Androgen Receptor Knockdown Research Articles

Male hormones activate EphA2 to facilitate Kaposi's sarcoma-associated herpesvirus infection: Implications for gender disparity in Kaposi's sarcoma.

There is increasing consensus that males are more vulnerable than females to infection by several pathogens. However, the underlying mechanism needs further investigation. Here, it was showed that knockdown of androgen receptor (AR) expression or pre-treatment with 5α-dihydrotestosterone, the AR agonist, led to a considerably dysregulated Kaposi’s sarcoma-associated herpesvirus (KSHV) infection. In endothelial cells, membrane-localized AR promoted the endocytosis and nuclear trafficking of KSHV. The AR interacted with ephrin receptor A2 (EphA2) and increased its phosphorylation at residue Ser897, which was specifically upregulated upon KSHV infection. This phosphorylation resulted from the AR-mediated recruitment of Src, which resulted in the activation of p90 ribosomal S6 kinase 1 (RSK1), which directly phosphorylates EphA2 at Ser897. Finally, the EphA2-mediated entry of KSHV was abolished in a Ser897Asn EphA2 mutant. Taken together, membrane-localized AR was identified as a KSHV entry factor that cooperatively activates Src/RSK1/EphA2 signaling, which subsequently promotes KSHV infection of both endothelial and epithelial cells.

Abstract 818: Unravelling the role of androgen targeted therapies in epithelial plasticity during prostate cancer progression

Abstract Recent evidence suggests that the use of potent androgen receptor (AR) antagonists have contributed to the increased prevalence of prostate cancer (PCa) tumors exhibiting therapy resistance. Androgen targeted therapies (ATT) inhibit the androgen/AR axis which PCa cells rely on for growth. ATT are initially effective however most men progress to metastatic castration resistant prostate cancer (mCRPC). Mechanisms of resistance include programs of epithelial plasticity (EP) such as epithelial-to-mesenchymal transition (EMT) and neuroendocrine transdifferentiation (NEtD). EMT enables PCa cells to become more motile and invasive. PCa cells that undergo NEtD gain neuroendocrine (NE)-like traits. Patients with NE prostate cancer (NEPC) usually present with visceral metastasis, are refractory to ATT and have short response to chemotherapies. We aim to characterize models of therapy-induced NEtD (tNEPC) and to investigate the role of tumor plasticity in the progression to mCRPC. To recapitulate ATT in vitro, androgen-dependent LNCaP cells were treated with either enzalutamide (Enz) (10μM) or shRNA targeting AR in culture medium containing fetal bovine serum (FBS) or charcoal stripped serum (CSS) with or without 10nM dihydrotestosterone (DHT). To model EMT, LNCaP cells were engineered to express doxycycline (Dox) inducible overexpression of EMT transcription factor Snail. Androgen deprivation in vitro induced expression of NE markers in LNCaP cells compared with FBS treated cells (mean fold increase ± SEM): neuron specific enolase (ENO2) (6.0 ± 2.5), neural cell adhesion molecule (NCAM) (10.4 ± 6.8) and midkine (MDK) (13.2 ± 2.4). Cells co-treated with Enz and DHT had increased expression of NE markers compared to DHT alone: ENO2 (2.7 ± 0.6), NCAM (2.9 ± 0.3) and MDK (2.3 ± 1.8). AR knockdown resulted in a potent induction of NE markers compared with control cells: ENO2 (8.3 ± 1.4), NCAM (8.3 ± 2.4) and MDK (9.0 ± 2.2). Dox-induced Snail enhanced androgen deprivation-mediated upregulation of NE markers compared with no-Dox cells: ENO2 (1.3 ± 0.1), NCAM (8.3 ± 2.4) and MDK (9.0 ± 2.2). To more accurately model a preclinical setting, LNCaP tumors were grown subcutaneously in severe combined immune deficiency mice. Once tumors grew to 200mm3, mice were castrated and upon tumor recurrence mice were treated with Enz (10mg/kg). At the ethical endpoint, expression of EMT and NE markers will be assessed. Patients treated with ATTs show an increase in NE-like traits. Thus, there is a need to identify molecular mechanisms involved in this adaptive response. In vitro, we have modelled the acquisition of NE-like features in PCa cells using ATTs, EMT drivers and AR knockdown. Further investigation using these models may lead to the development of biomarkers and therapeutic targets for the clinical management of advanced PCa. Citation Format: Ellca Ratther, Katrina G. Sweeney, Nataly Stylianou, Qiuhua Hu, Gregor Tevz, Colleen C. Nelson, Elizabeth D. Williams, Brett G. Hollier. Unravelling the role of androgen targeted therapies in epithelial plasticity during prostate cancer progression [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 818. doi:10.1158/1538-7445.AM2017-818

Abstract 5220: Chronic exposure to a novel AR-NTD inhibitor induces resistance via a selective metabolism pathway

