Increased Androgen Receptor Protein Research Articles

HC-1119, a deuterated Enzalutamide, inhibits Migration, Invasion and Metastasis of the AR-positive triple-negative breast Cancer cells

Triple-negative breast cancers (TNBCs) are aggressive, and they develop metastasis at earlier stages, relapse more frequently, and exhibits poorer prognosis than other subtypes of breast cancer. Due to the lack of estrogen receptor for endocrine therapy and HER2 for targeted therapy, new targeted therapies for TNBCs are urgently needed. Enzalutamide is a second-generation androgen receptor (AR) inhibitor, and HC-1119 is a new synthetic deuterated enzalutamide. Owing to the isotope effect, HC-1119 has many advantages over enzalutamide, including slow metabolism, high plasma concentration and low brain exposure. However, the efficacy of HC-1119 in inhibition of AR function in triple-negative breast cancer (TNBC) has not been studied. In this study, we found high-level AR expression in both Hs578T and SUM159PT TNBC cell lines. Activation of AR by dihydrotestosterone (DHT) in both cell lines increased AR protein, induced AR-nuclear localization, enhanced cell migration and invasion in culture, and promoted liver metastasis in mice. Importantly, cotreatment with HC-1119 of these cells efficiently abolished all of these effects of DHT on both Hs578T and SUM159PT cells. These results indicate that HC-1119 is a very effective new second-generation AR antagonist that can inhibit the migration, invasion and metastasis of the AR-positive TNBC cells.

Increased uterine androgen receptor protein abundance results in implantation and mitochondrial defects in pregnant rats with hyperandrogenism and insulin resistance

In this study, we show that during normal rat pregnancy, there is a gestational stage-dependent decrease in androgen receptor (AR) abundance in the gravid uterus and that this is correlated with the differential expression of endometrial receptivity and decidualization genes during early and mid-gestation. In contrast, exposure to 5α-dihydrotestosterone (DHT) and insulin (INS) or DHT alone significantly increased AR protein levels in the uterus in association with the aberrant expression of endometrial receptivity and decidualization genes, as well as disrupted implantation. Next, we assessed the functional relevance of the androgen-AR axis in the uterus for reproductive outcomes by treating normal pregnant rats and pregnant rats exposed to DHT and INS with the anti-androgen flutamide. We found that AR blockage using flutamide largely attenuated the DHT and INS-induced maternal endocrine, metabolic, and fertility impairments in pregnant rats in association with suppressed induction of uterine AR protein abundance and androgen-regulated response protein and normalized expression of several endometrial receptivity and decidualization genes. Further, blockade of AR normalized the expression of the mitochondrial biogenesis marker Nrf1 and the mitochondrial functional proteins Complexes I and II, VDAC, and PHB1. However, flutamide treatment did not rescue the compromised mitochondrial structure resulting from co-exposure to DHT and INS. These results demonstrate that functional AR protein is an important factor for gravid uterine function. Impairments in the uterine androgen-AR axis are accompanied by decreased endometrial receptivity, decidualization, and mitochondrial dysfunction, which might contribute to abnormal implantation in pregnant PCOS patients with compromised pregnancy outcomes and subfertility.Key messagesThe proper regulation of uterine androgen receptor (AR) contributes to anormal pregnancy process, whereas the aberrant regulation of uterine AR mightbe linked to polycystic ovary syndrome (PCOS)-induced pregnancy-relatedcomplications.In the current study, we found that during normal rat pregnancy there isa stage-dependent decrease in AR abundance in the gravid uterus and that thisis correlated with the differential expression of the endometrial receptivityand decidualization genes Spp1, Prl, Igfbp1,and Hbegf.Pregnant rats exposed to 5α-dihydrotestosterone (DHT) and insulin (INS)or to DHT alone show elevated uterine AR protein abundance and implantationfailure related to the aberrant expression of genes involved in endometrialreceptivity and decidualization in early to mid-gestation.Treatment with the anti-androgen flutamide, starting frompre-implantation, effectively prevents DHT + INS-induced defects in endometrialreceptivity and decidualization gene expression, restores uterine mitochondrialhomeostasis, and increases the pregnancy rate and the numbers of viablefetuses.This study adds to our understanding of the mechanisms underlying poorpregnancy outcomes in PCOS patients and the possible therapeutic use ofanti-androgens, including flutamide, after spontaneous conception.

