Androgen Receptor Response Elements Research Articles

Estrogen receptor beta suppresses the androgen receptor oncogenic effects in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer associated with poor prognosis and limited treatment options. The androgen receptor (AR) has emerged as a potential therapeutic target for luminal androgen receptor (LAR) TNBC. However, multiple studies have claimed that anti-androgen therapy for AR-positive TNBC only has limited clinical benefits. This study aimed to investigate the role of AR in TNBC and its detailed mechanism. Immunohistochemistry and TNBC tissue sections were applied to investigate AR and nectin cell adhesion molecule 4 (NECTIN4) expression in TNBC tissues. Then, in vitro and in vivo assays were used to explore the function of AR and estrogen receptor beta (ERβ) in TNBC. Chromatin immunoprecipitation sequencing (ChIP-seq), co-immunoprecipitation (co-IP), molecular docking method, and luciferase reporter assay were performed to identify key molecules that affect the function of AR. Based on the TNBC tissue array analysis, we revealed that ERβ and AR were positive in 21.92% (32/146) and 24.66% (36/146) of 146 TNBC samples, respectively, and about 13.70% (20/146) of TNBC patients were ERβ positive and AR positive. We further demonstrated the pro-tumoral effects of AR on TNBC cells, however, the oncogenic biology was significantly suppressed when ERβ transfection in LAR TNBC cell lines but not in AR-negative TNBC. Mechanistically, we identified that NECTIN4 promoter -42bp to -28bp was an AR response element, and that ERβ interacted with AR thus impeding the AR-mediated NECTIN4 transcription which promoted epithelial-mesenchymal transition in tumor progression. This study suggests that ERβ functions as a suppressor mediating the effect of AR in TNBC prognosis and cell proliferation. Therefore, our current research facilitates a better understanding of the role and mechanisms of AR in TNBC carcinogenesis.

Abstract C085: OPN-6602, a dual EP300/CBP bromodomain inhibitor modulates androgen-driven transcription, including MYC, in mCRPC

Abstract Metastatic castration-resistant prostate cancer (mCRPC) remains an urgent unmet medical need despite existing therapies that target androgen receptor (AR) signaling. Disease progression is commonly a result of acquired resistance to existing anti-androgen therapies. Biological models have revealed potential resistance mechanisms of CRPC through the reactivation of the AR pathway. Transcriptional coactivators CREB-binding protein (CBP) and E1A binding protein (EP300) are high value targets in mCRPC as they mediate androgen-dependent and -independent activity of the AR pathway. EP300 and CBP proteins harbor both histone acetyltransferase (HAT)- and bromo-domains, and the HAT activity of EP300 regulates AR function. EP300 bromodomain inhibition partially blocks its HAT activity, reducing AR-mediated gene expression and tumor growth. OPN-6602 and OPN-6742 are potent, selective, and orally active small molecule dual EP300/CBP bromodomain inhibitors which abrogate persistent AR signaling in CRPC by modulating H3K27 acetylation and the recruitment of EP300, AR full length (AR-FL), and AR variant 7 (AR-v7) to AR response elements. OPN-6602 and OPN-6742 obstruct the EP300 bromodomain leading to downregulation of the AR pathway and consequent anti-tumor effects in AR-dependent CRPC. OPN-6602 and OPN-6742 are highly selective (>100-fold) against other bromodomains based on a screening panel of 40 bromodomains. Biochemical screens using AlphaScreen technology show these compounds to be equipotent against EP300 and CBP. OPN-6602 is particularly potent by Surface Plasmon Resonance analysis, displaying a KD of 0.87 nM, with a fast association rate (1.5 E+06/Ms) and a slow dissociation rate (1.3 E-03/s). Consequently, OPN-6602 displays a noteworthy residence time of 13 minutes. Both compounds show an AR-selective mode of action with anti-proliferative activity in AR-dependent prostate cancer cell lines (e.g. VCaP, LNCaP, and 22Rv-1) but are inactive against AR-negative prostate cancer cell lines (e.g. DU145). VCaP and mCRPC patient-derived xenograft (PDX) RNA profiling provides evidence of a marked, dose-dependent, pharmacodynamic response indicative of EP300/CBP inhibition. In the VCaP xenograft model, OPN-6602 and OPN-6742 show dose-dependent tumor growth inhibition and corresponding pharmacodynamic responses with prostate-specific antigen level decreases and decreased H3K27 acetylation. In the QR (AR-v7+) PDX model of CRPC (post-androgen deprivation therapy), a synergistic effect was observed between OPN-6602 and enzalutamide. This combination further modulates the expression of AR-driven transcripts such as MYC compared to single agent treatments. The pharmacokinetic profiles of OPN-6602 and OPN-6742 are favorable for oral single daily dose administration due to a high Cmax and short half-life. Due to its higher potency, OPN-6602 has been chosen as clinical candidate and a first-in-human study will start in 2024. Citation Format: Bernice Matusow, Wayne Spevak, Chao Zhang, Yan Ma, Rafe Shellooe, Peipei Li, James Tsai, Pan- Yu Chen, Parmveer Singh, Jackie Walling, Christine Nichols, Jason Halladay, Kerry Inokuchi, Gaston Habets, Gideon Bollag. OPN-6602, a dual EP300/CBP bromodomain inhibitor modulates androgen-driven transcription, including MYC, in mCRPC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C085.

