Classical Androgen Receptor Research Articles

Testosterone/bicalutamide antagonism at the predicted extracellular androgen binding site of ZIP9

ZIP9 is a Zn2+ transporter, testosterone receptor, and mediator of signaling events through G-proteins. Despite these pivotal properties, however, its physiological and pathophysiological significance has not yet been comprehensively addressed. Using a cell line that lacks the classical androgen receptor we show that ZIP9-mediated phosphorylation of Erk1/2, CREB, or ATF-1 and expression of claudin-5 and zonula occludens-1 by testosterone can be completely antagonized by bicalutamide (Casodex), an anti-androgen of significant clinical impact.Computational modeling and docking experiments with ZIP9 reveal typical characteristics of ZIP transporters and an extracellular binding site for testosterone capable of accommodating bicalutamide. The presence of this site is verified by our demonstration that the membrane-impermeable testosterone analogue T-BSA-FITC labels the membrane only when ZIP9 is expressed and that this labeling is completely prevented by bicalutamide.The study connects structural features of ZIP9 to its functions and indicates a possible relevance of ZIP9 as a pharmacological target.

Rare genetic variants in the sodium-dependent organic anion transporter SOAT (SLC10A6): Effects on transport function and membrane expression

Sulfo-conjugated steroid hormones, such as dehydroepiandrosterone sulfate (DHEAS), pregnenolone sulfate or estrone-3-sulfate are abundant in the body, but are biologically inactive at classical androgen and estrogen steroid receptors. However, after carrier-mediated import and de-conjugation by the steroid sulfatase, these compounds participate in the overall steroid regulation of reproductive organs. The sodium-dependent organic anion transporter SOAT, coded by the SLC10A6 gene, is specific for the transport of steroid sulfates and is highly expressed in testicular germ cells, including pachytene spermatocytes, secondary spermatocytes, and round spermatids. Therefore, SOAT is supposed to be involved in the regulation of spermatogenesis and male fertility. In the present study, the SLC10A6 gene was analyzed for rare genetic variants, which might affect transport function or membrane expression of SOAT. Among the 31 SOAT variants analyzed, L44P, Q75R, P107L, G109S, S112F, N113K, S133F, G241D, G263E, G294R, and Y308N showed no transport activity for DHEAS at all. In the case of P107L, G241D, G263E, and Y308N, this was most likely due to significantly reduced expression in the plasma membrane. Other variants are located directly at (Q75R, S112F, N113K) or close to (G109S, S133F, and G263E) the supposed SOAT Na+ binding sites and thus could disable the sodium-coupled transport cycle. If these loss-of-function SOAT variants are more frequent in men with impaired spermatogenesis or infertility needs further investigation.

Androgens Regulate Ovarian Gene Expression Through Modulation of Ezh2 Expression and Activity

A substantial amount of evidence suggests that androgen signaling through classical androgen receptors is critical for both normal and pathologic ovarian physiology. Specifically, we and others have shown that, in mouse granulosa cells, androgen actions through both extranuclear and nuclear androgen receptor signaling are critical for normal follicle development and ovulation. Here, we show that androgens through the PI3K/Akt pathway rapidly (within minutes) phosphorylate and inhibit activity of the Polycomb group protein enhancer of zeste homolog 2 (Ezh2). Over the course of 24 to 48 hours, androgens then induce expression of the microRNA miR-101, which targets Ezh2 messenger RNA (mRNA), leading to a nearly complete loss of Ezh2 protein expression. This long-term androgen-induced loss of Ezh2 actions ultimately results in sustained reduction of the H3K27me3-repressive mark in the promoter region of the Runt-related transcription factor-1 (Runx1) gene, a luteinizing hormone (LH)-induced transcription factor essential for ovulation, leading to increased Runx1 mRNA expression. Accordingly, blocking androgen-induced inhibition of Ezh2 in vivo adversely affects LH-induced Runx1 mRNA expression and subsequent ovulation. Importantly, although estrogen treatment of granulosa cells similarly causes rapid activation of the PI3K/Akt pathway and short-term phosphorylation of Ezh2, it does not induce miR-101 expression and thereby does not reduce overall Ezh2 expression, demonstrating the androgen specificity of long-term Ezh2 suppression. Thus, this study provides insight regarding how androgen-induced extranuclear kinase signaling and intranuclear transcription through Ezh2 modifications may influence the expression pattern of genes, ultimately affecting various downstream physiological processes.

