Increased Androgen Receptor Activity Research Articles

JMJD2A promotes the development of castration-resistant prostate cancer by activating androgen receptor enhancer and inhibiting the cGAS-STING pathway.

Exploring targets for inhibiting androgen receptor (AR) activity is an effective strategy for suppressing the development of castration-resistant prostate cancer (CRPC). Upregulation of histone demethylase JMJD2A activity is an important factor in increasing AR expression in CRPC. Based on our research, we found that the binding affinity between JMJD2A and AR increases in CRPC, while the level of AR histone methylation decreases and the H3K27ac level increases in the AR enhancer region. Further investigations revealed that overexpression of the histone demethylase JMJD2A increased the binding affinity between JMJD2A and AR, decreased AR histone methylation levels, upregulated H3K27ac in the AR enhancer region, and increased AR activity. Conversely, knocking down JMJD2A effectively reversed these effects. Additionally, in CRPC, JMJD2A expression was upregulated, the tumor-intrinsic immune cGAS-STING signaling pathway was suppressed, the tumor microenvironment was altered, and AR expression was upregulated. However, both knocking down JMJD2A and inhibiting the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS-STING) signaling pathway reversed these effects. In summary, our study indicates that in CRPC, JMJD2A can directly bind to AR and activate residual AR enhancers through its demethylation activity, thereby promoting AR expression. Furthermore, upregulation of JMJD2A expression inhibits the innate immune cGAS-STING signaling pathway of the tumor, leading to a decrease in antitumor immune function, and further promoting AR expression.

Abstract ND03: Discovery of ARV-766, an androgen receptor degrading PROTAC® for the treatment of men with metastatic castration resistant prostate cancer

Abstract Prostate cancer is the second leading cause of cancer death in men in the United States. The androgen receptor (AR) plays critical roles in both early disease and advanced prostate cancer. Current therapeutic approaches targeting the androgen/AR axis are initially effective, but castration resistant prostate cancer (CRPC) inevitably develops. CRPC is linked with increased AR activity via gene overexpression, amplification, and gain-of-function mutations. To address these mechanisms of AR-dependent prostate tumor growth, we have developed a novel therapeutic agent, ARV-766, a proteolysis targeting chimera (PROTAC®) that induces a protein-protein interaction between the AR and specific E3 ubiquitin ligase complexes, leading to the ubiquitination of AR and its subsequent degradation via the proteasome. In vitro, ARV-766 degrades AR in various prostate cancer cell lines, including those harboring resistance-conferring, clinically relevant point mutations, with a half-maximal degradation concentration (DC50) of <1 nM in wild type VCaP. Importantly ARV-766 also maintains potency against the AR L702H mutant, which has been associated with resistance to some AR antagonists. In vivo, ARV-766 is orally bioavailable and robustly degrades AR with a >90% observed maximum degradation (Dmax) at efficacious doses. ARV-766 significantly and dose-dependently inhibits tumor growth in murine LNCaP and VCaP xenograft models, including an enzalutamide-insensitive non-castrated VCaP model. These preclinical data supported the clinical development of ARV-766 for the treatment of men with metastatic CRPC. Selected pre-clinical data along with the chemical structure of ARV-766 will be presented. Citation Format: Larry Snyder, S H. Lee, T K. Neklesa, X Chen, H Dong, C Ferraro, D A. Gordon, J Macaluso, J Pizzano, J Wang, R R. Willard, N Vitale, R Peck, M D. Moore, C M. Crews, J Houston, A P. Crew, I Taylor. Discovery of ARV-766, an androgen receptor degrading PROTAC® for the treatment of men with metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr ND03.

The Androgen Hormone-Induced Increase in Androgen Receptor Protein Expression Is Caused by the Autoinduction of the Androgen Receptor Translational Activity

The androgen receptor (AR) plays a central role in prostate, muscle, bone and adipose tissue. Moreover, dysregulated AR activity is a driving force in prostate cancer (PCa) initiation and progression. Consequently, antagonizing AR signalling cascades via antiandrogenic therapy is a crucial treatment option in PCa management. Besides, very high androgen levels also inhibit PCa cells’ growth, so this effect could also be applied in PCa therapy. However, on the molecular and cellular level, these mechanisms have hardly been investigated so far. Therefore, the present study describes the effects of varying androgen concentrations on the viability of PCa cells as well as localization, transactivation, and protein stability of the AR. For this purpose, cell viability was determined via WST1 assay. Alterations in AR transactivity were detected by qPCR analysis of AR target genes. A fluorescent AR fusion protein was used to analyse AR localization microscopically. Changes in AR protein expression were detected by Western blot. Our results showed that high androgen concentrations reduce the cell viability in LNCaP and C4-2 cell lines. In addition, androgens have been reported to increase AR transactivity, AR localization, and AR protein expression levels. However, high androgen levels did not reduce these parameters. Furthermore, this study revealed an androgen-induced increase in AR protein synthesis. In conclusion, inhibitory effects on cell viability by high androgen levels are due to AR downstream signalling or non-genomic AR activity. Moreover, hormonal activation of the AR leads to a self-induced stabilization of the receptor, resulting in increased AR activity. Therefore, in clinical use, a therapeutic reduction in androgen levels represents a clinical target and would lead to a decrease in AR activity and, thus, AR-driven PCa progression.

