Altered Androgen Receptor Research Articles

Prostate-Specific Antigen Response to Androgen Deprivation Therapy in the Neoadjuvant Setting for High-Risk Prostate Adenocarcinoma (PIRANHA): Pooled Analysis of Two Randomized Clinical Trials

A suboptimal prostate-specific antigen (PSA) response to neoadjuvant androgen deprivation therapy (ADT) among men who go on to receive definitive radiation therapy for prostate cancer might suggest the existence of castration-resistant disease or altered androgen receptor signaling. This in turn may portend worse long-term clinical outcomes, especially in men with high-risk disease. We set out to evaluate the prognostic impact of poor PSA response to neoadjuvant ADT in men with high-risk prostate cancer. This was a post hoc analysis of the multicenter TROG 03.04 RADAR and PCS IV randomized clinical trials. Inclusion criteria for this analysis were patients with high-risk prostate cancer (defined as Gleason score ≥8, initial PSA ≥20 ng/mL, or cT3a disease or higher) who received definitive radiation therapy, at least 18 months of ADT, and had a preradiation therapy PSA level drawn after at least 3 months of neoadjuvant ADT. Poor PSA response was defined as PSA >0.5 ng/mL. Cox regression and Fine-Gray models were used to test whether poor PSA response was associated with metastasis-free survival, biochemical recurrence, prostate-cancer specific mortality, and overall survival. Nine hundred thirty men met inclusion criteria for this analysis. Median follow-up was 130 months (interquartile range [IQR], 89-154 months). After a median of 3 months (IQR, 3-4.2 months) of neoadjuvant ADT, the median PSA was 0.60 ng/mL (IQR, 0.29-1.59). Overall, 535 men (57%) had a PSA >0.5 ng/mL. Poor PSA response was associated with significantly worse metastasis-free survival (hazard ratio [HR], 3.93; P = .02), worse biochemical recurrence (subdistribution HR, 2.39; P = .003), worse prostate-cancer specific mortality (subdistribution HR, 1.50; P = .005), and worse overall survival (HR, 4.51; P = .05). Patients with PSA >0.5 mg/mL after at least 3 months of neoadjuvant ADT had worse long-term clinical outcomes and should be considered for treatment intensification.

Significantly shortened telomere length and altered androgen receptor level in cumulus cells from women with polycystic ovary syndrome

ObjectiveThe aim of this study was to investigate the correlation between hormone receptor levels and telomere length (TL) in infertile women with and without polycystic ovary syndrome (PCOS). Materials and methodsThis prospective cohort study recruited a total of 431 cumulus oocyte complex (COC) from 88 infertile women between July 2012 and June 2014. The participants were divided into three groups: young age (<38 years, n = 42 and 227 COC), advanced age (≥38 years, n = 33 and 107 COC) and PCOS patients (n = 13 and 97 COC). Cumulus cells were collected from individual follicle during oocyte pick-up, and the mRNA levels of hormone receptors and TL were measured using real-time PCR. ResultsThe cumulus cells of PCOS patients demonstrated lower mRNA levels of LH receptor (75.57 ± 138.10 vs. 171.07 ± 317.68; p < 0.01) and androgen receptor (1.13 ± 1.52 vs. 4.08 ± 9.57; p < 0.01), as well as a shorter TL (2.39 ± 2.58 vs. 3.96 ± 4.72; p < 0.01) compared to those of the young age group. In the young age group, only androgen receptor mRNA level showed a significant association with TL (rho = 0.148, p = 0.026), while FSH receptor mRNA level was the only factor associated with TL (rho = 0.247, p = 0.015) in PCOS patients. For advanced-aged patients, no significant relationship was observed between hormone receptor mRNA levels and TL. Alternative splicing of androgen receptors was identified in some PCOS patients but not in young age controls. ConclusionThe findings suggest that the androgen receptor level and function may be altered in the cumulus cells of PCOS patients, leading to a shorter TL in cumulus cells in PCOS patients.