Natural extranuclear androgen receptor ligands as endocrine disruptors of cancer cell growth

Even though the term endocrine disruption primarily designates environmental chemicals that can interfere with the action of hormones, in recent years it has been extended to include also plant derived compounds that can reach the human body, naturally, or have been identified and studied as alternative pharmaceutical agents. In fact, for a large number of them, their antihormonal action was appreciated by different traditional herbal medicines. In the present review we report the majority of the plant derived compounds that exhibit an antiandrogenic effect and the known mechanisms of action. These include a disruption at testosterone production level and at the classical androgen receptor triggered pathways, including membrane initiated ones. Finally, for the first time we describe the possible involvement of alternative cell membrane androgen receptor systems and the lipid signaling disruption by natural androgen, providing hints about a novel class of therapeutic involvement of androgens.

The Master Neural Transcription Factor BRN2 Is an Androgen Receptor-Suppressed Driver of Neuroendocrine Differentiation in Prostate Cancer.

Understanding the contribution of the AR to the emergence of highly lethal, drug-resistant NEPC is critical for better implementation of current standard-of-care therapies and novel drug design. Our first-in-field data underscore the consequences of potent AR inhibition in prostate tumors, revealing a novel mechanism of AR-dependent control of neuroendocrine differentiation, and uncover BRN2 as a potential therapeutic target to prevent emergence of NEPC. Cancer Discov; 7(1); 54-71. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1.

Abstract 1824: Context dependent regulatory patterns of the androgen receptor (AR) and androgen receptor target genes

Abstract Background: Expression of the androgen receptor (AR) is associated with androgen-dependent proliferation arrest and terminal differentiation of normal prostate epithelial cells. Additionally, activation of the AR is required for survival of benign luminal epithelial cells and primary cancer cells. Androgen deprivation therapy (ADT) leads to apoptosis in both benign and cancerous tissue. Cancer cell escape from ADT is known as castration-resistant prostate cancer (CRPC). In the course of CRPC development the AR typically switches from being a cell-intrinsic inhibitor of normal prostate epithelial cell proliferation to becoming an oncogene that is critical for prostate cancer cell proliferation. Methods: Immortalized human prostate basal epithelial EP156T cells and progeny cells that underwent epithelial to mesenchymal transition (EMT), primary prostate epithelial cells (PrECs) and prostate cancer cell lines LNCaP, VCaP and 22Rv1 were used to examine context dependent restriction and activation of the AR and classical target genes, such as KLK3. Genome-wide gene expression analyses and single cell protein analyses were applied to study the effect of different contexts. Results: A variety of growth conditions were tested and found unable to activate AR expression and transcription of classical androgen-dependent AR target genes, such as KLK3, in prostate epithelial cells with basal cell features or in mesenchymal type prostate cells. The restriction of androgen and AR dependent transcription of classical target genes in prostate basal epithelial cells was at the level of AR expression. Exogenous AR expression was sufficient for androgen-dependent transcription of AR target genes in prostate basal epithelial EP156T-AR cells, but did not exert a positive feedback on endogenous AR expression. Mesenchymal type prostate EPT3-AR cells with exogenous AR expression, in contrast to epithelial type EP156T-AR cells, were androgen non-responsive and were unable to produce detectable PSA in the culture supernatants even with higher levels of exogenous AR protein than in EP156T-AR and LNCaP cells for up to 2 weeks in androgen containing growth medium. The restricted PSA expression in the mesenchymal context suggests that if ADT increases the pool of mesenchymal type prostate cancer cells, then this might go undetected during PSA monitoring of disease progression. Citation Format: Jan Roger Olsen, Waqas Azeem, Margrete R. Hellem, Kristo Marvyin, Yaping Hua, Yi Qu, Lisha Li, Biaoyang Lin, XiSong Ke, Anne Margrete Oyan, Karl-Henning Kalland. Context dependent regulatory patterns of the androgen receptor (AR) and androgen receptor target genes. [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 1824.

Context dependent regulatory patterns of the androgen receptor and androgen receptor target genes.