Abstract 43: Discovery of ARV-110, a first in class androgen receptor degrading PROTAC for the treatment of men with metastatic castration resistant prostate cancer

Abstract Prostate cancer is the second leading cause of cancer death in men in the United States. The androgen receptor (AR) plays critical roles in both early disease and advanced prostate cancer. Current therapeutic approaches targeting the androgen/AR axis are initially effective, but castration resistant prostate cancer (CRPC) inevitably develops. CRPC is tightly linked with increased AR activity via gene overexpression, amplification, and gain-of-function mutations. To address these mechanisms of AR-dependent prostate tumor growth, we have developed a novel therapeutic agent, ARV-110, a proteolysis targeting chimera (PROTAC®) that induces a protein-protein interaction between the AR and E3 ubiquitin ligase complexes, leading to the ubiquitination of AR and its subsequent degradation via the proteasome. In vitro, ARV-110 robustly degrades AR in various prostate cancer cell lines with a half-maximal degradation concentration (DC50) of ~1 nM. Whole cell lysate proteomics and ubiquitin proteomics demonstrated high selectivity for AR in VCaP cells. ARV-110 potently downregulates androgen responsive genes, inhibits proliferation and induces apoptosis in AR dependent prostate cancer cells. In vivo, ARV-110 is orally bioavailable and robustly degrades AR with a >90% observed maximum degradation (Dmax) at efficacious doses. ARV-110 significantly and dose-dependently inhibits tumor growth in murine LNCaP and VCaP xenograft models, including enzalutamide-resistant VCaP and enzalutamide-insensitive patient-derived xenograft models. These preclinical data supported the clinical development of ARV-110 for the treatment of men with metastatic CRPC. The discovery, chemical structure and initial clinical data of ARV-110 will be presented Citation Format: Lawrence B. Snyder, Taavi K. Neklesa, Xin Chen, Hanqing Dong, Caterina Ferraro, Deborah A. Gordon, Jennifer Macaluso, Jennifer Pizzano, Jing Wang, Ryan R. Willard, Nicholas Vitale, Ron Peck, Marcia Dougan Moore, Craig M. Crews, John Houston, Andrew P. Crew, Ian Taylor. Discovery of ARV-110, a first in class androgen receptor degrading PROTAC for the treatment of men with metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 43.

Increased androgen receptor levels and signaling in ovarian cancer cells by VEPH1 associated with suppression of SMAD3 and AKT activation

Studies indicate androgens contribute to initiation or progression of epithelial ovarian cancer through poorly understood mechanisms. We provide evidence that the androgen receptor (AR) interacts in a ligand-independent manner with the putative armadillo repeat domain of ventricular zone expressed PH domain-containing 1 (VEPH1). This interaction was increased by mutation of the two nuclear receptor-interacting LxxLL motifs present within the VEPH1 armadillo repeat domain. Androgen treatment did not result in nuclear co-localization of VEPH1 with AR, suggesting that VEPH1 does not function as a nuclear co-regulatory protein. VEPH1 expression decreased SMAD3 and activated AKT levels in ovarian cancer cell lines and increased AR activity and protein levels, consistent with an impact on receptor stability. Treatment of cells with dihydrotestosterone (DHT) increased AR protein levels measured 24 h after treatment, an effect augmented in VEPH1-transfected cells, and inhibited by knock-down of endogenous VEPH1. SMAD3 overexpression decreased AR protein levels and prevented the VEPH1-dependent increase in AR; however, silencing of SMAD3 paradoxically also decreased AR levels. DHT treatment led to a rapid and sustained decrease in phosphorylated AKT (pAKT) levels that was enhanced by VEPH1 expression. Inhibition of PI3K resulted in increased AR protein levels. These studies indicate that VEPH1 acts to enhance AR activity in ovarian cancer cells by decreasing SMAD3 and pAKT levels, resulting in increased levels of AR protein.