Paxillin Is Required for Androgen Receptor Expression in Granulosa Cells

Androgens are important in female reproduction, as evident from studies of mouse ovary-specific androgen receptor knockout models characterized by sub-fertility and diminished ovarian reserve. Androgen activity specifically promotes granulosa cell proliferation and follicle progression. However, the molecular mechanisms mediating androgen activity in granulosa cells are unknown. Our lab previously showed that the cytoplasmic adaptor protein paxillin is required for full transcriptional activity by the androgen receptor (AR) in prostate cancer cells, therefore we examined how paxillin affects the androgen receptor in granulosa cells. We found that paxillin knockdown results in significantly reduced AR protein levels, independently of AR gene transcription in human granulosa-derived KGN cells. Similar to previous data from other cell types, we found that paxillin directly interacts with poly-A binding protein (PABP) in KGN cells using proximity ligation assay. Ligand binding further increases AR protein expression by reducing its degradation. Using immunofluorescence, we confirm that in both KGN and primary mouse granulosa cells, similarly to the prostate, ligand-bound AR is primarily localized in the nucleus. Based on this and previous studies, we propose that paxillin enhances AR mRNA translation through interaction with PABP, and ligand binding further increases AR protein level by nuclear retention, protecting from degradation in the cytoplasm. Our findings highlight a previously unrecognized role of paxillin in granulosa cells, where it may be an important target for modulating androgen activity in androgen-related disorders of female reproduction.

18F]-FDHT PET/CT as a tool for imaging androgen receptor expression in high-grade glioma.

BackgroundG lioblastoma (GBM) is associated with poor overall survival. Recently, we showed that androgen receptor (AR) protein is overexpressed in 56% of GBM specimens and AR antagonists induced dose-dependent death in several GBM cell lines and significantly reduced tumor growth and prolonged the lifespan of mice implanted with human GBM. 16β-18F-fluoro-5α-dihydrotestosterone ([18F]-FDHT) is a positron emission tomography (PET) tracer used to detect AR expression in prostate and breast cancers. This study was aimed at exploring the ability of [18F]-FDHT-PET to detect AR expression in high-grade gliomas.MethodsTwelve patients with suspected high-grade glioma underwent a regular workup and additional dynamic and static [18F]-FDHT-PET/CT. Visual and quantitative analyses of [18 F]-FDHT kinetics in the tumor and normal brain were performed. Mean and maximum (max) standardized uptake values (SUVs) were determined in selected volumes of interest. The patients had surgery or biopsy after PET/CT. AR protein was analyzed in the tumor samples by western blot. Fold change in AR expression was calculated by densitometry analysis. Correlation between imaging and AR protein samples was determined.ResultsIn six of the 12 patients, [18 F]-FDHT uptake was significantly higher in the tumor than in the normal brain. These patients also had increased AR protein expression within the tumor. Pearson correlation coefficient analysis for the tumor-to-control normal brain uptake ratio in terms of SUVmean versus AR protein expression was positive and significant (R = 0.84; P = .002).Conclusion[18 F]-FDHT-PET/CT could identify increased AR expression in high-grade glioma.

A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

Triple-negative breast cancer (TNBC) is an aggressive subtype with peak recurrence as metastatic disease within the first few years of diagnosis. Androgen receptor (AR) expression is increased in anchorage-independent cells in TNBC preclinical models. Both AR knockdown and inhibition lead to reduced TNBC invasion in vitro, reduced tumorigenicity, and less recurrence in vivo in preclinical models. Transforming growth factor β (TGFβ) pathway gene signatures also increased during anchorage-independent survival both in vitro and in vivo in preclinical models and in circulating tumor cells (CTCs) from patients during emergence of chemo resistant disease. We hypothesized that a positive loop between AR and TGFβ signaling facilitates TNBC anchorage-independent survival. We find that multiple components of the TGFβ pathway, including TGFβ1 and 3, as well as pathway activity measured by nuclear localization and transcriptional activity of phosphorylated Smad3, are enhanced in anchorage-independent conditions. Further, exogenous TGFβ increased AR protein while TGFβ inhibition decreased AR and TNBC viability, particularly under anchorage-independent culture conditions. ChIP-seq experiments revealed AR binding to TGFB1 and SMAD3 regulatory regions in MDA-MB-453 cells. In clinical datasets, TGFB3 and AR positively correlate and high expression of both genes together corresponded to significantly worse recurrence-free and overall survival in both ER-negative and basal-like breast cancer. Finally, inhibiting both AR and TGFβ decreased cell survival, particularly under anchorage-independent conditions. These findings warrant further investigations into whether combined inhibition of AR and TGFβ pathways might decrease metastatic recurrence rates and mortality from TNBC.