BackgroundExpression of the androgen receptor (AR) is associated with androgen-dependent proliferation arrest and terminal differentiation of normal prostate epithelial cells. Additionally, activation of the AR is required for survival of benign luminal epithelial cells and primary cancer cells, thus androgen deprivation therapy (ADT) leads to apoptosis in both benign and cancerous tissue. Escape from ADT is known as castration-resistant prostate cancer (CRPC). In the course of CRPC development the AR typically switches from being a cell-intrinsic inhibitor of normal prostate epithelial cell proliferation to becoming an oncogene that is critical for prostate cancer cell proliferation. A clearer understanding of the context dependent activation of the AR and its target genes is therefore desirable.MethodsImmortalized human prostate basal epithelial EP156T cells and progeny cells that underwent epithelial to mesenchymal transition (EMT), primary prostate epithelial cells (PrECs) and prostate cancer cell lines LNCaP, VCaP and 22Rv1 were used to examine context dependent restriction and activation of the AR and classical target genes, such as KLK3. Genome-wide gene expression analyses and single cell protein analyses were applied to study the effect of different contexts.ResultsA variety of growth conditions were tested and found unable to activate AR expression and transcription of classical androgen-dependent AR target genes, such as KLK3, in prostate epithelial cells with basal cell features or in mesenchymal type prostate cells. The restriction of androgen- and AR-dependent transcription of classical target genes in prostate basal epithelial cells was at the level of AR expression. Exogenous AR expression was sufficient for androgen-dependent transcription of AR target genes in prostate basal epithelial cells, but did not exert a positive feedback on endogenous AR expression. Treatment of basal prostate epithelial cells with inhibitors of epigenetic gene silencing was not efficient in inducing androgen-dependent transcription of AR target genes, suggesting the importance of missing cofactor(s).ConclusionsRegulatory mechanisms of AR and androgen-dependent AR target gene transcription are insufficiently understood and may be critical for prostate cancer initiation, progression and escape from standard therapy. The present model is useful for the study of context dependent activation of the AR and its transcriptome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2453-4) contains supplementary material, which is available to authorized users.

Regulated expression of the prostacyclin receptor (IP) gene by androgens within the vasculature: Combined role for androgens and serum cholesterol

The prostanoid prostacyclin plays a key cardioprotective role within the vasculature. There is increasing evidence that androgens may also confer cardioprotection but through unknown mechanisms. This study investigated whether the androgen dihydrotestosterone (DHT) may regulate expression of the prostacyclin/I prostanoid receptor or, in short, the IP in platelet-progenitor megakaryoblastic and vascular endothelial cells. DHT significantly increased IP mRNA and protein expression, IP-induced cAMP generation and promoter (PrmIP)-directed gene expression in all cell types examined. The androgen-responsive region was localised to a cis-acting androgen response element (ARE), which lies in close proximity to a functional sterol response element (SRE) within the core promoter. In normal serum conditions, DHT increased IP expression through classic androgen receptor (AR) binding to the functional ARE within the PrmIP. However, under conditions of low-cholesterol, DHT led to further increases in IP expression through an indirect mechanism involving AR-dependent upregulation of SCAP expression and enhanced SREBP1 processing & binding to the SRE within the PrmIP. Chromatin immunoprecipitation assays confirmed DHT-induced AR binding to the ARE in vivo in cells cultured in normal serum while, in conditions of low cholesterol, DHT led to increased AR and SREBP1 binding to the functional ARE and SRE cis-acting elements, respectively, within the core PrmIP resulting in further increases in IP expression. Collectively, these data establish that the human IP gene is under the transcriptional regulation of DHT, where this regulation is further influenced by serum-cholesterol levels. This may explain, in part, some of the protective actions of androgens within the vasculature.

Similarities and Distinctions in Actions of Surface-Directed and Classic Androgen Receptor Antagonists.

The androgen receptor (AR) surface-directed antagonist MJC13 inhibits AR function and proliferation of prostate cancer (PC) cells. These effects are related to arrest of an AR/chaperone complex in the cytoplasm. Here, we compared MJC13 and classic AR antagonists such as flutamide and bicalutamide. Microarray analysis and confirmatory qRT-PCR reveals that MJC13 and flutamide inhibit dihydrotestosterone (DHT)-dependent genes in LNCaP PC cells. Both compounds are equally effective on a genome wide basis and as effective as second generation AR antagonists (MDV3100, ARN-509) at selected genes. MJC13 inhibits AR binding to the prostate specific antigen (PSA) promoter more strongly than flutamide, consistent with different mechanisms of action. Examination of efficacy of MJC13 in conditions that reflect aspects castrate resistant prostate cancer (CRPC) reveals that it inhibits flutamide activation of an AR mutant (ART877A) that emerges during flutamide withdrawal syndrome, but displays greatly restricted gene-specific activity in 22Rv1 cells that express a constitutively active truncated AR and is inactive against glucocorticoid receptor (GR), which can co-opt androgen-dependent signaling networks in CRPC. Importantly, MJC13 inhibits AR interactions with SRC2 and β-catenin in the nucleus and, unlike flutamide, strongly inhibits amplification of AR activity obtained with transfected SRC2 and β-catenin. MJC13 also inhibits DHT and β-catenin-enhanced cell division in LNCaP cells. Thus, a surface-directed antagonist can block AR activity in some conditions in which a classic antagonist fails and may display utility in particular forms of CRPC.