Androgen receptor-modulatory microRNAs provide insight into therapy resistance and therapeutic targets in advanced prostate cancer

Androgen receptor (AR) signalling is a key prostate cancer (PC) driver, even in advanced ‘castrate-resistant’ disease (CRPC). To systematically identify microRNAs (miRs) modulating AR activity in lethal disease, hormone-responsive and -resistant PC cells expressing a luciferase-based AR reporter were transfected with a miR inhibitor library; 78 inhibitors significantly altered AR activity. Upon validation, miR-346, miR-361-3p and miR-197 inhibitors markedly reduced AR transcriptional activity, mRNA and protein levels, increased apoptosis, reduced proliferation, repressed EMT, and inhibited PC migration and invasion, demonstrating additive effects with AR inhibition. Corresponding miRs increased AR activity through a novel and anti-dogmatic mechanism of direct association with AR 6.9 kb 3′UTR and transcript stabilisation. In addition, miR-346 and miR-361-3p modulation altered levels of constitutively active AR variants, and inhibited variant-driven PC cell proliferation, so may contribute to persistent AR signalling in CRPC in the absence of circulating androgens. Pathway analysis of AGO-PAR-CLIP-identified miR targets revealed roles in DNA replication and repair, cell cycle, signal transduction and immune function. Silencing these targets, including tumour suppressors ARHGDIA and TAGLN2, phenocopied miR effects, demonstrating physiological relevance. MiR-346 additionally upregulated the oncogene, YWHAZ, which correlated with grade, biochemical relapse and metastasis in patients. These AR-modulatory miRs and targets correlated with AR activity in patient biopsies, and were elevated in response to long-term enzalutamide treatment of patient-derived CRPC xenografts. In summary, we identified miRs that modulate AR activity in PC and CRPC, via novel mechanisms, and may represent novel PC therapeutic targets.

SRARP and HSPB7 are epigenetically regulated gene pairs that function as tumor suppressors and predict clinical outcome in malignancies.

Deletions of chromosome 1p36 are common in cancers; however, despite extensive studies, there has been limited success for discovering candidate tumor suppressors in this region. SRARP has recently been identified as a novel corepressor of the androgen receptor (AR) and is located on chromosome 1p36. Here, bioinformatics analysis of large tumor datasets was performed to study SRARP and its gene pair, HSPB7. In addition, using cancer cell lines, mechanisms of SRARP and HSPB7 regulation and their molecular functions were investigated. This study demonstrated that SRARP and HSPB7 are a gene pair located 5.2 kb apart on 1p36.13 and are inactivated by deletions and epigenetic silencing in malignancies. Importantly, SRARP and HSPB7 have tumor suppressor functions in clonogenicity and cell viability associated with the downregulation of Akt and ERK. SRARP expression is inversely correlated with genes that promote cell proliferation and signal transduction, which supports its functions as a tumor suppressor. In addition, AR exerts dual regulatory effects on SRARP, and although an increased AR activity suppresses SRARP transcription, a minimum level of AR activity is required to maintain baseline SRARP expression in AR+ cancer cells. Furthermore, as observed with SRARP, HSPB7 interacts with the 14‐3‐3 protein, presenting a shared molecular feature between SRARP and HSPB7. Of note, genome‐ and epigenome‐wide associations of SRARP and HSPB7 with survival strongly support their tumor suppressor functions. In particular, DNA hypermethylation, lower expression, somatic mutations, and lower copy numbers of SRARP are associated with worse cancer outcome. Moreover, DNA hypermethylation and lower expression of SRARP in normal adjacent tissues predict poor survival, suggesting that SRARP inactivation is an early event in carcinogenesis. In summary, SRARP and HSPB7 are tumor suppressors that are commonly inactivated in malignancies. SRARP inactivation is an early event in carcinogenesis that is strongly associated with worse survival, presenting potential translational applications.

Abstract 5498: Autoregulation of protein kinase D1 (PRKD1) expression in prostate cancer

Abstract Protein kinase D1 (PRKD1) is down regulated in gastric, breast and prostate cancer. Several regulatory mechanisms modulate PRKD1 activity in cancers. In colon cancer, it has been shown that PRKD1 is down regulated by nuclear beta-catenin. We explored whether beta-catenin is involved in regulation of PRKD1 expression in prostate cancer as well. A CHIP assay performed using prostate cancer LNCaP cell lysate and pull down with beta -catenin antibody, which demonstrated recruitment of beta -catenin to promoter of PRKD1 gene as well as at downstream regions in the gene suggesting that beta -catenin could be involved in PRKD1 regulation. To identify the exact binding site of beta-catenin to PRKD1 gene, we carried out CHIP sequencing. The beta -catenin protein complex was bound to a 166 bp sequence near exon 2 (chr14:29899631-29899796). Because beta -catenin is a coactivator and not a known transcription factor, we performed a transcription factor promoter array, which showed that the MYC/MAX transcription factor complex may be the mediator of the regulatory effect of beta -catenin on PRKD1 expression. We validated the results by performing CHIP assay using MYC and MAX antibodies which showed recruitment of both MYC and MAX to the same beta -catenin binding site to PRKD1. In order to assess the functional impact of beta -catenin regulation of PRKD1 in prostate cancer, beta-catenin mutated at threonine 120 residue (we have previously shown that PRKD1 is the only known kinase to phosphorylate the site) demonstrated increased nuclear translocation of beta-catenin and increase down regulation of PRKD1 and increased androgen receptor (AR) activity (increased AR activity is well established to be associated with PC progression). Our data has identified a novel auto-regulatory mechanism of PRKD1 expression through beta-catenin phosphorylation. Citation Format: Bita Nickkholgh, Sittadjody Sivanandane. Autoregulation of protein kinase D1 (PRKD1) expression in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5498. doi:10.1158/1538-7445.AM2017-5498