Androgen Receptor Transcriptionally Inhibits Programmed Death Ligand-1 Expression and Influences Immune Escape in Bladder Cancer

In multiple clinical trials, immune checkpoint blockade–based immunotherapy has shown significant therapeutic efficacy in bladder cancer (BCa). Sex is closely related to the incidence rate and prognosis of BCa. As one of the sex hormone receptors, the androgen receptor (AR) is a well-known key regulator that promotes the progression of BCa. However, the regulatory mechanism of AR in the immune response of BCa is still unclear. In this study, the expression of AR and programmed death ligand 1 (PD-L1) was negatively correlated in BCa cells, clinical tissues, and tumor data extracted from The Cancer Genome Atlas Bladder Urothelial Carcinoma cohort. A human BCa cell line was transfected to alter the expression of AR. The results show that AR negatively regulated PD-L1 expression by directly binding to AR response elements on the PD-L1 promoter region. In addition, AR overexpression in BCa cells significantly enhanced the antitumor activity of cocultured CD8+ T cells. Injection of anti–PD-L1 monoclonal antibodies into C3H/HeN mice significantly suppressed tumor growth, and stable expression of AR dramatically enhanced the antitumor activity in vivo. In conclusion, this study describes a novel role of AR in regulating the immune response to BCa by targeting PD-L1, thus providing potential therapeutic strategies for immunotherapy in BCa.

Ultrasensitive detection of the androgen receptor through the recognition of an androgen receptor response element and hybridization chain amplification.

An ultrasensitive electrochemical detection of the androgen receptor (AR) was developed based on the protection of a DNA duplex by the AR from restriction endonuclease-mediated digestion and a subsequent hybridization chain reaction (HCR). Two partially complementary DNA probes P1 and P2 were designed to form an androgen receptor binding probe (ARBP) through hybridization. The ARBP contains a duplex at one end and two single-stranded tails at the other end. The duplex part containing the recognition sites of the AR and NspI restriction endonuclease was immobilized on an Au electrode, whereas the single-stranded parts served as capture probes to activate the HCR. In the absence of the AR, NspI can cleave the duplex and release the capture probes, and thus, no HCR occurs. However, the AR can bind to the ARBP and protect the duplex from cleavage; therefore, the capture probes can trigger the HCR between four carefully designed G-quadruplex forming hairpin probes and the capture probes, resulting in the formation of numerous G-quadruplexes. Finally, differential pulse voltammetry (DPV) was carried out to quantify the AR. The assay revealed a detection limit of 7.64 fM. The verification of its high specificity and practicability in serum samples indicated its potential applications in the fields of clinical examination and disease diagnosis.

Radiosensitization by DNA, Binding Pyrrole, Imidazole Polyamides

Pyrrole (Py) - Imidazole (Im) polyamides are a class of non-genotoxic DNA-binding small molecules with modular sequence recognition and high affinity. A polyamide targeted to the sequence 5’-WGWWCW-3’, corresponding to the Androgen Receptor (AR) Response Element (ARE) half-site, suppresses AR positive prostate tumor xenografts. Evidence for inhibition of DNA dependent processes including allosteric inhibition of AR-DNA interactions and RNA Polymerase II transcription are observed in vitro. It is hypothesized that polyamides may displace DNA repair elements recruited after genotoxic insult and thereby sensitize cells to ionizing radiation.