Abstract 83: Comparison of surface-directed and classic androgen receptor antagonists

Abstract Background: The androgen receptor (AR) surface-directed antagonist MJC13 inhibits AR function and proliferation of prostate cancer (PC) cells and these effects are related to arrest of an AR/chaperone complex in the cytoplasm. Here, we compared activities of MJC13 and the classic AR antagonist flutamide. Method and Results: MJC13 and flutamide inhibit all dihydrotestosterone (DHT)-dependent genes in LNCaP PC cells and both compounds are equally effective. Control ChIP assays confirm that MJC13 inhibits AR binding to the prostate specific antigen (PSA) promoter more strongly than flutamide, consistent with different mechanisms of action. Examination of efficacy of MJC13 in conditions that reflect aspects of castrate resistant prostate cancer (CRPC) reveals that it inhibits a flutamide-dependent AR mutant (ART877A), but displays greatly restricted gene-specific activity in 22Rv1 cells that express a constitutively active truncated AR and is inactive against glucocorticoid receptor (GR), which can co-opt androgen-dependent signaling networks in CRPC. While flutamide inhibits AR interactions with the coactivator SRC2, it does not affect β-catenin binding. In contrast, MJC13 inhibits AR interactions with both coregulators and, unlike flutamide, strongly inhibits amplification of AR activity obtained with SRC2 and β-catenin. MJC13 also preferentially inhibits β-catenin-enhanced cell division in LNCaP cells relative to flutamide. Conclusion: Our findings suggest that a surface-directed antagonist can block AR activity in some conditions in which a classic antagonist fails and may display utility in some forms of CRPC. Citation Format: Ji Ho Suh, Arundhati Chattopadhyay, Douglas H. Sieglaff, Cheryl L. Storer, Marc B. Cox, Paul Webb. Comparison of surface-directed and classic androgen receptor antagonists. [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 83. doi:10.1158/1538-7445.AM2015-83

Inhibition of SGK1 enhances mAR-induced apoptosis in MCF-7 breast cancer cells

Functional membrane androgen receptors (mAR) have previously been described in MCF-7 breast cancer cells. Their stimulation by specific testosterone albumin conjugates (TAC) activate rapidly non-genomic FAK/PI3K/Rac1/Cdc42 signaling, trigger actin reorganization and inhibit cell motility. PI3K stimulates serum and glucocorticoid inducible kinase SGK1, which in turn regulates the function of mAR. In the present study we addressed the role of SGK1 in mAR-induced apoptosis. TAC-stimulated mAR activation elicited apoptosis of MCF-7 cells, an effect significantly potentiated by concomitant incubation of the cells with TAC and the specific SGK1 inhibitors EMD638683 and GSK650394. In line with this, TAC and EMD638683 activated caspase-3. These effects were insensitive to the classical androgen receptor (iAR) antagonist flutamide, pointing to iAR-independent, mAR-induced responses. mAR activation and SGK1 inhibition further considerably augmented the radiation-induced apoptosis of MCF-7 cells. Moreover, TAC- and EMD638683 triggered early actin polymerization in MCF-7 cells. Blocking actin restructuring with cytochalasin B abrogated the TAC- and EMD638683-induced pro-apoptotic responses. Further analysis of the molecular signaling revealed late de-phosphorylation of FAK and Akt. Our results demonstrate that mAR activation triggers pro-apoptotic responses in breast tumor cells, an effect significantly enhanced by SGK1 inhibition, involving actin reorganization and paralleled by down-regulation of FAK/Akt signaling.