Abstract Background: The androgen receptor (AR) is recognized as playing a crucial role in prostate cancer (PCa) maintenance and progression; therefore its inhibition has been the cornerstone of modern therapy for men who fail primary treatment. Current treatments are initially effective, however resistance ultimately develops and the disease progresses to a lethal form termed castration-resistant prostate cancer (CRPC). Our lab has discovered a new class of molecules (EPI) which inhibit the AR by binding to the N-terminal domain (NTD). The NTD interacts with transcriptional machinery, and its presence is vital for a transcriptionally functional receptor. We have previously shown that EPI-002 specifically inhibits both full-length AR and constitutively active AR splice variants. The efficacy of EPI-506, the prodrug of EPI-002, is currently being tested in a Phase I/II clinical trial for CRPC. Here we propose novel resistance mechanisms arising from sustained AR-NTD inhibition, with the goal of preemptively developing backup compounds. Methods: The androgen sensitive human PCa cell line LNCaP was used in all experiments. A resistant cell line (LNCaP-EPIR) was generated by passaging parental LNCaP cells weekly in media supplemented with EPI-002 beginning in September 2012. In vitro and in vivo studies using LNCaP and LNCaP-EPIR cells were employed to confirm biological resistance. A human affymetrix microarray identified possible resistance mechanisms and was validated using qRT-PCR, western blot and functional studies. LNCaP and LNCaP-EPIR cells were challenged with EPI-045, an EPI-analog predicted to remain effective in the context of EPI-002 resistance. Results: LNCaP-EPIR cells treated with 25 µM EPI-002 displayed similar growth rates to vehicle treatment, both in vitro and in vivo. Conversely, parental LNCaP cells showed significant growth inhibition in response to EPI-002. LNCaP-EPIR cells retained sensitivity to anti-androgens and AR knock-down by targeted siRNA, implying functional AR remains essential for growth. qRT-PCR data demonstrated that EPI-002 had reduced ability to block AR mediated gene transcription. Interrogation of microarray data revealed candidate genes (UGT2B family) which were specifically upregulated in the resistant line, and may function to metabolize EPI-002. Supporting this hypothesis EPI-045 which is predicted to be resistant to UGT2B metabolism, was able to significantly inhibit LNCaP-EPIR proliferation and AR transcriptional activity. Conclusions: Taken together these data suggest an EPI-specific mechanism of resistance, whereby EPI-002 is preferentially metabolized and removed from the cell. LNCaP-EPIR cells remain dependent upon AR signalling, and are sensitive to anti-androgens used clinically as well as a novel EPI-analog. This work highlights the potential for combination or sequential therapy in the context of drug-resistant CRPC. Citation Format: Jonathon K. Obst, Jun Wang, David Williams, Amy H. Tien, Nasrin R. Mawji, Yu Chi Yang, Raymond J. Andersen, Marianne D. Sadar. Chronic exposure to a novel AR-NTD inhibitor induces resistance via a selective metabolism pathway [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 5220. doi:10.1158/1538-7445.AM2017-5220

MP83-06 COMBINATION THERAPY WITH EPI-002 AND PARP INHIBITOR FOR CASTRATION-RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II1 Apr 2017MP83-06 COMBINATION THERAPY WITH EPI-002 AND PARP INHIBITOR FOR CASTRATION-RESISTANT PROSTATE CANCER Yusuke Ito, C. Adriana Banuelos, and Marianne D. Sadar Yusuke ItoYusuke Ito More articles by this author , C. Adriana BanuelosC. Adriana Banuelos More articles by this author , and Marianne D. SadarMarianne D. Sadar More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2574AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Following the results of the TOPARP-A Phase II trial, Olaparib, an oral PARP inhibitor was recently recognized as a FDA breakthrough therapy for metastatic castration recurrent prostate cancer (mCRPC) patients who have germline mutations in DNA repair genes. Although these results are noteworthy, the problem remains of how to treat the remaining mCRPC patients who do not have detectable germline mutations of DNA repair genes. Androgen receptor (AR) signaling regulates DNA repair in prostate cancer and AR modulating drugs induce DNA damage. Thus a combination approach using AR modulating drugs with a PARP inhibitor could be a promising option for the treatment of mCRPC. All currently approved AR modulating drugs, such as enzalutamide and abiraterone, either directly or indirectly target the AR C-terminus ligand-binding domain (LBD). Such drugs are often unsuccessful due to the emergence of AR splice variants (ARV-7, ARv567es) that are constitutively active and lack a LBD. EPI-002 is a first-in-class AR antagonist that targets both full-length AR and AR splice variants. Here we present data to support that a combination of Olaparib, a PARP inhibitor, and EPI-002 have beneficial effects in vitro. METHODS Combination therapy using EPI-002 and Olaparib were evaluated in vitro using human prostate cancer cells, LNCaP (androgen sensitive and expresses full-length AR) and LNCaP95, an androgen-independent cell line that expresses full-length AR and AR-V7 and is resistant to enzalutamide. The effects of monotherapy and combination therapy on cell cycle and DNA damage were analysed using FACS and Western blot. RESULTS Unexpectedly, EPI-002 caused an enormous decrease of Checkpoint kinase 1 (Chk1) protein levels in LNCaP and LNCaP95 cells. Chk1 is one of the important mediators in cell cycle checkpoint during the DNA damage response. Whereas, enzalutamide also decreased the expression of Chk1 in LNCaP, it had no effect on Chk1 levels in LNCaP95 cells. Consistent with these data, AR knockdown in LNCaP cells also decreased Chk1 levels. The PARP inhibitor, Olaparib, induced phosphorylation of Chk1. EPI-002 induced G1 cell cycle arrest whereas Olaparib induced G2/M cell cycle arrest. FACS analysis of γH2AX showed increased DNA damage with combination therapy compared to monotherapies. CONCLUSIONS EPI-002 decreased the expression of Chk1 in prostate cancer cells that expressed both full-length AR and AR-V7. Combination therapy of EPI-002 plus Olaparib may provide a therapeutic approach for prostate cancer that expresses AR-V7. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1108 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Yusuke Ito More articles by this author C. Adriana Banuelos More articles by this author Marianne D. Sadar More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Androgens and androgen receptor signaling contribute to ovarian development in the chicken embryo

