR-mediated transcriptional activity. ARB also exerted a mild additional effect on AR-mediated transcriptional activation by the ERβ up-regulation. An intervention study revealed that PSA progression was prolonged in prostate cancer patients given an ARB compared with placebo control. These data provide a new concept that ARBs are promising potential chemopreventive and chemotherapeutic agents for prostate cancer.

Central Role for PELP1 in Nonandrogenic Activation of the Androgen Receptor in Prostate Cancer

The ability of 17β-estradiol (E2) to regulate the proliferation of prostate cancer (PCa) cells in the absence of androgen is poorly understood. Here, we show the predominant estrogen receptor (ER) isoform expressed in PCa specimens and cell lines is ERβ. Our data indicate that E2 induces the formation of a complex between androgen receptor (AR), ERβ, and a proline-, glutamic acid-, and leucine-rich cofactor protein 1 (PELP1) in PCa cells. This protein complex is formed on AR's cognate DNA-responsive elements on the promoter in response to E2. Formation of this complex enables the transcription of AR-responsive genes in response to E2. Knockdown of PELP1, AR, or ERβ blocks the assembly of this complex, blocks E2-induced genomic activation of AR-regulated genes, and blocks E2-stimulated proliferation of PCa cells. Overall, this study shows that PELP1 may enable E2-induced AR signaling by forming a protein complex between AR, ERβ, and PELP1 on the DNA, leading to the proliferation of PCa cells in the absence of androgen. PELP1 may bridge the signal between E2 bound to ERβ and AR and thus allow for cross talk between these steroid receptors. These data suggest a novel mechanism of AR activation in the absence of androgens in PCa cells. Our data indicate that disruption of the complex between AR and PELP1 may be a viable therapeutic strategy in advanced PCa.

Abstract C48: Regulation of expression of CXCL12 and its receptors CXCR4 and CXCR7 by PTEN in prostate cancer

Abstract Introduction: Expression of the chemokine CXCL12, also known as SDF-1, and its receptors, CXCR4 and CXCR7, are associated with prostate cancer (PC) metastasis. Loss of the tumor suppressor PTEN (phosphatase and tensin homolog) occurs in 30-80% of primary PCs, and up to 50% of PC bone metastases. In mouse models, prostate-specific deletion of PTEN leads to Akt activation and increased expression of CXCR4 and CXCL12. Moreover, the level of PTEN expression is inversely associated with prostate tumorigenesis. Additionally, HIF-1α, a hypoxia-induced transcription factor previously shown to induce expression of CXCR4, is regulated by Akt. Thus, we hypothesize that the PTEN loss-mediated activation of HIF-1α regulates the function of CXCR4 and CXCR7 receptor pathways in PC progression. Methods: Prostate-specific deletion of floxed exon 5 of PTEN was achieved by Cre recombinase expressed under the control of an androgen-responsive probasin promoter. Stable cell lines developed from PTEN+/+, PTEN+/−, and PTEN−/− murine prostate epithelial cells that were isolated from the prostates of corresponding mice at 8 weeks of age, and the PTEN status in these cell lines was confirmed by genotyping and immunoblot analyses. Subsequently, these cells were treated with inhibitors of the PI3K/Akt pathway, and gene expression was determined by real time PCR. Results: We found that loss of PTEN resulted in increased expression of CXCR4, CXCR7, and CXCL12, suggesting that loss of PTEN may play a key role in the regulation of these chemokines in prostate cancer. Additionally, expression of HIF-1α was also upregulated upon PTEN loss. Upon treatment of PTEN−/− cells with inhibitors of the PI3K/Akt pathway, the induction of CXCL12 and CXCR7 was reversed, further demonstrating the role of the PTEN regulated pathway in the expression of these genes. Conclusions: These results suggest a molecular basis for activation of CXCL12 signaling through its receptors in prostate cancer driven by the loss of PTEN through the induction of HIF-1. Citation Format: M. Katie Conley-LaComb, Yong Q. Chen, Hyeong-Reh C. Kim, Michael L. Cher, Sreenivasa R. Chinni. Regulation of expression of CXCL12 and its receptors CXCR4 and CXCR7 by PTEN 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 C48.