Gender disparity in survival of early porcine fetuses due to altered androgen receptor or associated U2 spliceosome component

A single locus on the X chromosome codes for androgen receptor (AR) although this gene is subject to alternative splicing. AR is expressed in multiple tissues in males and females and is essential for reproductive success in the male. Since male and female mice are viable following naturally occurring and engineered loss of function with male mice infertile as anticipated, functional deletion of AR in pigs was hypothesized to provide a genetic containment strategy for males with edited genomes. In addition, deletion of AR might be a method to manage boar taint, hence contributing to a perceived improvement in animal welfare. The CRISPR/Cas9 technology was used to edit either exon 2 or exon 5 of the pig AR gene. Although pregnancies were established following embryo transfer of edited embryos, they were not maintained beyond day 25. Furthermore, normal M:F sex ratios were present in edited blastocysts and 19-day fetuses, but all fetuses recovered on day 21 or later were female. The pig AR gene differs from the mouse in having a U2 spliceosome component encoded in the intronic region. Hence, the absence of fetal survival beyond day 25 may be due to interference with the U2 component rather than AR.

HIRA-mediated loading of histone variant H3.3 controls androgen-induced transcription by regulation of AR/BRD4 complex assembly at enhancers.

Incorporation of histone variant H3.3 comprises active territories of chromatin. Exploring the function of H3.3 in prostate cancer (PC), we found that knockout (KO) of H3.3 chaperone HIRA suppresses PC growth in vitro and in xenograft settings, deregulates androgen-induced gene expression and alters androgen receptor (AR) binding within enhancers of target genes. H3.3 affects transcription in multiple ways, including activation of p300 by phosphorylated H3.3 at Ser-31 (H3.3S31Ph), which results in H3K27 acetylation (H3K27Ac) at enhancers. In turn, H3K27Ac recruits bromodomain protein BRD4 for enhancer-promoter interaction and transcription activation. We observed that HIRA KO reduces H3.3 incorporation, diminishes H3.3S31Ph and H3K27Ac, modifies recruitment of BRD4. These results suggest that H3.3-enriched enhancer chromatin serves as a platform for H3K27Ac-mediated BRD4 recruitment, which interacts with and retains AR at enhancers, resulting in transcription reprogramming. In addition, HIRA KO deregulates glucocorticoid- (GR) driven transcription of genes co-regulated by AR and GR, suggesting a common H3.3/HIRA-dependent mechanism of nuclear receptors function. Expression of HIRA complex proteins is increased in PC compared with normal prostate tissue, especially in high-risk PC groups, and is associated with a negative prognosis. Collectively, our results demonstrate function of HIRA-dependent H3.3 pathway in regulation of nuclear receptors activity.

(074) Association of Androgen Receptor Signaling in Human Corpus Cavernosum with Serum Testosterone Levels

Abstract Introduction Testosterone (T) plays an important role in properly developing and maintaining male reproductive tissues. The normal serum testosterone levels can vary between 300 to 1000 ng/dL or 10.4 to 34.7 nmol/L. However, it is unclear whether androgen receptor signaling can vary based on serum T levels or the half-life of testosterone therapy. What remains unknown is whether there is a uniform serum T level above which androgen receptor signaling within the tissue remains similar (saturation hypothesis). Objective We first evaluated whether varying serum levels of T alter androgen receptor (AR) signaling in human penile tissue. Then, we evaluated and compared AR signaling in corpus cavernosum tissue of men treated with short-acting vs. long-acting testosterone. We hypothesized that we would find similar AR signaling in the penile tissue of both eugonadal and hypogonadal men as well as those men receiving short and long-acting testosterone. Methods We obtained human corpus cavernosum (hCC) tissue biopsies during penile surgery (for erectile dysfunction) from 14 men. We recorded their serum T levels on week prior to surgery before 10AM. We used western blot to evaluate adrenergic receptor (AR) and VEGF protein levels. Men without erectile dysfunction undergoing penile surgery for Peyronie's disease were used as controls. Results The median age of participants was 61 (IQR 12) and the median T level was 325ng/dL (IQR 201.45). The results of western blot showed expression of similar levels of VEGF in hCC samples from men with T &amp;gt;200ng/dL. However, men with serum T levels lower than 200 ng/dL had decreased expression of VEGF and AR. Conclusions This finding is significant because it demonstrates that serum testosterone beyond a certain threshold may have a minimal impact on erectile function. If a serum testosterone threshold is identified, this threshold could be a valuable target for future studies on the effects of testosterone on ED. It may alter the treatment threshold for men who have ED as their primary symptom of hypogonadism. This data also suggests the saturation value for penile androgen receptors. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Acerus Pharmaceuticals, Boston Scientific, Coloplast, Endo Pharmaceuticals, Empower Pharmacy, Nestle Health, Olympus, Hims, Inc