Androgens and androgen receptor (AR) signaling play important roles throughout development. In the chicken, AR signaling is involved in reproduction; however, its specific role is unclear. We show that AR signaling is involved in the normal development of the female embryonic gonads. The AR mRNA level was detected in male and female embryonic gonads by quantitative RT-PCR, and its expression was higher in females than in males at all developmental stages examined. In female embryos, the AR localized to nuclei of cells in the left gonad. Although AR expression was low in the majority of the medulla, high expression was detected in cells of lacunae within the medulla. In addition, AR expression increased in cells of cortical cords within the cortex with the progression of development. AR expression in the right gonad was lower than that in left gonad throughout development. In the male gonad, the AR localized to the cytoplasm of cells in seminiferous tubules at all stages. Female AR knockdown (ARKD) embryos infected with a retrovirus expressing micro RNAs targeting the AR showed normal asymmetric gonads (development of the left and depression of the right gonads), whereas the number of lacunae decreased. Furthermore, there was a disruption in the structure of cortical cords. By contrast, the gonads of ARKD males developed normally during embryogenesis. These results indicate that androgens and AR signaling are essential for the development of lacunae and cortical cords in gonads of female embryos.

The Androgen Receptor Promotes Cellular Proliferation by Suppression of G-Protein Coupled Estrogen Receptor Signaling in Triple-Negative Breast Cancer

Background/Aims: The targeted therapy for triple-negative breast cancer (TNBC) is still challenging due to poor understanding on its molecular etiology. The androgen receptor (AR) has recently emerged as a prognostic and treatment-predictive marker in breast cancer. However, the role of AR in TNBC remained elusive. Methods: Immunohistochemistry (IHC) was used to detect AR and G-protein coupled estrogen receptor (GPER) expression in tissue microarrays of 165 TNBC patients. Microarray analysis of mRNAs was performed to identify downstream regulators of AR. TNBC cells were cultured with dihydrotestosterone (DHT) alone or in combination with AR knockdown performed with AR shRNA. Cell viability and colony formation were assessed. Western blotting and qRT-PCR were used to examine protein and mRNA expression, respectively. The potential mechanism of AR-mediated GPER suppression was identified by Chromatin immunoprecipitation (ChIP) assay. AR and GPER expressions were also assessed in nude mouse xenografts by IHC. Results: IHC staining showed that the expression of AR was positively associated with tumor size, lymph node metastasis and high-grade tumor in TNBC patients. AR activation triggered by DHT suppressed GPER expression, to promote cell growth of TNBC. G-1, a GPER agonist, inhibited DHT-stimulated proliferation. Further experiments illustrated that AR suppressed GPER activation via binding directly to the promoter of GPER. Moreover, a negative correlation between AR and GPER was observed in MDA-MB-231 tumor cell xenografts and TNBC patient samples. Conclusions: The suppression of GPER via AR may be involved in the positive actions towards the TNBC progression, making it a promising therapeutic target for TNBC treatment.

Up-Regulation of Follistatin-Like 1 By the Androgen Receptor and Melanoma Antigen-A11 in Prostate Cancer.

High affinity androgen binding to the androgen receptor (AR) activates genes required for male sex differentiation and promotes the development and progression of prostate cancer. Human AR transcriptional activity involves interactions with coregulatory proteins that include primate-specific melanoma antigen-A11 (MAGE-A11), a coactivator that increases AR transcriptional activity during prostate cancer progression to castration-resistant/recurrent prostate cancer (CRPC). Microarray analysis and quantitative RT-PCR were performed to identify androgen-regulated MAGE-A11-dependent genes in LAPC-4 prostate cancer cells after lentivirus shRNA knockdown of MAGE-A11. Chromatin immunoprecipitation was used to assess androgen-dependent AR recruitment, and immunocytochemistry to localize an androgen-dependent protein in prostate cancer cells and tissue and in the CWR22 human prostate cancer xenograft. Microarray analysis of androgen-treated LAPC-4 prostate cancer cells indicated follistatin-like 1 (FSTL1) is up-regulated by MAGE-A11. Androgen-dependent up-regulation of FSTL1 was inhibited in LAPC-4 cells by lentivirus shRNA knockdown of AR or MAGE-A11. Chromatin immunoprecipitation demonstrated AR recruitment to intron 10 of the FSTL1 gene that contains a classical consensus androgen response element. Increased levels of FSTL1 protein in LAPC-4 cells correlated with higher levels of MAGE-A11 relative to other prostate cancer cells. FSTL1 mRNA levels increased in CRPC and castration-recurrent CWR22 xenografts in association with predominantly nuclear FSTL1. Increased nuclear localization of FSTL1 in prostate cancer was suggested by predominantly cytoplasmic FSTL1 in benign prostate epithelial cells and predominantly nuclear FSTL1 in epithelial cells in CRPC tissue and the castration-recurrent CWR22 xenograft. AR expression studies showed nuclear colocalization of AR and endogenous FSTL1 in response to androgen. AR and MAGE-A11 cooperate in the up-regulation of FSTL1 to promote growth and progression of CRPC. Prostate 77:505-516, 2017. © 2016 Wiley Periodicals, Inc.