MicroRNA-185 downregulates androgen receptor expression in the LNCaP prostate carcinoma cell line.

The present study aimed to investigate whether microRNA (miR)‑185 downregulated androgen receptor expression in the LNCaP prostate carcinoma cell line. Human prostate cancer (PCa) LNCaPcells were cultured and transfected with synthetic has‑miR‑185 mimic or inhibitor. The transfected cells were subsequently evaluated with a viability assay, nuclear staining, reverse transcription quantitative polymerase chain reaction (RT‑qPCR), dual luciferase assay and western blot analysis. The results of the western blot analysis and RT‑qPCR indicated that transfection with an miR‑185 mimic markedly reduced the androgen receptor (AR) protein expression levels in LNCaPcells, whereas transfection with an miR‑185 inhibitor increased the protein expression of AR in the LNCaPcells. The results of the luciferase reporter assay demonstrated that the predicted target site in the AR 3' untranslated regions was a specific functional binding site for miR‑185, and that AR was a direct target of miR‑185. In addition, downregulation of AR by miR‑185 impaired the interaction between AR and androgen response element, and downregulated the expression of the AR target gene prostate specific antigen. Data also suggested that the downregulation of AR mediated by miR‑185, inhibited the proliferation and induced the apoptosis of the LNCaPcells. Therefore, the results of the present study suggested that miR‑185 may be a potential negative modulator of AR‑mediated signaling and may act as a tumor suppressor in prostate cancer cells.

SUMO-specific protease 1 modulates cadmium-augmented transcriptional activity of androgen receptor (AR) by reversing AR SUMOylation

Cadmium is a potential prostate carcinogen and can mimic the effects of androgen by a mechanism that involves the hormone-binding domain of the androgen receptor (AR), which is a key transcriptional factor in prostate carcinogenesis. We focused on transcriptional activity of AR to investigate the toxicity of cadmium exposure on human prostate cell lines. Cadmium increased the proliferative index of LNCaP and the proliferative effect was obstructed significantly by AR blocking agent. In luciferase assay, cadmium activated the transcriptional activity of AR in 293T cells co-transfected with wild-type AR and an ARE (AR response elements)-luciferase reporter gene. Cadmium also increased expression of PSA, a downstream gene of AR, whereas the metal had no significant effect on AR amount. AR is regulated by multiple posttranslational modifications including SUMOylation. SUMOylated AR shows a lower transcriptional activity. SUMO-specific protease 1 (SENP1) decreases AR SUMOylation by deconjugating AR-SUMO covalent bond. We detected that cadmium increased the amount of SENP1 in a dose and time dependent manner. Knocking down of SENP1 by RNAi led to decrease of PSA expression and transcriptional activity of AR in luciferase assay. Furthermore, co-immunoprecipitation (Co-IP) results showed that SUMOylation level of AR was decreased after cadmium treatment. In conclusion, our results indicated that cadmium-induced SENP1 enhanced AR transcriptional activity by decreasing AR SUMOylation.

Ligand-independent and tissue-selective androgen receptor inhibition by pyrvinium.

Pyrvinium pamoate (PP) is a potent noncompetitive inhibitor of the androgen receptor (AR). Using a novel method of target identification, we demonstrate that AR is a direct target of PP in prostate cancer cells. We demonstrate that PP inhibits AR activity via the highly conserved DNA binding domain (DBD), the only AR inhibitor that functions via this domain. Furthermore, computational modeling predicts that pyrvinium binds at the interface of the DBD dimer and the minor groove of the AR response element. Because PP acts through the DBD, PP is able to inhibit the constitutive activity of AR splice variants, which are thought to contribute to the growth of castration resistant prostate cancer (CRPC). PP also inhibits androgen-independent AR activation by HER2 kinase. The antiandrogen activity of pyrvinium manifests in the ability to inhibit the in vivo growth of CRPC xenografts that express AR splice variants. Interestingly, PP was most potent in cells with endogenous AR expression derived from prostate or bone. PP was able to inhibit several other hormone nuclear receptors (NRs) but not structurally unrelated transcription factors. PP inhibition of other NRs was similarly cell-type selective. Using dual-energy X-ray absorptiometry, we demonstrate that the cell-type specificity of PP manifests in tissue-selective inhibition of AR activity in mice, as PP decreases prostate weight and bone mineral density but does not affect lean body mass. Our results suggest that the noncompetitive AR inhibitor pyrvinium has significant potential to treat CRPC, including cancers driven by ligand-independent AR signaling.