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

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

Interference with the androgen receptor protein stability in therapy-resistant prostate cancer.

The androgen receptor (AR) plays a central role in the pathogenesis of prostate cancer (PCa). Most PCa cases develop eventually from an androgen-dependent stage to castration-resistant prostate cancer (CRPC) with AR-signaling still being active. Thus, inhibition of AR remains a well-established promising drug target in CRPC. However, despite the improvements of current treatment for CRPC by targeting the AR, the evolution of adaptive AR-signaling leads to therapy-resistant CRPC. Treatment failure is based mostly on the inability to keep AR under long-term restraint due to adaptive responses of AR-signaling. One underlying mechanism appears to be the increased AR protein stability. Therefore, the regulation of AR protein stability and its degradation is another interesting path that could enhance our knowledge of carcinogenesis and tumor evolution possibly leading to novel therapeutic targets. In this review, we discuss various molecular mechanisms and factors that stabilize AR protein levels directly or indirectly. We summarize novel approaches to interfere with AR stability including targeting the glucocorticoid receptor (GR), heat shock proteins, and co-chaperones as well as E3-ligases using small chimeric molecules. These novel approaches in combination with antiandrogen treatment inhibit PCa growth through the regulation of AR protein levels.

Targeting the androgen receptor (AR) with AR degradation enhancer ASC-J9® led to increase docetaxel sensitivity via suppressing the p21 expression

Chemotherapy with docetaxel remains the effective therapy to suppress castration resistant prostate cancer (CRPC) in some patients. However, most chemotherapy with docetaxel eventually fails with the development of docetaxel resistance after 18-weeks of treatment. Here we found docetaxel treatment might have an adverse effect of increasing the androgen receptor (AR) protein level in the CRPC cells, and combining docetaxel with anti-AR therapy using AR-shRNA or the AR degradation enhancer ASC-J9® may increase docetaxel sensitivity to better suppress the CRPC cell growth. Mechanism dissection found docetaxel might have the adverse effect of increasing the AR protein stability via suppressing the AR ubiquitination due to the increased AR phosphorylation. The consequence of such increased AR protein may then lead to increase p21 expression via transcriptional regulation. Preclinical studies with in vitro cells lines also demonstrated that targeting AR with ASC-J9® led to suppressing the AR-increased p21 expression to improve the docetaxel sensitivity in the CRPC cells that already developed docetaxel resistance. Together, these results suggest that a combined therapy of docetaxel and ASC-J9® is a novel therapy to better suppress CRPC in patients that already developed docetaxel resistance.

A reciprocal feedback between the PDZ binding kinase and androgen receptor drives prostate cancer

Elucidation of mechanisms underlying the increased androgen receptor (AR) activity and subsequent development of aggressive prostate cancer (PrCa) is pivotal in developing new therapies. Using a systems biology approach, we interrogated the AR-regulated proteome and identified PDZ binding kinase (PBK) as a novel AR-regulated protein that regulates full-length AR and AR variants (ARVs) activity in PrCa. PBK overexpression in aggressive PrCa is associated with early biochemical relapse and poor clinical outcome. In addition to its carboxy terminus ligand-binding domain, PBK directly interacts with the amino terminus transactivation domain of the AR to stabilise it thereby leading to increased AR protein expression observed in PrCa. Transcriptome sequencing revealed that PBK is a mediator of global AR signalling with key roles in regulating tumour invasion and metastasis. PBK inhibition decreased growth of PrCa cell lines and clinical specimen cultured ex vivo. We uncovered a novel interplay between AR and PBK that results in increased AR and ARVs expression that executes AR-mediated growth and progression of PrCa, with implications for the development of PBK inhibitors for the treatment of aggressive PrCa.

Association between cadmium and androgen receptor protein expression differs in prostate tumors of African American and European American men