Morphology and gene expression profile of the submandibular gland of androgen-receptor-deficient mice

ObjectivesIn the submandibular gland (SMG) of mice, the granular convoluted tubule (GCT) develops preferentially in males dependent on androgens. To clarify the molecular mechanism of androgen action in SMG, we examined the SMG of mice deficient for the androgen receptor (ARKO). DesignThe morphological features and gene expression in the SMG of control and ARKO mice with or without hormone treatments were analysed by immunohistochemistry, DNA microarray, and RT-PCR. ResultsThe development of GCT and expression of GCT-specific products such as NGF were even lower in ARKO male SMG than in control female SMG. The administration of androgens to ARKO males had no effect on SMG, whereas the administration of thyroid hormone (T4) caused the extensive conversion of striated duct cells to GCT cells with the increase of NGF mRNA. Gene expression profiles in control and ARKO male SMG were analysed by DNA microarrays, and genes with higher or lower expression in ARKO male SMG were determined. They were then classified into groups according to their responsiveness to the administration of dihydotestosterone (DHT) or T4 to ARKO males. RT-PCR revealed that, while no gene was responsive to DHT, expression of many genes was up- or down-regulated by T4. ConclusionsThese results confirmed that GCT cell differentiation induced by androgens is dependent on the classical androgen receptor (AR), whereas that by T4 is independent of AR. Differential reactivity of genes to androgens and thyroid hormone in ARKO mice may shed light on the mechanism of androgen action in the SMG.

Unzipping androgen action through ZIP9: a novel membrane androgen receptor.

Nearly 5 decades ago, the existence of an androgen receptor (AR) molecule was originally identified by Shutsung Liao at the University of Chicago with concordant findings published by Nicholas Bruchovsky and Jean Wilson (1–3). These seminal studies used rat prostate tissue and specifically described a protein with high affinity for 5 -dihydrotestosterone (DHT) that accumulated in the nucleus upon binding, initiating RNA synthesis and explaining the mode of action for androgens within target cells.Subsequentstudiesbymultiple laboratoriesdetermined that AR regulates the transcription of androgen-responsive genes and plays a pivotal role in male reproductive tract development, sexually dimorphic brain activities, and multiple other target tissues. Importantly, AR has an essential role in prostate carcinogenesis and progression, with blockade of its activity used as a primary therapeutic target. Classical nuclear AR signaling (also known as genomic actions) in target cells involves the diffusion of testosterone through the cell membrane and into the cytoplasm, where it or its metabolite DHT binds to the AR, triggering conformational changes, dissociation from heat shock protein 90, dimerization, and translocation to the cell nucleus. There, the AR complex binds to DNA at androgen-response elements, recruits coregulators, and activates transcription of genes that are then translated into proteins. This classic or genomic mechanism of androgen action is relatively slow, requiring approximately 1–6 hours for alterations in gene transcriptional activity. It is noteworthy that as early as 1975, Liao also described rapid androgen actions occurring within 10 minutes of DHT administration that were independent of RNA synthesis, speculating that “androgens may affect known protein factors or others that are yet to be identified” (4). While research attention remain focused on nuclear steroid receptor signaling pathways, culminating in the cloning of steroid receptor genes in the 1980s, including that for AR (5, 6), evidence began to emerge in the 1990s that androgens are also capable of exerting rapid effects by interacting with AR in the cytoplasm or at the plasma membrane. Membrane-associated ARs were initially described in cell types such as T lymphocytes, monocytes, and osteoblasts to effect influx of calcium (7–9). Subsequent studies found that similar to membrane estrogen receptors, membrane ARs can also interactwithsignalingmolecules torapidlyactivatesignaling pathways, including the MAPK/ERK (10) and focal adhesion kinase 1 (FAK) /phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathways (11, 12). ARs along the cell surface have also been shown more recently to regulate actin cytoskeletal reorganization and prostate-specific antigen secretion in prostate cancer cells (13). The genomic AR signaling pathway has long been the standard therapeutic target for prostate cancer, and initially blockade of nuclear AR activity is very effective in slowing prostate tumor growth and progression. However, tumors eventually become resistant to nuclear AR antagonists and resume growth and rapid metastatic progression. Nongenomic androgen signaling represents an alternative focus for the development of potential treatment strategies that could enhance tumor response to antiandrogen treatment, particularly in delaying the development of castrate-resistant prostate cancer (14). Progress on studies of nongenomic androgen signaling in prostate cancer cells has been hampered by the challenges associated with identification of novel membrane ARs in these

Abstract 5152: Mechanism and therapeutic potential of the histone demethylase GASC1 in castration-resistant prostate cancer