Up-Regulation of Hepatic Alpha-2-HS-Glycoprotein Transcription by Testosterone via Androgen Receptor Activation

Background/Aims: Fetuin-A (alpha-2-HS-glycoprotein, AHSG), a liver borne plasma protein, contributes to the prevention of soft tissue calcification, modulates inflammation, reduces insulin sensitivity and fosters weight gain following high fat diet or ageing. In polycystic ovary syndrome, fetuin-A levels correlate with free androgen levels, an observation pointing to androgen sensitivity of fetuin-A expression. The present study thus explored whether the expression of hepatic fetuin-A is modified by testosterone. Methods: HepG2 cells were treated with testosterone and androgen receptor antagonist flutamide, and were silenced with androgen receptor siRNA. To test the in vivo relevance, male mice were subjected to androgen deprivation therapy (ADT) for 7 weeks. AHSG mRNA levels were determined by quantitative RT-PCR and fetuin-A protein abundance by Western blotting. Results: In HepG2 cells, AHSG mRNA expression and fetuin-A protein abundance were both up-regulated following testosterone treatment. The human alpha-2-HS-glycoprotein gene harbors putative androgen receptor response elements in the proximal 5 kb promoter sequence relative to TSS. The effect of testosterone on AHSG mRNA levels was abrogated by silencing of the androgen receptor in HepG2 cells. Moreover, treatment of HepG2 cells with the androgen receptor antagonist flutamide in presence of endogenous ligands in the medium significantly down-regulated AHSG mRNA expression and fetuin-A protein abundance. In addition, ADT of male mice was followed by a significant decrease of hepatic Ahsg mRNA expression and fetuin-A protein levels. Conclusions: Testosterone participates in the regulation of hepatic fetuin-A expression, an effect mediated, at least partially, by androgen receptor activation.

The prostate cancer-up-regulated myc-associated zinc-finger protein (MAZ) modulates proliferation and metastasis through reciprocal regulation of androgen receptor

Prostate cancer (PCa) is one of the most commonly diagnosed malignancies in men and the second leading cause of male cancer mortality. MAZ (Myc-associated zinc-finger protein) is a transcription factor that regulates the transcription of oncogenes, and the deregulated MAZ expression is closely related to the development and progression of a variety of cancers. In the present study, the role of MAZ in PCa tumorigenesis and its interaction with androgen receptor (AR), which is essential to PCa development in humans, were investigated. MAZ expression was found to be higher in clinical PCa specimens than in benign prostatic hyperplasia (BPH) and adjacent normal tissues, and MAZ expression was positively correlated with AR expression, which was also observed in PCa cell lines. After knockdown of MAZ by siRNA, cell proliferation was notably inhibited, colony formation declined, the cell cycle was arrested at G0/G1 phase, and the number of cells in S phase decreased (p < 0.05). MAZ knockdown resulted in a significant decline in the migration and invasion capacity of the LNCaP-AD cell line. siRNA knockdown of AR significantly decreased MAZ expression, and knockdown of MAZ significantly increased the expression of AR and DHT-induced androgen response element (ARE). These results suggest that MAZ and AR are interrelated and that MAZ plays an important role in PCa pathogenesis, which could be a potential therapeutic target.

Genome-Wide Analysis of Androgen Receptor Targets Reveals COUP-TF1 as a Novel Player in Human Prostate Cancer