Androgen Receptor Signaling is Similar in Human Corpus Cavernosum in Men with Different Serum Testosterone Levels

BackgroundTestosterone (T) plays an important role in male reproductive function and tissue development. Normal serum T levels vary between 300 and 1000 ng/dl. It is not known, however, if varying serum T levels alter androgen receptor (AR) signaling in tissue. ObjectiveTo measure AR signaling levels in human corpus cavernosal tissue in males with different serum T levels. Design, setting, and participantsParticipants were selected from a group of males undergoing surgical management for erectile dysfunction (ED; penile prosthesis placement). T levels were measured 1 week before surgery and a sample of corpus cavernosal tissue was procured during surgery. The tissue was homogenized, measured for protein concentration, and used for western blot analysis. VEGF was selected as an AR marker and actin was used for protein normalization. Outcome measurements and statistical analysisVEGF and actin expression levels were analyzed using western blot analysis and ImageJ was used for quantification of antibody expression. Results and limitationsAR signaling was measured in terms of VEGF expression. Above a T level of 200 ng/dl, there was no significant difference found in VEGF expression. Only one patient had a T level less than 200 ng/dl, limiting the generalizability of these results. In addition, all patients had a history of ED, and controls (patients without ED) were not included in the study. ConclusionsAbove a serum T level of 200 ng/dl, there was no significant difference in AR signaling. This finding suggests that there could be a saturation level present in corpus cavernosal tissue that is approximately 200 ng/dl. Patient summarySerum testosterone levels above a certain threshold may not be necessary for biological functions. Instead, it is most likely that there is an approximate serum testosterone level that fully saturates tissue androgen receptors and results in peak function in men.

EVALUATION OF ANDROGEN SATURATION IN HUMAN CORPUS CAVERNOSUM

Testosterone (T) plays an essential role in properly developing and maintaining male reproductive tissues (1). The normal serum testosterone levels can vary between 300 to 1000 ng/dL. However, it is unclear whether androgen receptor signaling can vary based on serum T levels. What remains unknown is whether there is a consistent serum T level above which androgen receptor signaling within the tissue remains similar. Therefore, we evaluated whether varying serum levels of T alter androgen receptor (AR) signaling in human penile tissue.

Abstract 6243: RAR gamma interactome and cistrome guide the androgen receptor enhancer usage in prosate cancer

Abstract Epigenetic mechanisms in prostate cancer (PCa) alter androgen receptor (AR) enhancer access resulting in re-wired AR signaling and cancer progression and/or therapy resistance. Previously, we established miR-96 targets RARγ and its coactivator TACC1, which in turn impacts AR signaling associated with PCa progression. In the current study we sought to define the RARγ complex, its interactions with the AR, and in turn how this controls gene expression. Increased RARγ levels through miR-96 treatments increased both ENZA antiproliferative sensitivity associated with an increase in G1 and G2/M. IMPACT-seq identified miR-96 recognition elements (MREs) in HPr1AR (~1950 at 24h) and LNCaP cells (~ 350) with ~ 150 shared. In parallel, m6A-Seq revealed that the epitranscriptome mark m6A differed significantly between cells. For example, m6A was more significantly enriched on TACC1 mRNA in LNCaP compared to HPr1AR and m6A-marked MRE resulted in genes being more signficantly down-regulated by miR96, including TACC1. RIME revealed that RARγ significantly interacts miR-96 differentially expressed transcripts (DETs) and proteins (DEPs) including m6A regulators (METTL3) and QSER1 complex that protects against enhancer methylation. Reflecting this, restoring RARγ levels in 22Rv1 cells, increased AR genomic binding 12-fold to greater than 9,500 sites and further by TACC1. These gained AR sites were enriched for GATA and Homeobox motifs, significantly overlapped with H3K27ac and super-enhancers, and also ChromHMM-defined Poised Enhancers (logPV = 163) and Transcribed regions (logPV = 179). RNA-Seq experiments revealed that RARγ/TACC1 significantly altered the frequency and fold change of DHT-regulated genes. Finally, miR-96 antagomir/ENZA treatments drove a unique transcriptome that triggered cell cycle arrest and significantly associated with worse survival in the SU2C cohort. To summarize these data suggest that miR-96 is directed to control the RARγ/TACC1 complex in part by m6A marks. Restoring the RARγ/TACC1 complex increased association with potential bookmarking factor like QSER1 and redirects AR to active enhancers that promote ENA-regulated genes and increase propensity for cells to undergo cell arrest. Citation Format: Sajad A. Wani, Jaimie S. Gray, Moray J. Campbell. RAR gamma interactome and cistrome guide the androgen receptor enhancer usage in prosate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6243.