Downregulation of miR-17-92a cluster promotes autophagy induction in response to celastrol treatment in prostate cancer cells

Celastrol has potential application for the treatment of prostate cancer. However it causes autophagy as a protective response in prostate and other types of cancers, thus unveiling the underlying mechanisms may benefit its future application. In the present study, we demonstrate that the miR-17-92a cluster plays a negative role in celastrol induced-autophagy. Dissection of miR-17-92a cluster revealed the role of miR-17 seed family (miR-20a and miR-17) in autophagy inhibition in the context of prostate cancer cells. Autophagy-related gene ATG7 was validated as a target of miR-17 seed family by dual-luciferase assay and qPCR. Celastrol induced autophagy was inhibited by miR-20a or miR-17, while the inhibitory effects were rescued in the presence of pcDNA-ATG7 lacking 3′ UTR, demonstrating that these two members target ATG7 to inhibit celastrol-induced autophagy. As celastrol degrades androgen receptor (AR), a key transcription factor in prostate cancer cells, we further investigated whether AR affected miR-17-92a expression in prostate cancer cells. AR binding sites were found in the promoter and two introns of miR-17-92a. In addition, higher expression levels of miR-17-92a were observed in AR positive cells compared with AR negative cells. Ectopic expression of AR could enhance the expression of miR-17-92a cluster in AR-negative prostate cancer cells while knockdown of AR decreased miR-17-92a expression in AR-positive cells, demonstrating the regulation of AR on miR-17-92a transcription. In summary, our results demonstrate that celastrol downregulates AR and its target miR-17-92a, leading to autophagy induction in prostate cancer cells.

Androgen-induced Long Noncoding RNA (lncRNA) SOCS2-AS1 Promotes Cell Growth and Inhibits Apoptosis in Prostate Cancer Cells

Long noncoding RNAs (lncRNA) have been associated with the development of cancer. However, the interplay between lncRNAs and androgen receptor (AR) signaling in prostate cancer is still unclear. Here, we identified lncRNAs induced by androgen in AR-positive prostate cancer cells, where induction was abolished by AR knockdown as well as an anti-androgen, bicalutamide. By combining these data, we identified an androgen-regulated lncRNA, suppressor of cytokine signaling 2-antisense transcript 1 (SOCS2-AS1), the expression of which was higher in castration-resistant prostate cancer model cells, i.e long-term androgen-deprived (LTAD) cells, than in parental androgen-dependent LNCaP cells. SOCS2-AS1 promoted castration-resistant and androgen-dependent cell growth. We found that SOCS2-AS1 knockdown up-regulated genes related to the apoptosis pathway, including tumor necrosis factor superfamily 10 (TNFSF10), and sensitized prostate cancer cells to docetaxel treatment. Moreover, we also demonstrated that SOCS2-AS1 promotes androgen signaling by modulating the epigenetic control for AR target genes including TNFSF10 These findings suggest that SOCS2-AS1 plays an important role in the development of castration-resistant prostate cancer by repressing apoptosis.

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.

Abstract 2898: Elucidating the role of BHLHE40/DEC1/SHARP2/STRA13 in prostate cancer