Androgen activity plays a key role in prostate cancer progression. Androgen receptor (AR) is the main mediator of androgen activity in the prostate, through its ability to act as a transcription mediator. Here we performed a genome-wide analysis of human AR binding to promoters in the presence of an agonist or antagonist in an androgen dependent prostate cancer cell line. Many of the AR bound promoters are bound in all examined conditions while others are bound only in the presence of an agonist or antagonist. Several motifs are enriched in AR bound promoters, including the AR Response Element (ARE) half-site and recognition elements for the transcription factors OCT1 and SOX9. This suggests that these 3 factors could define a module of co-operating transcription factors in the prostate. Interestingly, AR bound promoters are preferentially located in AT rich genomic regions. Analysis of mRNA expression identified chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1) as a direct AR target gene that is downregulated upon binding by the agonist liganded AR. COUP-TF1 immunostaining revealed nucleolar localization of COUP-TF1 in epithelium of human androgen dependent prostate cancer, but not in adjacent benign prostate epithelium. Stromal cells both in human and mouse prostate show nuclear COUP-TF1 staining. We further show that there is an inverse correlation between COUP-TF1 expression in prostate stromal cells and the rising levels of androgen with advancing puberty. This study extends the pool of recognized putative AR targets and identifies a negatively regulated target of AR – COUP-TF1 – which could possibly play a role in human prostate cancer.

Dibutyltin (DBT) an endocrine disrupter in zebrafish

Dibutyltin (DBT) is a plastic stabilizer and metabolite of tributyltin (TBT), other than its immune and neurological effects, little else is known about DBT. We have examined the actions of DBT as an endocrine disruptor in zebrafish (Danio rerio). In HeLa cells transiently co-transfected with plasmid constructs containing zebrafish estrogen receptors (zfERα, zfERβ1 and zfERβ2) and zebrafish estrogen response element (zfERE-luc), ethinylestradiol (EE2) induced luciferase activity 2 to 6-fold and DBT increased that activity. DBT inhibited testosterone induced luciferase activity, in HeLa cells transiently co-transfected with zebrafish androgen receptor (zfAR) and murine androgen receptor response element (ARE-slp-luc). In HeLa cells co-transfected with zfERβ1 and zfERβ2 and a plasmid containing zebrafish aromatase (zfCyp19a-luc), DBT increased EE2-induced luciferase activity. Real-time PCR analysis of sexual differentiation markers and phase II metabolism markers in fish exposed to 0.1 to 0.5mg/kg DBT in vivo revealed a masculinizing effect. In brain there was decreased production of FST. In the liver there was decreased production of FST, zfCYP19a, and zfERβ1 mRNA and in the gonads there was decreased production of FST, DAX1, and zfCYP19a. DBT also increased UGT expression and decreased GST following exposure. DBT in vivo has an overall masculinizing effect but in vitro has a feminizing effect, indicating that DBT has complex actions within zebrafish, which may be due to the metabolism of DBT to monobutyltin (MBT) and action of other reactive metabolites which promote masculinization and mask the feminizing effect of DBT alone. In vitro results show a clear feminizing effect in a system with fewer complexities. These results are discussed in the context of DBT as an endocrine disruptor in zebrafish.

Testosterone, not 5alpha-dihydrotestosterone Stimulates Liver Receptor homolog-1 Expression by Activating the Androgen Receptor in Primary Rat Granulosa Cells.

Androgens are crucial for normal folliculogenesis and female fertility. Using primary rat granulosa cells, we recently demonstrated that in the absence of gonadotropins, testosterone increases aromatase (Cyp19) and P450 side change cleavage (P450scc) expression. Interestingly, 5alpha-dihydrotestosterone (DHT), a more potent activator of the AR, does not stimulate the expression of Cyp19 and P450scc to the same extent as testosterone. An explanation for this unique effect of testosterone remains elusive. Here, we have explored the nature of the mechanisms involved in the stimulation of gene expression by testosterone in granulosa cells (GCs), and the differential response of these cells to testosterone and DHT. Using primary GCs, we showed that testosterone, but not DHT, stimulates liver receptor homolog-1 (LRH-1) mRNA expression. LRH-1 is a transcription factor known to be involved in the up-regulation of Cyp19 expression. The effects of testosterone on LRH-1 expression are mediated by an increase in the transcription of this gene since testosterone also increased LRH-1 promoter activity. In agreement with the lack of stimulation of LRH-1 mRNA expression by DHT, this androgen did not increase the activity of the LRH-1 promoter in GCs. Based on these results; we reasoned that if DHT and T occupy the same androgen receptor (AR), then treatment with increasing concentrations of DHT might inhibit the ability of testosterone to stimulate LRH-1. This presupposes that the DHT pretreatment saturates and substantially reduces the number of ARs available to testosterone. The results of this experiment demonstrated that the stimulation of LRH-1 expression by testosterone could be out competed in a concentration-dependent manner not only by DHT but also by R1881, a potent AR agonist. This result suggests that the effects of testosterone are indeed mediated by the activation of the AR. Interestingly, both testosterone and DHT stimulated the activity of a reporter construct containing three consensus AR response elements. Moreover, in contrast to the selective response of the LRH-1 promoter for testosterone observed in ovarian GCs, both testosterone and DHT stimulated the activity of the LRH-1 promoter in human embryonic kidney cells (293T cells). These results indicate that only granulosa cells provide the appropriated milieu of coregulators needed for the differential stimulation of LRH-1 expression by testosterone and DHT. To begin to understand the mechanisms by which testosterone regulates LRH-1 expression in GCs, studies using progressive mutant deletions of the LRH-1 promoter were performed. These experiments identified the region between -348bp and -266bp of the LRH-1 promoter as the target for the testosterone-AR complex. Within this region a selective AR response element was found. These results suggest strongly that in ovarian granulosa cells, pathways activated primarily by testosterone may control the expression of LRH-1. Supported by NIH R01HD057110 and R21HD066233. (poster)