MP38-18 ANDROGEN RECEPTOR SIGNALING IN CORPUS CAVERNOSUM IS SIMILAR IN MEN WITH SERUM TESTOSTERONE LEVELS ABOVE 200 ng/dL

You have accessJournal of UrologyCME1 May 2022MP38-18 ANDROGEN RECEPTOR SIGNALING IN CORPUS CAVERNOSUM IS SIMILAR IN MEN WITH SERUM TESTOSTERONE LEVELS ABOVE 200 ng/dL Kajal Khodamoradi, Alexandra Dullea, Roei Golan, Jesse Ory, Himanshu Arora, and Ranjith Ramasamy Kajal KhodamoradiKajal Khodamoradi More articles by this author , Alexandra DulleaAlexandra Dullea More articles by this author , Roei GolanRoei Golan More articles by this author , Jesse OryJesse Ory More articles by this author , Himanshu AroraHimanshu Arora More articles by this author , and Ranjith RamasamyRanjith Ramasamy More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002592.18AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Testosterone (T) plays an important role in the proper development and maintenance of male reproductive tissues. The normal serum testosterone levels can vary between 300 to 1000 ng/dL. However, it is unclear whether androgen receptor signaling can vary based on serum T levels. What remains unknown is also whether there is a uniform serum T level above which androgen receptor signaling within the tissue remains similar (saturation hypothesis). Therefore, we evaluated whether varying serum levels of T alters androgen receptor (AR) signaling in human penile tissue. METHODS: We obtained human corpus cavernosum (CC) tissue biopsies during penile surgery (either for erectile dysfunction or Peyronie’s disease). We recorded their serum T levels one week prior to surgery. After mechanical dispersion of CC tissue with a homogenizer, the total protein was extracted using RIPA buffer, and quantitative detection of vascular endothelial growth factor (VEGF) was evaluated by western blot. RESULTS: The mean age of participants was 64±10 and the mean T level was 487.46ng/dL±377.20. The results of western blot showed that all corpus cavernosum samples in men with T >200ng/dL expressed similar levels of VEGF in men. However, men with a low serum T level (<200 ng/dL) had decreased expression of VEGF. The VEGF expression from men undergoing penile surgery for Peyronie's disease without erectile dysfunction was used as controls. CONCLUSIONS: Despite a wide range of serum T levels (200-1594 ng/dl), androgen receptor signaling was similar in penile tissue. This data suggests that the saturation value for penile androgen receptors appears to be around approximately 200ng/dL. Understanding androgen receptor saturation hypothesis provides important context necessary for appropriate prescribing of exogenous T to optimize patient outcomes. Source of Funding: This work was supported by Acerus Pharmaceuticals, National Institutes of Health Grant R01 DK130991 and Clinician Scientist Development Grant from the American Cancer Society to RR © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e625 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kajal Khodamoradi More articles by this author Alexandra Dullea More articles by this author Roei Golan More articles by this author Jesse Ory More articles by this author Himanshu Arora More articles by this author Ranjith Ramasamy More articles by this author Expand All Advertisement PDF DownloadLoading ...