Cadmium is a known carcinogen that has been implicated in prostate cancer, but how it affects prostate carcinogenesis in humans remains unclear. Evidence from basic science suggests that cadmium can bind to the androgen receptor causing endocrine disruption. The androgen receptor is required for normal prostate development and is the key driver of prostate cancer progression. In this study, we examined the association between cadmium content and androgen receptor protein expression in prostate cancer tissue of African American (N = 22) and European American (N = 30) men. Although neither overall tumor cadmium content (log transformed) nor androgen receptor protein expression level differed by race, we observed a race-cadmium interaction with regard to androgen receptor expression (P = 0.003) even after accounting for age at prostatectomy, smoking history, and Gleason score. African American men had a significant positive correlation between tumor tissue cadmium content and androgen receptor expression (Pearson correlation = 0.52, P = 0.013), while European Americans showed a non-significant negative correlation between the two (Pearson correlation = −0.19, P = 0.31). These results were unchanged after further accounting for tissue zinc content or dietary zinc or selenium intake. African American cases with high-cadmium content (>median) in tumor tissue had more than double the androgen receptor expression (0.021 vs. 0.008, P = 0.014) of African American men with low-cadmium level. No difference in androgen receptor expression was observed in European Americans by cadmium level (high 0.015 vs. low 0.011, P = 0.30). Larger studies are needed to confirm these results and if upheld, determine the biologic mechanism by which cadmium increases androgen receptor protein expression in a race-dependent manner. Our results suggest that cadmium may play a role in race disparities observed in prostate cancer.

Abstract 1854: Generation and characterization of mouse model of Pmepa1 conditional knockout in prostate epithelia

Abstract Introduction and Objectives: Prostate cancer (CaP) is the most common non-skin malignancy diagnosed in American men and desfunctions of androgen receptor (AR) plays an essential role in prostate tumorigenesis. PMEPA1 is an androgen and TGF-β -induced gene abundant in prostate, which was identified to degrade AR protein via NEDD4 E3 ligase mediated pathway. Reduced or loss of PMEPA1 expression, commonly detected in prostate tumors, led to increased AR protein and activated AR signaling. PMEPA1 inhibited TGF-β receptor 1 meditated signaling by a negative feedback loop. It was reported that loss of PMEPA1 facilitates bone metastasis of CaP through blocking TGF-β signaling. To further investigating the biological function of PMEP1 gene in CaP tumorigenesis, particularly via AR and TGF-β, we generated Pmepa1 prostate conditional knockout mouse model. Methods: C57BL/6 mice were utilized for generation of Pmepa1 conditional knockout model. Pmepa1 gene was conditionally deleted in mouse prostate epithelium by ARR2PB-Cre-Lox system. Male mice of genotypes of wild-type, Pmepa1 flox/wild-type-ARR2PB-Cre, Pmepa1 flox/flox-ARR2PB-Cre were euthanized at the age of 3 months for analysis. The prostate tissue was collected for frozen and formalin fixation for histology analysis, and other major organs including heart, lung, liver, spleen, bladder and testis were also collected for control. The tissue were sectioned and stained with Hematoxylin & Erosin (H&E). Total RNA and protein were harvested by homogenizing tissue. The protein levels of Pmepa1, Ar and Nkx3.1 were analyzed by immunohistochemistry (IHC) and immunoblotting, and the transcript levels of these genes were evaluated by in situ hybridization (ISH) and quantitative-PCR (Q-PCR). Results: The expression of Pmepa1 protein was found to focus on lateral lobe, consistent with Ar protein expression pattern. The data of Q-PCR and ISH showed that transcript level of Pmepa1 was dramatically suppressed in Pmepa1 flox/flox and flox/wild-type ARR2PB-Cre mice. Compared to wild-type ones, Pmepa1 protein was shown significantly decreased in heterozygous and homozygous knockout mice by immunoblotting and IHC staining. And the protein level of Ar was stronger in Pmepa1 conditional deleted mouse. There was no dramatic morphology change in mouse prostate gland by H&E staining. Conclusions: Conditional deletion of Pmepa1 gene in mouse prostate epithelium led to enhanced Ar protein and activating Ar signaling. The effects of Pmepa1 gene loss in mouse prostate gland on other signaling such as TGF-β and prostate tumorigenesis are needed to be further evaluated. Funding: This study was supported by CPDR, USUHS, HU0001-10-2-0002 to DGM. Citation Format: Hua Li, Shashwat Sharad, Talai Barbiev, Yingjie Song, Denise Young, Taduru Sreenath, Albert Dobi, Shiv Srivastava. Generation and characterization of mouse model of Pmepa1 conditional knockout in prostate epithelia [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 1854. doi:10.1158/1538-7445.AM2017-1854