Abstract 1968: Obesity-induced activation of the androgen receptor

Abstract Background: Prostate cancer is the second leading cause of cancer deaths for men in the US. Obesity increases the risk of dying from prostate cancer by almost 50%. The mechanisms by which obesity promotes a more aggressive disease remain largely unknown, hindering the development of effective approaches for improving patient outcome. It is hypothesized that because obesity is associated with elevated circulating levels of growth factors, activation of the androgen receptor may be responsible for the increased risk of a more aggressive cancer in obese men. This current study investigates the effects of obese sera on androgen receptor activity in a highly novel prostate cancer cell line. Methods: LNCaP prostate cancer cells were stably infected with a lentiviral Dual-Luciferase reporter plasmid containing an androgen receptor response element (ARE). These stable ARE-reporter cells were then exposed to sera from either obese or control weight mice. Specific signal transduction inhibitors were used to determine the role of growth factor signaling in mediating the effects observed in response to exposure to the different sera. Results: Exposure to obese sera activated the androgen receptor to levels three-fold greater than those seen with control sera. Intriguingly, use of a PI3 kinase inhibitor reduced obesity-induced activation of the AR by 75%, but only by 64% in the control sera. Studies are on-going to determine the role of both the Akt and MAPK pathways in these results, as well as the relative contribution of circulating androgens in activating the AR. Downstream targets of the AR induced by exposure to the obese sera are being identified both by Western blot and microarray analyses. Conclusions: These results suggest that obesity-related circulating factors induce activation of the androgen receptor in prostate cancer cells. This activation appears to be both ligand dependent (androgens) and ligand independent (growth factors). These data suggest that one mechanism by which obesity influences time to progression and hormone independence is through activation of the androgen receptor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1968. doi:10.1158/1538-7445.AM2011-1968

Elucidating the mechanism of action of tributyltin (TBT) in zebrafish

Tributyltin (TBT), an antifouling agent, has been implicated in the masculinization of fish species worldwide, but the masculinizing mechanism is not fully understood. We have examined the actions of TBT as an endocrine disruptor in zebrafish ( Danio rerio). In HeLa cells transiently co-transfected with plasmid constructs containing the zebrafish estrogen receptors (zfERα, zfERβ 1 and zfERβ 2) and the zebrafish estrogen response element (zfERE-tk-luc), ethinyl estradiol (EE2) induced luciferase activity 4 to 6-fold and was inhibited by TBT. In HeLa cells transiently co-transfected with the zebrafish androgen receptor (zfAR) and the murine androgen receptor response element (ARE-slp-luc), testosterone induced luciferase activity was not inhibited by TBT. In HeLa cells co-transfected with zfERα, zfERβ 1 and zfERβ 2 and a plasmid containing zebrafish aromatase (zfCyp19b-luc), TBT inhibited luciferase activity. In zebrafish exposed to 1 mg/kg and 5 mg/kg TBT in vivo, there was a increase in liver sulfotransferase and a decrease acyl-CoA testosterone acyltransferase activity. Real-time PCR analysis of sexual differentiation markers in fish exposed to TBT in vivo revealed a tissue-specific response. In brain there was increased production of Sox9, Dax1, and SF1 mRNA, an androgenizing effect, while in the liver there was increased production of Dax1, Cyp19a and zfERβ 1 mRNA but decreased production of Sox9 mRNA, a feminizing effect. In the gonads there was increased production of zfERα and zfCyp19a mRNA, again a feminizing effect. TBT has an overall masculinizing effect but the masculinizing effect is tempered by a feminizing effect on gene transcription in certain tissues. These results are discussed in the context of TBT as an endocrine disruptor in zebrafish.