Abstract Background: Basic-helix-loop-helix proteins (BHLH) are transcription factors that play important roles in critical developmental processes in many organisms by repressing the transcription of various target genes. Previous studies showed that overexpression of the BHLHE40 (also known as DEC1, STRA13 or SHARP2) transcriptional repressor results in growth suppression, cell cycle arrest, and cellular senescence indicating that BHLHE40 may have important tumor suppressor functions. Studies showed that the expression of BHLHE40 is indeed downregulated in some tumors, intriguingly however, it is also overexpressed in many such as lung cancer, breast cancer. Our overall goal is to elucidate the function of BHLHE40 in prostate cancer (CaP) and gain an understanding of how it is regulated. Methods: Initial studies were based on qPCR analysis to compare the expression levels of BHLHE40 in different cell lines ranging from androgen-dependent LNCaP cells to the castration resistant C4, C4-2, C4-2b, R1, Rv1, PC3 and PC3 cells that stably express the androgen receptor (AR) (PC3-AR). Further analysis were done by treating LNCaP and C4-2 cells with 1 nM dihydrotestosterone (DHT) in media containing fetal bovine serum (FBS), or charcoal stripped serum (CSS). Western blot analysis was done to check the level of BHLHE40 protein in these cell lines. Knock-down of AR, or the use of AR inhibitors Enzalutamide and/or Bicalutamide were used to further assess the regulation of BHLHE40 by the AR. Results: qPCR comparison of BHLHE40 transcript levels in LNCaP versus C4, C4-2, R1, Rv1, PC3 cells showed that while LNCaP cells have high transcript levels of BHLHE40, the CRPC cells all had very low transcript levels of this transcription factor. When comparing LNCaP to PC3 and PC3-AR cells, it was interesting to note that PC3-AR cells showed significantly higher transcript levels of BHLHE40 than PC3 implying that BHLHE40 may be regulated by the AR pathway. Treatment with 1 nM DHT on these cells showed that BHLHE40 increases over time following DHT treatment. In contrast, inhibition of AR by Enzalutamide (2 μM) showed reduced BHLHE40 levels, although Bicalutamide (10 μM) did not significantly altered the expression level of BHLHE40 in LNCaP cells. Conclusions: Our results indicate that BHLHE40 is regulated by the AR pathway, although whether this is direct or indirect is yet to be determined. This observation is significant, since it indicates that the AR may be utilizing this transcription factor to suppress the transcription of other genes. Future studies will attempt to determine if BHLHE40 may have an androgen-responsive-element (ARE) in its promoter and via ChIP assay to identify possible binding sites for AR in conjunction with luciferase assay to assess androgen-receptor mediated regulation. Citation Format: Zsofia Kiss, Paramita Ghosh. Elucidating the role of BHLHE40/DEC1/SHARP2/STRA13 in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2898.

MP90-04 A ROLE FOR HISTONE DEMETHYALASE JMJD1A IN THE DNA DAMAGE RESPONSE OF PROSTATE CANCER CELLS

INTRODUCTION AND OBJECTIVES: The major therapies for prostate cancer (PCa) such as radio-, chemoor androgen deprivation therapy induce DNA damage. Thus the DNA damage response (DDR) and expression of DDR genes are key factors in determining outcome of PCa therapies. Histone demethylase JMJD1A regulates the gene expression by demethylating H3K9. Our previous work shows that JMJD1A is essential for the long-term proliferation and survival of PCa cells in vitro or in the xenograft prostate tumor model, partly via its effect on the activities of androgen receptor (AR) and c-Myc transcription factors. Here, we find a new role for JMJD1A in the expression of DDR genes and sensitivity of PCa cells to irradiation and androgen deprivation. METHODS: Real-time PCR was used to measure the mRNA levels of some DDR genes in the PCa cells (control or siRNA knockdown (KD) of JMJD1A, AR or c-Myc). Chromatin immunoprecipitation (ChIP) was used to evaluate the binding of JMJD1A to the AR or MYC binding sites on these DDR genes. JMJD1A-KD PCa cells were treated with or without ionizing radiation (IR), and analyzed by the r-H2AX staining (a widely used marker for the DNA double strand breaks (DSBs)), western blot analysis of DNA damage signaling proteins, flow cytometry analysis of cell cycle, and GFP reporter assays for the DSB repair via the homologous recombination (HR) or non-homologous end joining (NHEJ) pathways. The growth of these PCa cells was also measured under the IR and androgen deprivation conditions. RESULTS: JMJD1A was found to promote the expression of some DDR genes by co-activating AR and/or c-Myc. The DDR genes regulated by JMJD1A encode factors mediating the DSB repair by both HR and NHEJ pathways. JMJD1A KD led to increased r-H2AX foci in the non-irradiated cells, and the delayed clearance of r-H2AX foci in the irradiated cells. JMJD1A KD also increased the levels of phospho-DNAPK or phospho-CHEK2 in the western blot analysis and caused G2 cell cycle arrest. Consistently, JMJD1A KD reduced the percentage of GFPpositive cells that reflects the DSB repair by both HR and NHEJ pathways. JMJD1A-KD cells showed a reduced growth under IR and androgen deprivation conditions. CONCLUSIONS: JMJD1A promotes the DSB repair via regulation of DDR gene expression through AR and/or c-Myc. The role of JMJD1A in the DSB repair may contribute to the resistance of PCa cells to the radioand androgen deprivation therapies.

The Androgen Receptor Regulates PPARγ Expression and Activity in Human Prostate Cancer Cells.