Abstract 3326: Identification of a novel androgen receptor co-regulator in prostate cancer

Abstract Prostate cancer (PC) is one of the most frequently diagnosed tumors in the United States, affecting one in six men during their lifetime and second only to lung cancer as a cause of cancer death in men. One of the major drivers of prostate cancer is the androgen receptor (AR). It has been shown that deregulation of AR co-regulators play a crucial role in PC through modulating the transcriptional activity of the receptor. While the importance of the AR complex is well established, there is still an incomplete understanding of what all the associated co-regulators are and how they modulate AR signaling. Here, we have identified that the inhibitor of growth family member 4 (ING4), a protein that has been implicated in the pathogenesis of several solid tumors such as gastric and breast cancer, is a novel AR co-regulator. Overexpression of ING4 increased AR activity in an androgen-dependent manner as assessed using luciferase reporter gene assays. Supporting a role as a potential AR co-activator, RNA Next-Generation Sequencing of prostate cancer cells with ING4 knocked down revealed a repression of endogenous AR/R1881-activated genes and expression of AR/R1881 suppressed genes. Further, mining of existing clinical datasets revealed that the ING4 gene expression is de-regulated in PCs compared relative to normal tissue and tracks with poor patient prognosis. Collectively, the current findings strongly support the involvement of ING4 in AR signaling and PC progression and development. Citation Format: Aymen A. Shatnawi, Sridhar Acharya Malkaram, Efrosini Tsouko, Daniel E. Frigo. Identification of a novel androgen receptor co-regulator in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3326. doi:10.1158/1538-7445.AM2017-3326

The molecular underpinnings of prostate cancer: impacts on management and pathology practice.

Prostate cancer (PCa) is a clinically heterogeneous disease and current treatment strategies are based largely on anatomical and pathological parameters. In the recent past, several DNA sequencing studies of primary and advanced PCa have revealed recurrent patterns of genomic aberrations that expose mechanisms of resistance to available therapies and potential new drug targets. Suppression of androgen receptor (AR) signalling is the cornerstone of advanced prostate cancer treatment. Genomic aberrations of the androgen receptor or alternative splicing of its mRNA are increasingly recognised as biomarkers of resistance to AR-targeted therapies such as abiraterone or enzalutamide. Genomic aberrations of the PI3K-AKT axis, in particular affecting PTEN, are common in PCa, and compounds targeting different kinases in this pathway are showing promise in clinical trials. Both germline and somatic defects in DNA repair genes have been shown to sensitise some patients to therapy with PARP inhibition. In addition, abnormalities in mismatch-repair genes are associated with response to immune checkpoint inhibition in other solid tumours and present a tantalising therapeutic avenue to be pursued. Aberrations in CDK4/6-RB1 pathway genes occur in a subset of PCas, may associate with differential sensitivity to treatment, and are likely to have clinical implications beyond prognostication. Inhibitors of CDK4/6 are already being tested in prostate cancer clinical trials. Furthermore, deletions of RB1 are strongly associated with a neuroendocrine phenotype, a rare condition characterized by a non-AR-driven transcriptomic profile. Finally, aberrations in genes involved in regulating the chromatin structure are an emerging area of interest. Deletions of CHD1 are not infrequent in PCa and may associate with increased AR activity and genomic instability, and these tumours could benefit from DNA-damaging therapies. This review summarises how genomic discoveries in PCa are changing the treatment landscape of advanced CRPC, both by identifying biomarkers of resistance and by identifying vulnerabilities to be targeted. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract 4679: Silencing of PMEPA1, a NEDD4 E3 ubiquitin ligase binding protein, stabilizes androgen receptor and confers resistance to AR inhibitors