Identification of ETS-like transcription factor 4 as a novel androgen receptor target in prostate cancer cells

Transcriptional control by androgens via androgen receptor (AR) is strongly involved in prostate cancer development, but the critical target genes have remained elusive. We have characterized E twenty-six-like transcription factor 4 (ELK4) (also known as serum response factor accessory protein 1) as a novel AR target in human prostate cancer cells. In-silico screening identified three putative AR response elements (AREs) within -10 kb from the transcription start site of ELK4. Both ARE1 at -167/-153 and ARE2 at -481/-467 bound AR in vitro and mediated androgen induction as isolated elements in transcription assays in non-prostate cells. However, merely the ARE2 that cooperates with a proximal forkhead box A1-binding site was critical for the AR-dependent activation of ELK4 promoter in prostate cancer cells. Preferential loading of holo-AR onto the ARE2 and concomitant recruitment of RNA polymerase II onto the ELK4 promoter was confirmed in prostate cancer cells by chromatin immunoprecipitation. Database searches indicated that the expression of ELK4 is markedly increased in prostate cancers relative to normal prostates. Moreover, prostate cancer tissue immunostainings showed that nuclear ELK4 levels are significantly increased in androgen-refractory prostate cancers compared to untreated tumours. Reduction of the amount of ELK4 in LNCaP cells by RNAi retarded cell growth. In conclusion, ELK4 is a direct AR target in prostate cancer cells. Androgens may thus contribute to the growth of prostate cancer via influencing ELK4 levels.

Probasin promoter assembles into a strongly positioned nucleosome that permits androgen receptor binding

The promoter of the murine probasin (PB) gene exhibits strong androgen receptor (AR)-specific and tissue-specific regulation and is considered a promising candidate for gene therapy treatment of advanced prostate cancer. To characterize the determinants of chromatin specificity of the PB promoter with the AR we initially investigated the in vitro interactions of recombinant AR DNA binding domain (AR-DBD) with reconstituted nucleosomes incorporating the proximal PB promoter (nucleotides −268 to −76). We demonstrate that a DNA fragment of this promoter region exhibits strong nucleosome positioning. The phased DNA sequence protected by the histone octamer includes four androgen receptor response elements (AREs) which are arranged as two sets of class I and class II sites spaced approximately 90 bp apart. Class I AREs form classical contacts with the AR, whereas class II AREs contain atypical binding sequences and have been shown to stabilize AR binding to adjacent class I sites, resulting in synergistic transcriptional activation and increased hormone sensitivity. We used DNase 1 footprinting and electrophoretic mobility shift assays (EMSA) to show that the AR-DBD binds to its cognate sequences independently of their nucleosomal organization. In addition, we show that the ability of the AR-DBD to interact with the nucleosomal PB promoter is not affected by histone acetylation. Thus the AR-DBD is able to bind to its cognate sequences within the PB promoter in a way that is indifferent to the presence or absence of histones and nucleosomal structure.

Androgen Receptor–Mediated Regulation of the α-Subunit of the Epithelial Sodium Channel in Human Kidney