TBECH, 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane, alters androgen receptor regulation in response to mutations associated with prostate cancer

Point mutations in the AR ligand-binding domain (LBD) can result in altered AR structures leading to changes of ligand specificity and functions. AR mutations associated to prostate cancer (PCa) have been shown to result in receptor activation by non-androgenic substances and anti-androgenic drugs. Two AR mutations known to alter the function of anti-androgens are the ART877A mutation, which is frequently detected mutation in PCa tumors and the ARW741C that is rare and has been derived in vitro following exposure of cells to the anti-androgen bicalutamide. AR activation by non-androgenic environmental substances has been suggested to affect PCa progression. In the present study we investigated the effect of AR mutations (ARW741C and ART877A) on the transcriptional activation following exposure of cells to an androgenic brominated flame retardant, 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane (TBECH, also named DBE-DBCH). The AR mutations resulted in higher interaction energies and increased transcriptional activation in response to TBECH diastereomer exposures. The ART877A mutation rendered AR highly responsive to low levels of DHT and TBECH and led to increased AR nuclear translocation. Gene expression analysis showed a stronger induction of AR target genes in LNCaP cells (ART877A) compared to T-47D cells (ARWT) following TBECH exposure. Furthermore, AR knockdown experiments confirmed the AR dependency of these responses. The higher sensitivity of ART877A and ARW741C to low levels of TBECH suggests that cells with these AR mutations are more susceptible to androgenic endocrine disrupters.

Prenatal exposure to sodium valproate alters androgen receptor expression in the developing cerebellum in a region and age specific manner in male and female rats

Valproic acid (VPA) is an anti-epileptic drug with teratogenicity activity that has been related to autism. In rodents, exposure to VPA in utero leads to brain abnormalities similar than those reported in the autistic brain. Particularly, VPA reduces the number of Purkinje neurons in the rat cerebellum parallel to cerebellar abnormalities found in autism. Thus, we injected pregnant females on embryonic day 12 either with VPA (600mg/kg, i.p.) or 0.9% saline solution and obtained the cerebellum from their offspring at different postnatal time points. Testosterone has been linked to autism and plays an important role during brain development. Therefore, we identified and analyzed the androgen receptor (AR) by immunohistochemistry and densitometry, respectively. We found VPA decreases AR density in the superficial Purkinje layer only in cerebellar lobule 8 at PN7, but increased it at PN14 compared to control in males. In females, VPA decreased AR density in the superficial Purkinje layer in cerebellar lobule 6 at PN14, but increased it in lobule 9 at the same time point. No differences were found in the deep Purkinje layer of any cerebellar lobule in terms of AR density neither in males nor females. We additionally found a particular AR density decreasing in both superficial and deep regions across development in the majority of cerebellar lobules in males, but in all cerebellar lobules in females. Thus, our results indicate that VPA disrupts the AR ontogeny in the developing cerebellum in an age and region specific manner in male and female rats. Future epigenetic studies including the evaluation of histone deacetylases (HDAC’s) might shed light these results as HDAC’s are expressed by Purkinje neurons, interact with the AR and are VPA targets. This work contributes to the understanding of the cerebellar development and it might help to understand the role of the cerebellum in neurodevelopmental disorders such as autism.

Kinase modulation of androgen receptor signaling: implications for prostate cancer.

Androgens and androgen receptors play essential roles in the development and progression of prostate cancer, a disease that claims roughly 28,000 lives annually. In addition to androgen biding, androgen receptor activity can be regulated via several post-translational modifications such as ubiquitination, acetylation, phosphorylation, methylation & SUMO-ylation. Off these modifications, phosphorylation has been the most extensively studied. Modification by phosphorylation can alter androgen receptor localization, protein stability and transcriptional activity, ultimately leading to changes in the biology of cancer cells and cancer progression. Understanding, role of phosphorylated androgen receptor species holds the key to identifying a potential therapeutic drug target for patients with prostate cancer and castrate resistant prostate cancer. Here, we present a brief review of recently discovered protein kinases phosphorylating AR, focusing on the functional role of phosphorylated androgen receptor species in prostate cancer and castrate resistant prostate cancer.