Abstract INTRODUCTION: The androgen receptor (AR) dysfunctions contribute to prostate cancer (CaP) development and progression. Earlier we defined the regulation of AR protein levels by a negative feed-back loop between AR and the androgen regulated PMEPA1, a NEDD4 E3 ubiquitin ligase binding protein. Our data also revealed decreased or absent PMEPA1 transcripts in 65% prostate tumors. This study focuses on the relationship of PMEPA1 protein with AR and its transcriptional targets (PSA) in human CaP, as well as the impact of PMEPA1 silencing on growth of PMEPA1-shRNA LNCaP cells derived tumors in naïve and castrated nude mice. METHODS: Ten weeks old athymic Ncr-nu/nu mice were evaluated for the growth of tumor xenografts derived from PMPEA1-shRNA LNCaP (N = 20) and control-shRNA LNCaP cells (N = 20). Each mouse received 4×106 cells by subcutaneous injection to right flank side. The mice with tumors were castrated at 9th week post-injection. The tumor sizes were monitored twice per week until 16th week post-injection. The xenograft tissues were assayed by immunohistochemical (IHC) staining for PMEPA1, AR and PSA protein levels. The AR, PMEPA1 and PSA expressions in whole mount human prostate specimens were evaluated by IHC staining and QRT-PCR. RESULTS: The PMEPA1 shRNA-LNCaP and control shRNA-LNCaP cells started to form subcutaneous tumors at 4 weeks after injections. However, the growth rate of PMEPA1 shRNA-LNCaP derived tumors was significantly faster than control derived tumors (P<0.05). PMEPA1 shRNA-LNCaP tumors expressed higher levels of AR and PSA proteins as assessed by IHC. At 7 weeks after castration the tumor sizes increased by 69% in the control group in contrast to 304% in the PMEPA1-shRNA harboring xenografts (P<0.05). In human CaP, decreased PMEPA1 mRNA expression significantly correlated with increased levels of AR transcription target PSA, as a surrogate for elevated AR. Focally increased PSA and AR protein levels were detected in a subset of low PMEPA1 expressing tumors.CONCLUSIONS: PMEPA1 silencing in CaP cells leads to gain of AR, increased tumor growth with potential to induce resistance to AR inhibition targeted therapy. Decreased PMEPA1 in human CaP correlated with increased AR activity. Thus, monitoring PMEPA1 expression in CaP cells offers new opportunities in therapeutic stratification of CaP. Further, restoring PMEPA1 in CaP cells may provide a new strategy for AR degradation focused therapy. Citation Format: Hua Li, Elizabeth Umeda, Yingjie Song, Denise Young, Lakshmi Ravindranath, Ahmed Mohamed, Shashwat Sharad, Gyorgy Petrovics, David McLeod, Isabell Sesterhenn, Taduru Sreenath, Albert Dobi, Shiv Srivastava. Silencing of PMEPA1, a NEDD4 E3 ubiquitin ligase binding protein, stabilizes androgen receptor and confers resistance to AR inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4679. doi:10.1158/1538-7445.AM2015-4679

MP61-10 SILENCING OF PMEPA1 IS ASSOCIATED WITH ACTIVATION OF AR SIGNALING IN HUMAN PROSTATE CANCER AND CASTRATION RESISTANT TUMOR GROWTH IN NUDE MOUSE

You have accessJournal of UrologyProstate Cancer: Basic Research IV1 Apr 2015MP61-10 SILENCING OF PMEPA1 IS ASSOCIATED WITH ACTIVATION OF AR SIGNALING IN HUMAN PROSTATE CANCER AND CASTRATION RESISTANT TUMOR GROWTH IN NUDE MOUSE Hua Li, Elizabeth Umeda, Yingjie Song, Denise Young, Lakshmi Ravindranath, Ahmed A. Mohamed, Yongmei Chen, Shashwat Sharad, Gyorgy Petrovics, David G. McLeod, Isabell Sesterhenn, Taduru Sreenath, Albert Dobi, and Shiv Srivastava Hua LiHua Li More articles by this author , Elizabeth UmedaElizabeth Umeda More articles by this author , Yingjie SongYingjie Song More articles by this author , Denise YoungDenise Young More articles by this author , Lakshmi RavindranathLakshmi Ravindranath More articles by this author , Ahmed A. MohamedAhmed A. Mohamed More articles by this author , Yongmei ChenYongmei Chen More articles by this author , Shashwat SharadShashwat Sharad More articles by this author , Gyorgy PetrovicsGyorgy Petrovics More articles by this author , David G. McLeodDavid G. McLeod More articles by this author , Isabell SesterhennIsabell Sesterhenn More articles by this author , Taduru SreenathTaduru Sreenath More articles by this author , Albert DobiAlbert Dobi More articles by this author , and Shiv SrivastavaShiv Srivastava More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2191AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The androgen receptor (AR) dysfunctions contribute to prostate cancer (CaP) development and progression. Earlier we defined the regulation of AR protein levels by a negative feed-back loop between AR and the androgen regulated PMEPA1, a NEDD4 E3 ubiquitin ligase binding protein. Our data also revealed decreased or absent PMEPA1 transcripts in 65% prostate tumors. This study focuses on the relationship of PMEPA1 protein with AR and its transcriptional targets (PSA) in human CaP, as well as the impact of PMEPA1 silencing on growth of PMEPA1-shRNA LNCaP cells derived tumors in naïve and castrated nude mice. METHODS Ten weeks old athymic Ncr-nu/nu mice were evaluated for the growth of tumor xenografts derived from PMPEA1-shRNA LNCaP (N=20) and control-shRNA LNCaP cells (N=20). Each mouse received 4×106 cells by subcutaneous injection to right flank side. The mice with tumors were castrated at 9th week post-injection. The tumor sizes were monitored twice per week until 16th week post-injection. The xenograft tissues were assayed by immunohistochemical (IHC) staining for PMEPA1, AR and PSA protein levels. The AR, PMEPA1 and PSA expressions in whole mount human prostate specimens were evaluated by IHC staining and QRT-PCR. RESULTS The PMEPA1 shRNA-LNCaP and control shRNA-LNCaP cells started to form subcutaneous tumors at 4 weeks after injections. However, the growth rate of PMEPA1 shRNA-LNCaP derived tumors was significantly faster than control derived tumors (P<0.05). PMEPA1 shRNA-LNCaP tumors expressed higher levels of AR and PSA proteins as assessed by IHC. At 7 weeks after castration the tumor sizes increased by 69% in the control group in contrast to 304% in the PMEPA1-shRNA harboring xenografts (P<0.05). In human CaP, decreased PMEPA1 mRNA expression significantly correlated with increased levels of AR transcription target PSA, as a surrogate for elevated AR. Focally increased PSA and AR protein levels were detected in a subset of low PMEPA1 expressing tumors. CONCLUSIONS PMEPA1 silencing in CaP cells leads to gain of AR, increased tumor growth with potential to induce resistance to AR inhibition targeted therapy. Decreased PMEPA1 in human CaP correlated with increased AR activity. Thus, monitoring PMEPA1 expression in CaP cells offers new opportunities in therapeutic stratification of CaP. Further, restoring PMEPA1 in CaP cells provide a new strategy for AR degradation focused therapy. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e750 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hua Li More articles by this author Elizabeth Umeda More articles by this author Yingjie Song More articles by this author Denise Young More articles by this author Lakshmi Ravindranath More articles by this author Ahmed A. Mohamed More articles by this author Yongmei Chen More articles by this author Shashwat Sharad More articles by this author Gyorgy Petrovics More articles by this author David G. McLeod More articles by this author Isabell Sesterhenn More articles by this author Taduru Sreenath More articles by this author Albert Dobi More articles by this author Shiv Srivastava More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Increased androgen receptor activity and cell proliferation in aromatase inhibitor-resistant breast carcinoma