The peroxisome proliferator activated receptor gamma (PPARγ) is a ligand‐activated transcription factor that regulates growth and differentiation within normal prostate and prostate cancers. However the factors that control PPARγ within the prostate cancers have not been characterized. The goal of this study was to examine whether the androgen receptor (AR) regulates PPARγ expression and function within human prostate cancer cells. qRT‐PCR and Western blot analyses revealed nanomolar concentrations of the AR agonist dihydrotestosterone (DHT) decrease PPARγ mRNA and protein within the castration‐resistant, AR‐positive C4‐2 and VCaP human prostate cancer cell lines. The AR antagonists bicalutamide and enzalutamide blocked the ability of DHT to reduce PPARγ levels. In addition, siRNA mediated knockdown of AR increased PPARγ protein levels and ligand‐induced PPARγ transcriptional activity within the C4‐2 cell line. Furthermore, proteasome inhibitors that interfere with AR function increased the level of basal PPARγ and prevented the DHT‐mediated suppression of PPARγ. These data suggest that AR normally functions to suppress PPARγ expression within AR‐positive prostate cancer cells. To determine whether increases in AR protein would influence PPARγ expression and activity, we used lipofectamine‐based transfections to overexpress AR within the AR‐null PC‐3 cells. The addition of AR to PC‐3 cells did not significantly alter PPARγ protein levels. However, the ability of the PPARγ ligand rosiglitazone to induce activation of a PPARγ‐driven luciferase reporter and induce expression of FABP4 was suppressed in AR‐positive PC‐3 cells. Together, these data indicate AR serves as a key modulator of PPARγ expression and function within prostate tumors. J. Cell. Physiol. 231: 2664–2672, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

Androgens downregulate miR-21 expression in breast cancer cells underlining the protective role of androgen receptor.

Although the protective role of androgen receptor (AR) in breast cancer (BC) is well established, the mechanisms involved remains largely unexplored. MicroRNAs play fundamental roles in many biological processes, including tumor cell development and metastasis. Herein, we report that androgens reduce BC cells proliferation acting as a negative modulator of the onco-miRNA-21.The synthetic androgen miboleron (Mib) decreases BC cell proliferation induced by miR-21 over-expression and AR knockdown evidenced the requirement of AR in the down-regulation of miR-21 expression. These effects seem to be a general mechanism occurring in BC tissues.Chromatin immune-precipitation (ChIP) analysis disclosed the binding of AR to a specific ARE sequence in miR-21 proximal promoter and recognizes the recruitment of HDAC3 as component for AR-mediated transcriptional repression. Such event is associated to a significantly reduced PolII binding in Mib treated extracts confirming that activated AR is a transcriptional repressor of miR-21 expression, providing further insight into the protective role of androgens in breast cancer cells.Collectively, our data and the widespread AR expression in primary and metastatic breast tumours, suggest a careful examination of the therapeutic potential of androgens also in potentiating the effectiveness of anti-oestrogen adjuvant therapies.

Correlation of galeterone-induced degradation of the androgen receptor with inhibition of a deubiquitinating enzyme.

345 Background: Galeterone is a highly selective oral small molecule drug candidate that disrupts androgen receptor (AR) signaling. It degrades the AR (IC50 ~1µM), is a potent CYP17 lyase inhibitor ( < 50nM), and possesses AR antagonist activity (~600nM). Galeterone-induced AR degradation was observed in models having either full-length AR or known constitutively active truncated forms of the AR receptor that lack the ligand binding domain (LBD), AR-V7 and AR567es. The LBD of the AR is not required for galeterone-dependent AR degradation. Galeterone-induced degradation activity is blocked by co-administration of the proteasome inhibitor MG132. Furthermore, galeterone-induced degradation of AR can be blocked by selective knock-down of the E3 ligases, Mdm2 and CHIP. Methods: We utilized a series of biochemical and cell-based in vitro studies to further elucidate and characterize additional signaling molecules in the proteasomal dependent mechanism of galeterone-induced AR degradation. Results: We screened a panel of 22 deubiquitinating enzymes (DUBs) in vitro and demonstrated that galeterone inhibited enzymatic activity of the DUB, USP12 with IC50 in the single digit micromolar range. In addition, we used surface plasmon resonance to demonstrate that dose-dependent inhibition of USP12 activity involves direct binding of galeterone to USP12 and USP12/UAF1 complexes with a KD of ≤ 10µM. Conclusions: Interestingly, USP12 is a co-activator of AR and selective knock-down of this DUB has been shown to increase AR degradation. USP12 has been linked to regulation of the phosphatases PHLPPs through ubiquitination. PHLPPs dephosphorylate AKT, providing an important regulatory mechanism for controlling the PI3K/AKT pathway. Since it is known that galeterone induces an increase in pAKT and pMdm2, the latter being a substrate of activated AKT, this suggests that inhibition of USP12 regulates pAKT levels through enhanced degradation of PHLPPs via increased ubiquitination. These data suggest that a differentiating mechanism of galeterone from other AR targeting agents is through inhibition of USP12, leading to enhanced AR degradation.

Autophagic degradation of the androgen receptor mediated by increased phosphorylation of p62 suppresses apoptosis in hypoxia

Prostate cancer grows under hypoxic conditions. Hypoxia decreases androgen receptor (AR) protein levels. However, the molecular mechanism remains unclear. Here, we report that p62-mediated autophagy degrades AR protein and suppresses apoptosis in prostate cancer LNCaP cells in hypoxia. In LNCaP cells, hypoxia decreased AR at the protein level, but not at the mRNA level. Hypoxia-induced AR degradation was inhibited not only by knockdown of LC3, a key component of the autophagy machinery, but also by knockdown of p62. Depletion of p62 enhanced hypoxia-induced poly(ADP-ribose) polymerase cleavage and caspase-3 cleavage, markers of apoptosis, whereas simultaneous knockdown of p62 and AR suppressed hypoxia-induced apoptosis. Hypoxia increased the formation of a cytosolic p62–AR complex and enhanced sequestration of AR from the nucleus. Formation of this complex was promoted by the increased phosphorylation of serine 403 in the ubiquitin-associated domain of p62 during hypoxia. An antioxidant and an AMP-activated protein kinase (AMPK) inhibitor reduced hypoxia-induced p62 phosphorylation at serine 403 and suppressed hypoxia-induced complex formation between AR and p62. These results demonstrate that hypoxia enhances the complex formation between p62 and AR by promoting phosphorylation of p62 at serine 403, probably through activating AMPK, and that p62-mediated autophagy degrades AR protein for cell survival in hypoxia.