Bone stroma-derived cells change coregulators recruitment to androgen receptor and decrease cell proliferation in androgen-sensitive and castration-resistant prostate cancer cells

Prostate cancer (CaP) bone metastasis is an early event that remains inactive until later-stage progression. Reduced levels of circulating androgens, due to andropause or androgen deprivation therapies, alter androgen receptor (AR) coactivator expression. Coactivators shift the balance towards enhanced AR-mediated gene transcription that promotes progression to androgen-resistance. Disruptions in coregulators may represent a molecular switch that reactivates latent bone metastasis. Changes in AR-mediated transcription in androgen-sensitive LNCaP and androgen-resistant C4-2 cells were analyzed for AR coregulator recruitment in co-culture with Saos-2 and THP-1. The Saos-2 cell line derived from human osteosarcoma and THP-1 cell line representing human monocytes were used to display osteoblast and osteoclast activity. Increased AR activity in androgen-resistant C4-2 was due to increased AR expression and SRC1/TIF2 recruitment and decreased SMRT/NCoR expression. AR activity in both cell types was decreased over 90% when co-cultured with Saos-2 or THP-1 due to dissociation of AR from the SRC1/TIF2 and SMRT/NCoR coregulators complex, in a ligand-dependent and cell-type specific manner. In the absence of androgens, Saos-2 decreased while THP-1 increased proliferation of LNCaP cells. In contrast, both Saos-2 and THP-1 decreased proliferation of C4-2 in absence and presence of androgens. Global changes in gene expression from both CaP cell lines identified potential cell cycle and androgen regulated genes as mechanisms for changes in cell proliferation and AR-mediated transactivation in the context of bone marrow stroma cells.

Norfloxacin drug induces reproductive toxicity and alters androgen receptor gene expression in testes and cloacal gland of male Japanese quail (Coturnix Japonica)

In an attempt to investigate the reproductive toxicity of norfloxacin in Japanese quail, male quail were given norfloxacin at 20 mg/kg body weight for 14 d. Then reproductive function and androgen receptor (AR) gene expression was examined in treated and control birds. The results of the present study indicate that fertility, cloacal gland area, sperm concentration, and serum testosterone were reduced significantly (p < 0.05) on day 14 in the norfloxacin-treated birds. Upregulation (p < 0.05) of AR mRNA was also seen in the testes on the 14th d of treatment. A trend toward downregulation of AR mRNA was seen in the cloacal gland of norfloxacin-treated birds. Histological observations revealed that norfloxacin induces cellular atrophy in testes and changes in glandular tissue in the cloacal gland. The results of the present study demonstrate that norfloxacin induces testicular toxicity in Japanese quail.

The role of DAB2IP in androgen receptor activation during prostate cancer progression

Altered androgen-receptor (AR) expression and/or constitutively active AR are commonly associated with prostate cancer (PCa) progression. Targeting AR remains a focal point for designing new strategy of PCa therapy. Here, we have shown that DAB2IP, a novel tumor suppressor in PCa, can inhibit AR-mediated cell growth and gene activation in PCa cells via distinct mechanisms. DAB2IP inhibits the genomic pathway by preventing AR nuclear translocation or phosphorylation and suppresses the non-genomic pathway via its unique functional domain to inactivate c-Src. Also, DAB2IP is capable of suppressing AR activation in an androgen-independent manner. In addition, DAB2IP can inhibit several AR splice variants showing constitutive activity in PCa cells. In DAB2IP(-/-) mice, the prostate gland exhibits hyperplastic epithelia, in which AR becomes more active. Consistently, DAB2IP expression inversely correlates with AR activation status particularly in recurrent or metastatic PCa patients. Taken together, DAB2IP is a unique intrinsic AR modulator in normal cells, and likely can be further developed into a therapeutic agent for PCa.