Aromatase inhibitors (AI) are commonly used to treat postmenopausal estrogen-receptor (ER)-positive breast carcinoma. However, resistance to AI is sometimes acquired, and the molecular mechanisms underlying such resistance are largely unclear. Recent studies suggest that AI treatment increases androgen activity during estrogen deprivation in breast carcinoma, but the role of the androgen receptor (AR) in breast carcinoma is still a matter of controversy. The purpose of this study is to examine the potential correlation between the AR- and AI-resistant breast carcinoma. To this end, we performed immunohistochemical analysis of 21 pairs of primary breast carcinoma and corresponding AI-resistant recurrent tissue samples and established two stable variant cell lines from ER-positive T-47D breast carcinoma cell line as AI-resistance models and used them in in vitro experiments. Immunohistochemical analysis demonstrated that the expression of prostate-specific antigen (PSA) and Ki-67 were significantly higher and ER and progesterone receptor (PR) were lower in recurrent lesions compared to the corresponding primary lesions. Variant cell lines overexpressed AR and PSA and exhibited neither growth response to estrogen nor expression of ER. Androgen markedly induced the proliferation of these cell lines. In addition, the expression profile of androgen-induced genes was markedly different between variant and parental cell lines as determined by microarray analysis.These results suggest that in some cases of ER-positive breast carcinoma, tumor cells possibly change from ER-dependent to AR-dependent, rendering them resistant to AI. AR inhibitors may thus be effective in a selected group of patients.

Transcriptomic analysis reveals inhibition of androgen receptor activity by AMPK in prostate cancer cells.

Metabolic alterations contribute to prostate cancer development and progression; however, the role of the central metabolic regulator AMP-activated protein kinase (AMPK) remains controversial. The androgen receptor (AR), a key driver of prostate cancer, regulates prostate cancer cell metabolism by driving the expression of a network of metabolic genes and activates AMPK through increasing the expression of one of its upstream kinases. To more clearly define the role of AMPK in prostate cancer, we performed expression profiling following pharmacologic activation of this kinase. We found that genes down-regulated upon AMPK activation were over-expressed in prostate cancer, consistent with a tumour suppressive function of AMPK. Strikingly, we identified the AR as one of the most significantly enriched transcription factors mediating gene expression changes downstream of AMPK signalling in prostate cancer cells. Activation of AMPK inhibited AR transcriptional activity and reduced androgen-dependent expression of known AR target genes. Conversely, knock-down of AMPK increased AR activity. Modulation of AR expression could not explain these effects. Instead, we observed that activation of AMPK reduced nuclear localisation of the AR. We thus propose the presence of a negative feedback loop in prostate cancer cells whereby AR activates AMPK and AMPK feeds back to limit AR-driven transcription.

18F-16β-fluoro-5α-dihydrotestosterone (FDHT) PET as a prognostic biomarker for survival in patients with metastatic castrate-resistant prostate cancer (mCRPC).