Estrogen induces androgen-repressed SOX4 expression to promote progression of prostate cancer cells.

The sex determing region Y-box 4 (SOX4) gene is a critical developmental transcriptional factor that is overexpressed in prostate cancer (PCa). While we and others have investigated the role of SOX4 overexpression in PCa, the molecular mechanism underlying its aberrant expression remains unclear. Immunohistochemistry were utilized to detect SOX4 expression and the correlation between estrogen receptor β (ERβ), androgen receptor (AR) and SOX4 in a cohort of 94 clinical specimens. Real-time quantitative PCR and Western blotting were used to study the transcript and protein expression levels. Immunofluorescence staining and co-immunoprecipitation were performed to assess the interaction and subcellular location of ERβ and AR. Chromatin immunoprecipitation (ChIP) assays and Luciferase reporter assays were performed to explore the binding and transcriptional activities of ERβ and AR to the SOX4 promoter. Cellular function was evaluated by MTS, invasion and wound healing assays. SOX4 expression is up-regulated in Castration-Resistant Prostate Cancer (CRPC) tumors compared to hormone-dependent PCa (HDPC) cases. Increased expression was also observed in PCa cells after long-term androgen-deprivation treatment (ADT). In vitro data indicated that SOX4 is an AR transcriptional target and down-regulated by dihydrotestosterone (DHT) via AR. 17β-estradiol (E2) up-regulates SOX4 expression in the absence of androgen through the formation of a protein complex between ERβ and AR. Knockdown of AR or ERβ blocks the E2-induced SOX4 expression. ChIP assays confirmed that both ERβ and AR bind to the SOX4 promoter in response to E2. Functionally, silencing SOX4 significantly attenuates the proliferative effect, as well as the capacity of migration and invasion of E2 on PCa cells. Clinically, overexpression of SOX4 is significantly associated with ERβ expression in PCa. In addition, this association is still retained in CRPC patients with poor prognosis. These findings suggest that SOX4 is a novel DHT-repressed AR-target gene. E2 could promote proliferation of PCa cells through the up-regulation of SOX4 under androgen-depleted environment. Our data provides a possible molecular basis for the overexpression of SOX4 in CRPC and may facilitate the detection and prevention of the emergence of CRPC.

Abstract P3-05-21: Investigation into the oncogenic potential of the androgen receptor in aromatase inhibitor resistant breast cancer

Abstract The heterogenous nature of breast cancer, exemplified by a wide range of subtype classifications, has been well documented. The identification of the novel molecular apocrine breast tumours (ER-, PR-ve, HER2-ve, AR+), with a gene expression profile reflective of ER+ luminal breast cancer, has led to the proposition of an alternative role for the androgen receptor as a pseudo ER. This proposition was borne out of global expression profiling and AR recruitment of molecular apocrine cells, demonstrating that more than 50% of AR binding to the genome mirrors the pattern of ER binding suggesting that in the absence of ER, AR can mimic ER in its DNA binding capabilities. Aromatase Inhibitor (AI) therapy is the gold standard first line therapy for postmenopausal breast cancer. AIs function by abrogating the activity of the enzyme Cyp 19 (aromatase), which is responsible for converting circulating androgens to estrogen. By preventing this conversion step, this eradicates the main steroid driving breast cancer growth and in turn results in a more androgenic environment. We hypothesise that AR may also act as a pseudo ER in our AI resistant (MCF7-letrozole resistant) cells. Preliminary data demonstrates that AR is recruited to some but not all classical ER target genes as well as some AR target genes in MCF7-LetR cells, therefore highlighting the potential of AR to act as an oncogene in this setting. Analysis of AR protein expression also demonstrates higher levels of AR in AI resistant cells versus sensitive cells. In the evaluation of the responsiveness of a range of breast cancer cell lines to the AR antagonist Bicalutamide, it was noted that our AI resistant cells demonstrated responsiveness to Bicalutamide treatment. RNA sequencing experiments following AR knockdown in AI resistant cells also provide crucial information as to the key genes differentially regulated by AR. Given that resistance to AI therapy is an emerging clinical issue, further elucidation of the potential mechanisms of AI resistance and potential AR targets is highly warranted. Citation Format: Leonie Young, Laura Creevey, Azlena Ali, Arnold DK Hill, Marie McIlroy. Investigation into the oncogenic potential of the androgen receptor in aromatase inhibitor resistant breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-21.