Androgen receptor splice variants are resistant to inhibitors of Hsp90 and FKBP52, which alter androgen receptor activity and expression

Androgen ablation therapy is the most common treatment for advanced prostate cancer (PCa), but most patients will develop castration-resistant prostate cancer (CRPC), which has no cure. CRPC is androgen-depletion resistant but androgen receptor (AR) dependent. AR is a nuclear receptor whose transcriptional activity is regulated by hormone binding to the ligand-binding domain (LBD). Constitutively active AR splice variants that lack LBDs often are expressed in CRPC. The expression of these variants indicates that methods to inhibit AR activity that do not rely on inactivating the LBD are needed. Heat shock protein 90 (Hsp90), a potential therapeutic target in PCa, is an AR chaperone crucial for proper folding, hormone binding and transcriptional activity of AR. We generated LNCaP cell lines with regulated expression of the AR-V7 variant as well as a cell line expressing artificially truncated AR (termed AR-NTD) to characterize splice variant function. Using an Hsp90 inhibitor, Geldanamycin (GA), and an AR-Hsp90-FKBP52 specific inhibitor, MJC13, we sought to determine if the AR variants also require Hsp90 and associated co-chaperone, FKBP52, for their activity. GA inhibits AR transcriptional activity but has little effect on AR-V7 activity. Moreover, GA decreases the stability of AR protein, with no effect on AR-V7 levels. Full-length AR activity is strongly inhibited by MJC13 while AR-V7 is unaffected. Thus, the variants are resistant to inhibitors of the Hsp90-AR heterocomplex. Although Hsp90 inhibitors will continue to inhibit growth promoting kinases and signaling through activated full-length AR in CRPC, AR signaling through variants will be retained.

Does changing androgen receptor status during prostate cancer development impact upon cholesterol homeostasis?

BackgroundRecent evidence associates prostate cancer with high cholesterol levels, with cholesterol being an important raw material for cell-growth. Within the cell, cholesterol homeostasis is maintained by two master transcription factors: sterol-regulatory element-binding protein 2 (SREBP-2) and liver X receptor (LXR). We previously showed that the androgen receptor, a major player in prostate cell physiology, toggles these transcription factors to promote cholesterol accumulation. Given that prostate cancer therapy targets the androgen receptor, selecting for cells with altered androgen receptor activity, how would this affect SREBP-2 and LXR activity? Using a novel prostate cancer progression model, we explored how this crosstalk between the androgen receptor and cholesterol homeostasis changes during prostate cancer development.Methodology/Principal FindingsFirstly, we characterised our progression model, which involved 1) culturing LNCaP cells at physiological testosterone levels to generate androgen-tolerant LNCaP-305 cells, and 2) culturing LNCaP-305 with the anti-androgen casodex to generate castration-resistant LNCaP-364 cells. This progression was accompanied by upregulated androgen receptor expression, typically seen clinically, and a reduction in androgen receptor activity. Although this influenced how SREBP-2 and LXR target genes responded to androgen treatment, cellular cholesterol levels and their response to changing sterol status was similar in all LNCaP sub-lines.Conclusion/SignificanceOverall cholesterol homeostasis is unaffected by changing androgen receptor activity in prostate cancer cells. This does not negate the relationship between androgens and cholesterol homeostasis, but rather suggests that other factors compensate for altered androgen receptor activity. Given that cholesterol regulation is maintained during progression, this supports the growing idea that cholesterol metabolism is a suitable target for prostate cancer.