4517 Background: At present there is no imaging biomarker for patients with mCRPC as the disease primarily metastasizes to bone, which is difficult to visualize and quantify using standard techniques. We have conducted clinical trials in which patients with progressive mCRPC are scanned using FDG-PET to examine glucose metabolism, and FDHT-PET. FDHT is a novel tracer and structural analog of dihydrotestosterone that binds to the androgen receptor (AR) to demonstrate AR overexpression, a key biologic feature of mCRPC. We previously reported an association between baseline FDG-PET and overall survival in mCRPC. We now present the prognostic utility of baseline FDHT-PET. Methods: We prospectively scanned mCRPC patients with FDG and FDHT-PET prior to a change in therapy as part of imaging clinical trials. PET results were represented as the hottest lesion (SUVmax), or average of the five hottest lesions (SUVmaxavg). Five lesions per patient were recorded. If less than 5 lesions were captured, a value of 1 was imputed for the remaining lesions. Clinical and lab parameters were collected. Univariate analysis was performed on PET, clinical and lab variables for association with overall survival (OS). Multivariate models of prognostic factors were constructed. Results: 170 mCRPC patients were imaged with FDG and 116 also had FDHT-PET at baseline. Median survival was 21.2 months (95%CI: 19.1-24.0). On univariate analysis, FDHTmax, FDHTmaxavg, FDGmaxavg, PSA, hemoglobin, alkaline phosphatase, and LDH were associated with OS. FDHTmax and FDHTmaxavg were significantly correlated with one another. In a multivariate model, FDHTmaxavg and LDH were prognostic of survival. In a separate model assessing FDG, FDGmaxavg and LDH were also prognostic. Conclusions: FDHT not only allows for a visual assessment of the AR axis in prostate cancer, but appears to associate with overall survival in mCRPC. Increased AR activity as measured by FDHT SUVmaxavg held greater prognostic value than PSA or Gleason score. FDHT is also being evaluated as a clinical response indicator and pharmacodynamic measure with AR directed therapies.

Role of androgen receptor CAG repeat polymorphism length in hypothalamic progesterone sensitivity in hyperandrogenic adolescent girls

Polycystic ovary syndrome (PCOS) is characterized by oligo/anovulation, hyperandrogenism, and in many cases, polycystic ovaries. Women with PCOS often exhibit persistently increased gonadotropin-releasing hormone (GnRH) pulse frequency compared to normal cycling women, contributing to enhanced luteinizing hormone (LH) secretion and increased androgen production [1]. Administration of progesterone (P) decreases GnRH pulse frequency in normal women, but the GnRH pulse generator in women with PCOS is relatively insensitive to P negative feedback [2]. Treatment with the androgen-receptor antagonist flutamide restores GnRH pulse generator sensitivity to P inhibition in women with PCOS [3]. Hyperandrogenemia in adolescence can represent a forerunner of adult PCOS, though the etiology of PCOS remains unclear. As a group, adolescent girls with HA display impaired GnRH pulse generator sensitivity to progesterone inhibition (i.e., reduced P-sensitivity). However, P-sensitivity is varied in HA girls, despite comparable levels of plasma free testosterone (T) [4] . The androgen receptor (AR) gene contains a polymorphic trinucleotide (CAG) repeat sequence located in the N-terminal transactivation domain of the AR [5] , and the number of CAG repeats influences AR activity. In vitro, expression of AR alleles with shorter CAG repeat lengths is associated with increased AR activity [6]. We measured AR CAG repeat length in a subgroup of subjects previously shown to have varied GnRH pulse generator sensitivity to P-mediated slowing [4] , hypothesizing that shorter AR CAG repeat length would be associated with reduced P-sensitivity in HA girls.

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.

Cyclin D1 Is a Selective Modifier of Androgen-dependent Signaling and Androgen Receptor Function

D-type cyclins regulate cellular outcomes in part through cyclin-dependent, kinase-independent mechanisms that modify transcription factor action, and recent in vivo studies showed that cyclin D1 associates with a large number of transcriptional regulators in cells of the retina and breast. Given the frequency of cyclin D1 alterations in cancer, it is imperative to delineate the molecular mechanisms by which cyclin D1 controls key transcription factor networks in human disease. Prostate cancer was used as a paradigm because this tumor type is reliant at all stages of the disease on androgen receptor (AR) signaling, and cyclin D1 has been shown to negatively modulate AR-dependent expression of prostate-specific antigen (KLK3/PSA). Strategies were employed to control cyclin D1 expression under conditions of hormone depletion, and the effect of cyclin D1 on subsequent androgen-dependent gene expression was determined using unbiased gene expression profiling. Modulating cyclin D1 conferred widespread effects on androgen signaling and revealed cyclin D1 to be a selective effector of hormone action. A subset of androgen-induced target genes, known to be directly regulated by AR, was strongly suppressed by cyclin D1. Analyses of AR occupancy at target gene regulatory loci of clinical relevance demonstrated that cyclin D1 limits AR residence after hormone stimulation. Together, these findings reveal a new function for cyclin D1 in controlling hormone-dependent transcriptional outcomes and demonstrate a pervasive role for cyclin D1 in regulating transcription factor dynamics.