Abstract P3-04-06: Inhibiting androgen receptor nuclear localization decreases estrogen receptor (ER) activity and tumor growth in ER+ breast cancer

Abstract Background: Androgen receptor (AR) is widely expressed in breast tumors, but the role of AR in estrogen receptor (ER)+ tumors is controversial. In the absence of estradiol (E2), dihydrotestosterone (DHT) increases growth of ER+ breast cancer cells in vitro and in vivo. Anti-androgens such as bicalutamide (Bic) and enzalutamide (ENZ) inhibit this DHT-mediated proliferation. Surprisingly we have found that ENZ, which impairs nuclear entry of liganded AR, also inhibits E2-mediated proliferation of ER+ breast cancer cells, while Bic does not. Hypothesis: We hypothesize that nuclear localization of AR is necessary for maximal E2-mediated proliferation in ER+/AR+ breast cancer cells, and targeting AR with ENZ or other agents that impede AR nuclear entry or cause AR degradation will inhibit growth of ER+/AR+ human breast cancer cell lines and decrease tumor burden in preclinical models. Methods: ER+/AR+ MCF7, BCK4, and ZR-75-1 cells were treated with E2 plus or minus ER and AR antagonists and proliferation was measured by crystal violet staining or Incucyte live cell imaging. Nuclear AR was assessed by immunocytochemistry or nuclear/cytoplasmic fractionation. For in vivo experiments, 1x10^6 luciferase-expressing MCF7 cells were injected into the mammary fat pad of nu/nu mice and tumor growth monitored by caliper and IVIS imaging. Results: ENZ blocked E2-induced proliferation and showed synergistic activity with the ER antagonists 4-hydroxy-Tamoxifen (OH-Tam) and Fulvestrant (Fulv) in vitro. E2-induced expression of ER target genes including PR and SDF1 was also inhibited by ENZ, but not by Bic. Similarly, AR knockdown decreased baseline and E2-induced proliferation of MCF7 cells and E2-induced ER target gene expression. Both DHT and E2 treatment induced nuclear translocation of AR, which was decreased by ENZ. Nuclear translocation of AR in response to E2 occurs only in ER+ cell lines, further supporting a role for nuclear AR in E2-induced ER activity. In vivo, ENZ inhibited E2-induced growth of MCF7 tumors as effectively as Tamoxifen (Tam), and the combination of ENZ plus Tam was more effective than either drug alone. ENZ also inhibited growth of Tam-resistant MCF7 cells in vitro. Conclusions: Our results suggest that nuclear localization of AR plays a previously-unrecognized role in E2-mediated ER activity in ER+/AR+ breast cancer cells. Because of its ability to inhibit nuclear entry of liganded AR, ENZ may serve as an effective therapeutic in ER+/AR+ breast cancers. Importantly, ENZ may be particularly useful in combination with current anti-estrogen therapies (Tam or Fulv) since it affects ER, but via an indirect mechanism acting through AR. ENZ may also be effective in tumors resistant to ER-directed therapy based on in vitro data and published clinical data indicating that many such tumors express more AR protein than ER. Funded by: DOD BCRP Clinical Translational Award BC120183 to JKR, American Cancer Society Postdoctoral Fellowship PF-13-314-01 – CDD to NCD. Citation Format: Nicholas C D'Amato, Britta M Jacobsen, Dawn R Cochrane, Nicole S Spoelstra, Beatrice L Babbs, Anthony Elias, Jennifer K Richer. Inhibiting androgen receptor nuclear localization decreases estrogen receptor (ER) activity and tumor growth in ER+ breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-04-06.

Androgen Receptor Silences Thioredoxin‐interacting Protein and Competitively Inhibits Glucocorticoid Receptor‐Mediated Apoptosis in Pancreatic β‐Cells

Androgen receptor (AR) is known to bind to the same cis-element that glucocorticoid receptor (GR) binds to. However, the effects of androgen signaling on glucocorticoid signaling have not yet been elucidated. Here, we investigated the effects of testosterone on dexamethasone (DEX, a synthetic glucocorticoid)-induced apoptosis of pancreatic β-cells, which might be involved in the pathogenesis of type 2 diabetes mellitus in males. We used INS-1 #6 cells, which were isolated from the INS-1 pancreatic β-cell line and which express high levels of AR. Testosterone and dihydrotestosterone inhibited apoptosis induced by DEX in INS-1 #6 cells. AR knockdown and the AR antagonist hydroxyflutamide each diminished the anti-apoptotic effects of testosterone. AR was localized in the nucleus of both INS-1 #6 cells and pancreatic β-cells of male rats. Induction of thioredoxin-interacting protein (TXNIP) is known to cause pro-apoptotic effects in β-cells. Testosterone suppressed the DEX-induced increase of TXNIP at the transcriptional level. A Chromatin immunoprecipitation assays showed that both AR and GR competitively bound to the TXNIP promoter in ligand-dependent manners. Recombinant DNA-binding domain of AR bound to the same cis-element of the TXNIP promoter that GR binds to. Our results show that AR and GR competitively bind to the same cis-element of TXNIP promoter as a silencer and enhancer, respectively. These results indicate that androgen signaling functionally competes with glucocorticoid signaling in pancreatic β-cell apoptosis.