Abstract The histone lysine demethylase, GASC1 (Gene Amplified in Squamous Cell Carcinoma 1) is an epigenetic regulatory protein both highly expressed during mammalian embryogenesis and overexpressed in several types of cancer, including aggressive Castration Resistant Prostate Cancer (CRPC). GASC1 mainly catalyzes demethylation of tri- and di-methylated forms of histone H3 lysine 9 (H3K9me3/me2) epigenetic repressive marks, regulating gene expression and chromatin architecture. This research aims to elucidate the fundamental mechanism by which GASC1 dysregulation contributes to CRPC progression, as well as to evaluate its potential as a therapeutic target against aggressive prostate cancer. Previously, our lab and others have shown that GASC1 interacts with the androgen receptor (AR) and functions as a co-activator of AR-induced transcription in prostate cancer. We have used an shRNA approach to determine whether GASC1 knockdown affects the proliferation and transformation of CRPC cells. We found that knocking down GASC1 inhibits the growth and colony formation of C4-2B and CWR22Rv1 cells in vitro. Knockdown also reduced transcript levels of classical and CRPC-specific AR target genes in either the presence or absence of the AR ligand. As a result, we are evaluating the therapeutic potential of novel and highly specific lysine demethylase inhibitors towards abating GASC1 function in prostate cancer cells in vitro. We have recently determined that one of these novel inhibitors is highly effective in modulating the survival and proliferation of several prostate cancer cell lines including CRPC lines with IC50 values, all well under 1 uM. Together, our data demonstrates that GASC1 is a therapeutically relevant target in controlling and preventing the emergence of prostate cancers, with particular application against aggressive CRPC subtypes. Citation Format: Roselyne M. Labbe, Qin Ye, Andreana Holowatyj, Lihong Zhang, Zeng Quan Yang. Mechanism and therapeutic potential of the histone demethylase GASC1 in castration-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5152. doi:10.1158/1538-7445.AM2014-5152

Testosterone suppresses the expression of regulatory enzymes of fatty acid synthesis and protects against hepatic steatosis in cholesterol-fed androgen deficient mice

AimsNon-alcoholic fatty liver disease and its precursor hepatic steatosis is common in obesity and type-2 diabetes and is associated with cardiovascular disease (CVD). Men with type-2 diabetes and/or CVD have a high prevalence of testosterone deficiency. Testosterone replacement improves key cardiovascular risk factors. The effects of testosterone on hepatic steatosis are not fully understood. Main methodsTesticular feminised (Tfm) mice, which have a non-functional androgen receptor (AR) and very low serum testosterone levels, were used to investigate testosterone effects on high-cholesterol diet-induced hepatic steatosis. Key findingsHepatic lipid deposition was increased in Tfm mice and orchidectomised wild-type littermates versus intact wild-type littermate controls with normal androgen physiology. Lipid deposition was reduced in Tfm mice receiving testosterone treatment compared to placebo. Oestrogen receptor blockade significantly, but only partially, reduced the beneficial effects of testosterone treatment on hepatic lipid accumulation. Expression of key regulatory enzymes of fatty acid synthesis, acetyl-CoA carboxylase alpha (ACACA) and fatty acid synthase (FASN) were elevated in placebo-treated Tfm mice versus placebo-treated littermates and Tfm mice receiving testosterone treatment. Tfm mice on normal diet had increased lipid accumulation compared to littermates but significantly less than cholesterol-fed Tfm mice and demonstrated increased gene expression of hormone sensitive lipase, stearyl-CoA desaturase-1 and peroxisome proliferator-activated receptor-gamma but FASN and ACACA were not altered. SignificanceAn action of testosterone on hepatic lipid deposition which is independent of the classic AR is implicated. Testosterone may act in part via an effect on the key regulatory lipogenic enzymes to protect against hepatic steatosis.

Targeting Androgen Receptor/Src Complex Impairs the Aggressive Phenotype of Human Fibrosarcoma Cells