Rodents studies suggest that androgens are involved in sex-specific differences in blood pressure. In humans, there is no difference in blood pressure between boys and girls, but after puberty, blood pressure increases more in men than in women. We investigated androgen-dependent regulation of the alpha-subunit of the epithelial sodium channel (alphaEnaC) in human kidney and in the human renal cell line immortalized human renal proximal tubular cell line (HKC-8). We used microarray technique to analyze androgen-dependent gene regulation and performed quantitative RT-PCR for verification. Promoter constructs for human alphaENaC were used in transfection studies to analyze the regulation by testosterone. We investigated the in vivo effect of testosterone on alphaENaC in a rat model and used the mouse collecting duct cell line M-1 for transepithelial electrophysiological measurements. The androgen receptor (AR) was expressed in male kidney and HKC-8 cells. AlphaENaC mRNA expression increased 2- to 3-fold after treatment with testosterone in HKC-8 cells. The induction by testosterone was completely blocked by adding the AR antagonist flutamide. Analysis of the alphaENaC promoter sequence identified a putative AR response element (ARE) located 140 nucleotides upstream from the transcription start site. HKC-8 cell transfection studies showed that testosterone directly upregulated gene expression via this ARE. In vivo, testosterone treatment of orchiectomized rats resulted in an increased renal alphaENaC mRNA expression. In testosterone-treated mouse M-1 cells, amiloride caused a significant stronger decrease in short circuit current than in control cells. These data show that alphaENaC expression is directly regulated by androgens in vitro and in vivo and highlight a potential mechanism explaining the reported gender differences in blood pressure.

Androgen Receptor Activation by Gs Signaling in Prostate Cancer Cells

The androgen receptor (AR) is activated in prostate cancer patients undergoing androgen ablative therapy and mediates growth of androgen-insensitive prostate cancer cells, suggesting it is activated by nonandrogenic factors. We demonstrate that activated alpha subunit of heterotrimeric guanine nucleotide-binding G(s) protein activates the AR in prostate cancer cells and also synergizes with low concentration of androgen to more fully activate the AR. The G alpha(s) activates protein kinase A, which is required for the nuclear partition and activation of AR. These data suggest a role for G alpha(s) and PKA in the transactivation of AR in prostate cancer cells under the environment of reduced androgen levels.

JNK interacting protein 1 (JIP-1) protects LNCaP prostate cancer cells from growth arrest and apoptosis mediated by 12-0-tetradecanoylphorbol-13-acetate (TPA).

12-0-tetradecanoylphorbol-13-acetate (TPA) stimulates protein kinase C (PKC) which mediates apoptosis in androgen-sensitive LNCaP human prostate cancer cells. The downstream signals of PKC that mediate TPA-induced apoptosis in LNCaP cells are unclear. In this study, we found that TPA activates the c-Jun NH2-terminal kinase (JNK)/c-Jun/AP-1 pathway. To explore the possible role that the JNK/c-Jun/AP-1 signal pathway has on TPA-induced apoptosis in LNCaP cells, we stably transfected the scaffold protein, JNK interacting protein 1 (JIP-1), which binds to JNK inhibiting its ability to phosphorylate c-Jun. TPA (10−9–10−7 mol l−1) caused phosphorylation of JNK in both wild-type and JIP-1-transfected (LNCaP-JIP-1) cells. It resulted in phosphorylation and upregulation of expression of c-Jun protein in the wild-type LNCaP cells, but not in the JIP-1-transfected LNCaP cells. In addition, upregulation of AP-1 reporter activity by TPA (10−9 mol l−1) occurred in LNCaP cells but was abrogated in LNCaP-JIP-1 cells. Thus, TPA stimulated c-Jun through JNK, and JIP-1 effectively blocked JNK. TPA (10−12–10−8 mol l−1) treatment of LNCaP cells caused their growth inhibition, cell cycle arrest, upregulation of p53 and p21waf1, and induction of apoptosis. All of these effects were significantly attenuated when LNCaP-JIP-1 cells were similarly treated with TPA. A previous study showed that c-Jun/AP-1 blocked androgen receptor (AR) signaling by inhibiting AR binding to AR response elements (AREs) of target genes including prostate-specific antigen (PSA). Therefore, we hypothesised that TPA would not be able to disrupt the AR signal pathway in LNCaP-JIP-1 cells. Contrary to expectation, TPA (10−9–10−8 mol l−1) inhibited DHT-induced AREs reporter activity and decreased levels of PSA in the LNCaP-JIP-1 cells. Taken together, TPA, probably by stimulation of PKC, phosphorylates JNK, which phosphorylates and increases expression of c-Jun leading to AP-1 activity. Growth control of prostate cancer cells can be mediated through the JNK/c-Jun pathway, but androgen responsiveness of these cells can be independent of this pathway, suggesting that androgen independence in progressive prostate cancer may not occur through activation of this pathway.