779 Androgen Receptor Phosphorylation is Associated With Clinical Outcome Measures in Hormone Naive Prostate Cancer

Introduction: Although prostate cancer (Pca) can be readily diagnosed, estimation of likely outcome remains problematical as many will have a benign course and will not die of their disease, while some patients progress rapidly to castrate resistant disease and die within 18−24 months. Ideally, there would be a factor or factors which would evidently set apart those patients who will, in due course succumb to the disease from those who will not. In Pca activation of signal transduction cascades has been shown to alter androgen receptor (AR) activity. Although it has been suggested that changes in AR phosphorylation might be directly responsible, the candidate kinases mediating phosphorylation is not conclusive. Recent evidence suggests that Protein kinase C (PKC) phosphorylates AR at Serine 578 (pAR). Materials and Methods: The aim of the current study was to investigate if the relationship between the AR and PKC is upheld in clinical prostate cancer specimens. Scansite 2.0 predicted PKC as a strong candidate mediating phosphorylation of AR at serine residue 578. Immunohistochemical analysis was performed to assess protein expression levels of PKC and pAR in a cohort of 90 patients with hormone naive prostate adenocarcinoma. Results and Discussion: High cytoplasmic pAR expression (> median) was significantly associated with time to biochemical relapse (p = 0.034) and with decreased time to death from relapse (p = 0.034). Additionally, high expression of cytoplasmic pAR was significantly associated with decreased disease-specific survival (p = 8.6×10−5), as was high nuclear pAR expression (p = 0.036). Protein expression of PKC was not significantly associated with survival. However, nuclear PKC correlated strongly with pAR expression, in both the cytoplasm (c.c 0.426, p = 0.002) and nucleus (c.c. 0.469, p = 0.001). Conclusion: These results suggest that the phosphorylation of the AR by PKC may be of functional importance in the clinical setting. Consistent with predictions by Scansite expression of PKC significantly correlated with phosphorylation of pAR. Phosphorylation pAR is implicated in Pca progression and survival. Further work investigating the functional consequences of inhibition of AR phosphorylation at serine 578 is warranted.

A hypothesis-generating exploratory analysis of efficacy and safety of abiraterone acetate (AA) in African American (Af Am) patients (pts) with metastatic castration-resistant prostate cancer (mCRPC).

55 Background: The reported increased prostate cancer lethality in Af Ams has been attributed by some to altered androgen receptor (AR) signaling. We compared toxicity, PSA response, time-to-PSA progression (TTPP), and radiographic progression-free survival (rPFS) in Af Am vs non Af Am pts with CRPC treated with AA + prednisone (P) vs placebo + P. We hypothesized that differences in response to AA may be observed if differences in AR signaling exist in Af Ams. Methods: COU-AA-301 is a randomized double blind study of AA (1000 mg + P 5 mg po BID) vs placebo + P post-docetaxel. Results: TTPP, rPFS, and PSA response rate were higher with AA vs placebo. In Af Am pts, treatment emergent AEs (TEAEs) for AA vs placebo occurred in 96.4% vs 100.0% of pts (50.0% and 66.7%, respectively, grade 3/4); serious TEAEs occurred in 42.9% and 33.3% of pts (28.6% and 26.7%, respectively, grade 3/4). The safety profile of AA appears comparable between the Af Am and overall study populations. Conclusions: Although the small number of Af Am pts in this study precludes formal conclusions regarding efficacy and safety of AA in this pt population, the overall trend suggests these pts experienced clinical benefit from AA with a safety profile comparable to the overall study population. These findings do not appear to support the hypothesis that AR signaling accounts for the increased lethality of prostate cancer seen in Af Ams. Further studies of AA in Af Am pts are planned to understand the potential benefit in this population. Additional efforts are needed to increase participation of Af Am pts in clinical trials. [Table: see text]

Inhibition of the Androgen Receptor Activity by Coprinus comatus Substances

Prostatic adenocarcinoma is the second leading cause of death from cancer in Western men. The common prostate cancer treatments are effective in the early stages; however, advanced prostate cancer is resilient to most of these treatments. Altered androgen receptor (AR) activity caused by point mutations or signaling mechanisms that regulate AR function has been proposed as a key mechanism in the transition to the androgen-independent stage. Our previous results demonstrated that hexane extract prepared from Coprinus comatus (C. comatus) strain 734 was able to interfere with AR activity. The current study was made to further evaluate the antiandrogenic activity of the C. comatus mushroom strain 734. Activity-guided chromatography was conducted and 2 active fractions, F-32-and F-33, were found to contain substances that were able to inhibit AR-mediated reporter activity and reduce the levels of AR and prostate-specific antigen (PSA) transcripts in LNCaP cells. Fraction F-32 also inhibited the proliferation and clonigenicity of LNCaP cells. Furthermore, F-32 was able to inhibit the binding of AR to the PSA enhancer region and to inhibit Akt-mediated AR phosphorylation at Ser 213. This study illustrated the potential of substances from the C. comatus mushroom to serve as natural antiandrogenic modulators for the treatment of prostatic disorders.