BackgroundHormones and growth factors influence the proliferation and invasiveness of human mesenchymal tumors. The highly aggressive human fibrosarcoma HT1080 cell line harbors classical androgen receptor (AR) that responds to androgens triggering cell migration in the absence of significant mitogenesis. As occurs in many human cancer cells, HT1080 cells also express epidermal growth factor receptor (EGFR).Experimental Findings: We report that the pure anti-androgen Casodex inhibits the growth of HT1080 cell xenografts in immune-depressed mice, revealing a novel role of AR in fibrosarcoma progression. In HT1080 cultured cells EGF, but not androgens, robustly increases DNA synthesis. Casodex abolishes the EGF mitogenic effect, implying a crosstalk between EGFR and AR. The mechanism underlying this crosstalk has been analyzed using an AR-derived small peptide, S1, which prevents AR/Src tyrosine kinase association and androgen-dependent Src activation. Present findings show that in HT1080 cells EGF induces AR/Src Association, and the S1 peptide abolishes both the assembly of this complex and Src activation. The S1 peptide inhibits EGF-stimulated DNA synthesis, cell matrix metalloproteinase-9 (MMP-9) secretion and invasiveness of HT1080 cells. Both Casodex and S1 peptide also prevent DNA synthesis and migration triggered by EGF in various human cancer-derived cells (prostate, breast, colon and pancreas) that express AR.ConclusionThis study shows that targeting the AR domain involved in AR/Src association impairs EGF signaling in human fibrosarcoma HT1080 cells. The EGF-elicited processes inhibited by the peptide (DNA synthesis, MMP-9 secretion and invasiveness) cooperate in increasing the aggressive phenotype of HT1080 cells. Therefore, AR represents a new potential therapeutic target in human fibrosarcoma, as supported by Casodex inhibition of HT1080 cell xenografts. The extension of these findings in various human cancer-derived cell lines highlights the conservation of this process across divergent cancer cells and identifies new potential targets in the therapeutic approach to human cancers.

Physiological Testosterone Retards Cardiomyocyte Aging in Tfm Mice via Androgen Receptor-independent Pathway

To determine whether testosterone modulates markers of cardiomyocytes aging via its classic androgen receptor (AR)-dependent pathway or conversion to estradiol. Male littermates and testicular feminized (Tfm) mice were randomly separated into 4 experimental groups littermate controls (n=8), Tfm mice (n=7), testosterone-treated Tfm mice (n=8), and Tfm mice treated with testosterone in combination with the aromatase inhibitor anastrazole (n=7). Cardiomyocytes were isolated from mouse left ventricles, the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the amount of malondialdehyde (MDA) were measured using colorimetry method, and expression of p16(INK4α) and retinoblastoma (Rb) proteins were detected by Western blotting. The SOD and GSH-Px enzyme activities of cardiomyocytes were decreased, and the MDA levels and the expression of p16(INK4α) and Rb proteins were increased in Tfm mice compared with control mice. An increase was observed in the activities of SOD and GSH-Px enzyme as well as a decrease in MDA levels and the expression of p16(INK4α) and Rb proteins in the testosterone-treated Tfm mice. After co-treatment with anastrazole in Tfm mice, these improvement were partly inhibited. Physiological testosterone replacement can delay cardiomyocyte aging in Tfm mice, an effect that is independent of the AR pathway and in part conversion to estradiol.

Abstract 2966: The role of histone demethylase GASC1 in promoting prostate cancer progression.

Abstract Prostate cancer affects 1 in 6 American men, and is the second leading cause of cancer-related deaths in males. The defiance of castration-resistant prostate cancer (CRPC) to currently available treatments represents the most challenging aspect of this disease. Although resistant to androgen deprivation treatment, most CRPCs continue to express the androgen receptor (AR) and remain dependent on AR signaling for growth and proliferation. Previously, we cloned a novel gene, GASC1 (gene amplified in squamous cell carcinoma 1), from 9p24 amplified cancer cells. Recently, the GASC1 protein has been identified as a key histone demethylase that controls chromatin-mediated regulation of gene expression. Importantly, the GASC1 protein interacts with the AR and serves as a co-activator of AR-induced transcription in prostate cells. In this study, we demonstrated that various aggressive tumors, including CRPCs, have upregulated GASC1. We used an shRNA approach to determine whether GASC1 knockdown would affect the proliferation and transformation of CRPC cell lines. We found that knocking down GASC1 inhibits growth and colony formation of CRPC cells (C4-2B and CWR22Rv1) in vitro. Previously, it was reported that the GASC1 protein interacts with the AR and functions as a co-activator of AR-induced transcription in LNCaP cells. The GASC1 protein contains N-terminal Jumonji catalytic domains and C-terminal Tudor and PHD domains; however, which domains are responsible for the association of GASC1 with the AR have not yet been identified. To address this question, we generated stable LNCaP cell lines expressing V5-tagged N-terminal Jumonji catalytic domains and C-Terminal PHD/Tudor domains, and performed Co-IP analyses. We found that the C-terminal region is essential for GASC1 association with the AR in prostate cancer cells. To explore the contribution of GASC1 to endogenous AR-regulated gene expression in AR-positive CRPC cells, we performed quantitative RT-PCR assays to measure the expression of both classical AR target genes (including PSA/KLK3, KLK2, FKBP5, and NDRG1) and CRPC-specific genes (including UBE2C, CDK1, CDC20, and CCNA2) in C4-2B cells with or without GASC1 knockdown. We found that knocking down GASC1 significantly reduced transcript levels of a set of classical and CRPC-specific AR target genes in either the presence or absence of the AR ligand. Our data demonstrates that GASC1 is a target of substantial relevance to aggressive prostate cancers, including CRPCs. As such, targeting GASC1 is a promising strategy to control CRPC or prevent its emergence. Citation Format: Lihong Zhang, Jinling Hou, Andreana Holowatyj, Zeng-Quan Yang. The role of histone demethylase GASC1 in promoting prostate cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2966. doi:10.1158/1538-7445.AM2013-2966