Abstract 1702: Androgen receptor antagonist bicalutamide sensitizes androgen-independent prostate cancer cells to mTOR inhibitor rapamycin by regulating phosphorylation of translation initiation suppressor eIF4E-binding protein 1 (4E-BP1)

Abstract We previously showed that resistance of prostate cancer cells to the mTOR inhibitor rapamycin results from its ability to induce androgen receptor (AR) transcriptional activity, whereas co-treatment with the anti-androgen bicalutamide prevented this effect. Based on our data, collaborators at UC Davis Cancer Center are conducting a clinical trial to test the efficacy of this combination in patients with advanced prostate cancer. However, the mechanism by which the combination of rapamycin and bicalutamide work remains unknown. Investigation of the cause for resistance of prostate cancer cells to rapamycin showed that rapamycin-induced growth inhibition as well as AR transcriptional activity is mediated primarily by p70S6 kinase (S6K1), a downstream target of mTOR, but not by the 4E-BP1/eIF4E/eIF4G pathway, another downstream target of mTOR. Additionally, 4E-BP1 phosphorylation at Ser 65 was dose dependently inhibited by rapamycin in androgen-dependent LNCaP, but not in androgen-independent C4-2 cells, although rapamycin equally affected S6K1phosphorylation in both cells. Comparison of AR expression and 4E-BP1 phosphorylation in various prostate cancer cell lines demonstrated that those expressing high levels of the full-length AR also had increased 4E-BP1 phosphorylation, whereas no such relationship existed with S6K1. Hence, we investigated whether increased AR activity contributed to resistance of C4-2 cells to rapamycin-induced 4E-BP1 dephosphorylation. Bicalutamide treatment as well as culture in medium containing decreased levels of androgens repressed 4E-BP1 phosphorylation, showing that the AR is a positive regulator of 4E-BP1 phosphorylation. The combination of bicalutamide and rapamycin, but not rapamycin alone, inhibited 4E-BP1 phosphorylation in C4-2 cells. Additionally, bicalutamide sensitized PC-3 cells stably transfected with the AR, but not wild-type PC-3 cells that are AR null, to rapamyin-induced 4E-BP1 dephosphorylation. We also compared the effect of bicalutamide in two cell lines derived from CWR22 relapsed tumors: in CWR22R1 cell, which expressed significant levels of the full-length AR, 4E-BP1 phosphorylation was inhibited by bicalutamide, whereas in CWR22Rv1 cells, where the AR lack the ligand-binding domain (LBD), bicalutamide was unable to affect 4E-BP1 phosphorylation. Thus, in cells where bicalutamide is able to bind the AR, this drug sensitizes the cells to the effects of rapamycin. These results were verified in a CWR22 mouse xenograft model of prostate cancer progression. Taken together, our data show that rapamycin treatment increased AR activity by inhibiting S6K1, and that this increase in AR activity in turn induced resistance to rapamycin's ability to repress 4E-BP1 phosphorylation. 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 1702.

Manipulating prohibitin levels provides evidence for an in vivo role in androgen regulation of prostate tumours

Current hormonal therapies for prostate cancer are effective initially, but inevitably tumours progress to an advanced, metastatic stage, often referred to as ‘androgen independent’. However, the androgen receptor (AR) signalling pathway is still key for their growth. It is speculated that tumours escape hormonal control via reduction in corepressor proteins. Manipulating such proteins is thus a potential therapeutic strategy to halt or even reverse tumour progression. We aimed to elucidate the effects of altering levels of the AR corepressor and androgen-target protein prohibitin (PHB) on prostate tumour growth. Prostate cancer cells incorporating an integrated androgen-responsive reporter gene and stably expressing vectors to inducibly overexpress or knockdown PHB were generated and used to assess effects on androgen signalling (by real time imaging) and tumour growth both in culture and in vivo. PHB overexpression inhibited AR activity and prostate-specific antigen (PSA) expression as well as androgen-dependent growth of cells, inducing rapid accumulation in G0/G1. Conversely, reduction in PHB increased AR activity, PSA expression, androgen-mediated growth and S-phase entry. In vivo, doxycycline-induced PHB regulation resulted in marked changes in AR activity, and showed significant effects upon tumour growth. Overexpression led to tumour growth arrest and protection from hormonal starvation, whereas RNAi knockdown resulted in accelerated tumour growth, even in castrated mice. This study provides proof of principle that i) reduction in PHB promotes both androgen-dependent and ‘androgen-independent’ tumour growth, and ii) altering AR activity via increasing levels or activity of corepressors is a valid therapeutic strategy for advanced prostate cancer.