Androgen receptor decreases the cytotoxic effects of chemotherapeutic drugs in upper urinary tract urothelial carcinoma cells

Upper urinary tract urothelial carcinomas (UUTUCs) represent relatively uncommon yet devastating tumors that affect more males than females. However, the correlation between gender difference and disease progression remains unclear. Androgen and the androgen receptor (AR) were previously hypothesized to account for the gender difference in the incidence of urothelial carcinomas; however, the role of AR in the development and progression of UUTUCs is not well understood. In addition, although UUTUCs are responsive to chemotherapy, various responses are presented among patients. Therefore, the aim of the present study was to determine the role of AR in the response of UUTUC cells to chemotherapeutic drugs. In this study, AR overexpression in UUTUC cells (BFTC 909) was identified to reduce the cytotoxic effect of chemotherapeutic drugs, including doxorubicin, cisplatin and mitomycin C and protected cells from drug-induced death. The expression of ABCG2, an ATP-binding cassette half-transporter associated with multidrug resistance, was increased in AR-overexpressing BFTC cells. In addition, use of the AR degradation enhancer, ASC-J9®, repressed the AR effect on increasing cell viability under drug treatment. In summary, results of the present study indicate that the status of AR expression levels in UUTUCs may be a significant factor in affecting the efficacy of chemotherapy and classic chemotherapeutic drugs and AR targeted therapy may provide a novel potential therapeutic approach to improve treatment of UUTUCs.

Dihydrotestosterone regulating apolipoprotein M expression mediates via protein kinase C in HepG2 cells

BackgroundAdministration of androgens decreases plasma concentrations of high-density lipid cholesterol (HDL-C). However, the mechanisms by which androgens mediate lipid metabolism remain unknown. This present study used HepG2 cell cultures and ovariectomized C57BL/6 J mice to determine whether apolipoprotein M (ApoM), a constituent of HDL, was affected by dihydrotestosterone (DHT).MethodsHepG2 cells were cultured in the presence of either DHT, agonist of protein kinase C (PKC), phorbol-12-myristate-13-acetate (PMA), blocker of androgen receptor flutamide together with different concentrations of DHT, or DHT together with staurosporine at different concentrations for 24 hrs. Ovariectomized C57BL/6 J mice were treated with DHT or vehicle for 7d or 14d and the levels of plasma ApoM and livers ApoM mRNA were measured. The mRNA levels of ApoM, ApoAI were determined by real-time RT-PCR. ApoM and ApoAI were determined by western blotting analysis.ResultsAddition of DHT to cell culture medium selectively down-regulated ApoM mRNA expression and ApoM secretion in a dose-dependent manner. At 10 nM DHT, the ApoM mRNA levels were about 20% lower than in untreated cells and about 40% lower at 1000 nM DHT than in the control cells. The secretion of ApoM into the medium was reduced to a similar extent. The inhibitory effect of DHT on ApoM secretion was not blocked by the classical androgen receptor blocker flutamide but by an antagonist of PKC, Staurosporine. Agonist of PKC, PMA, also reduced ApoM. At 0.5 μM PMA, the ApoM mRNA levels and the secretion of ApoM into the medium were about 30% lower than in the control cells. The mRNA expression levels and secretion of another HDL-associated apolipoprotein AI (ApoAI) were not affected by DHT. The levels of plasma ApoM and liver ApoM mRNA of DHT-treated C57BL/6 J mice were lower than those of vehicle-treated mice.ConclusionsDHT directly and selectively down-regulated the level of ApoM mRNA and the secretion of ApoM by protein kinase C but independently of the classical androgen receptor.