Antidepressive effects of ginsenoside Rg1 via regulation of HPA and HPG axis

BackgroundHypothalamic–pituitary–adrenal (HPA) axis hyperactivity is a well-established pathological feature of major depression, accompanied by the persistent increase of glucocorticoid level and the dysfunction of hypothalamic–pituitary–gonadal (HPG) axis. Ginsenoside Rg1 (Rg1) is one of the most active ingredients of Panax ginseng, which has various biological activity. ObjectiveThis study aimed to investigate the antidepressive effects of Rg1 and elucidate its impact on neuroendocrine system. MethodsThe antidepressive effects of Rg1 were first analysed in mice, and was further identified in the chronic-unpredictable-mild-stress (CUMS) model and the gonadectomized (GDX) model. The effects of Rg1 on depression-like behaviour were analysed by the forced swimming test (FST), tail suspension test (TST), sucrose preference test, and measurement of pentobarbital-induced sleep. The serum corticosterone and testosterone levels were detected by ELISA. The protein levels of glucocorticoid receptor (GR) and androgen receptor (AR) were analysed by western blot and immunohistochemistry analysis. ResultsRg1 significantly decreased the immobility time of mice in FST and TST. Furthermore, Rg1 alleviated anhedonia and hopelessness, decreased serum corticosterone level, and increased serum testosterone level, and the GR protein level in the PFC and hippocampus of the CUMS-treated rats. Moreover, Rg1 improved sleep disruption, down-regulated the serum corticosterone level, and increased AR protein level in the PFC of the GDX-treated mice. ConclusionTogether, these studies suggest that Rg1 displayed antidepressant activity through the modulation of the HPA and the HPG axis. These findings provide new mechanism involved in the antidepressive effects of Rg1 and propose theoretical clues for clinical therapies.

A drug-drug interaction (DDI) study to assess the effect of oral galeterone on the pharmacokinetics (PK) of oral midazolam.

316 Background: Galeterone (gal) is a semisynthetic steroid that targets androgen receptor (AR) signaling via increased AR protein degradation, inhibition of CYP17 activity, and inhibition of androgen binding to AR. Gal is a novel potential treatment for prostate cancer. In vitro, gal competitively inhibits CYP3A4 (IC50 = 5.5 μM; midazolam as substrate). This clinical study evaluated whether multiple daily doses of gal alter the single-dose PK of midazolam, a sensitive probe substrate for functional CYP3A4 intestinal and hepatic activity. Methods: In an open-label, fixed sequence, DDI study, 18 healthy male volunteers received midazolam 2 mg Day 1 and Day 5 and gal 2550 mg Days 2-5. Midazolam plasma PK was determined on Days 1 and 5. The effect of gal on the natural log-transformed Cmax, AUC0-inf, and AUC0-t of midazolam was assessed with a linear mixed-effects model; point estimates for geometric means, geometric mean ratios with 90% confidence intervals were determined. Safety was also monitored. Results: The Tmax of midazolam was not altered by gal coadministration. The < 2-fold increases in midazolam Cmax and AUCs were statistically significant. For Cmax, the point estimate of the geometric least squares (LS) mean ratio between the 2 treatments was 1.25 (90% CI: 1.05, 1.48). Higher ratios were observed for the AUC ratios, with LS mean ratio for AUC0-t of 1.57 (90% CI: 1.43, 1.73) and for AUC0-inf of 1.58 (90% CI: 1.42, 1.75). The mean midazolam t1/2 was approximately 76% higher with gal coadministration. Multiple doses of gal were well tolerated with no apparent effect on the safety of midazolam. Conclusions: Per FDA guidance, < 2-fold increases in midazolam Cmax and AUCs observed with gal coadministration support its classification as a weak inhibitor of CYP3A4. Given the similar or higher in vitro IC50’s for CYP2C8 and CYP2C19, respectively, a comparable or lesser degree of enzyme inhibition is expected for gal 2550 mg daily. Based on these results, concurrent CYP3A4, CYP2C8, and CYP2C19 substrates are not contraindicated in patients taking gal; caution is advised in patients receiving or initiating sensitive CYP3A4, CYP2C8, and CYP2C19 substrates, especially those with a narrow therapeutic range.

Reciprocal feedback inhibition of the androgen receptor and PI3K as a novel therapy for castrate-sensitive and -resistant prostate cancer.

Gain-of-function of the androgen receptor (AR) and activation of PI3K/AKT/mTOR pathway have been demonstrated to correlate with progression to castration-resistant prostate cancer (CRPC). However, inhibition of AR or PI3K/mTOR alone results in a reciprocal feedback activation. Therefore, we hypothesized that dual inhibition of the AR and PI3K/mTOR pathway might lead to a synergistic inhibition of cell growth and overcome drug resistance in CRPC. Here, we reported that androgen-depletion increased AR protein level and Akt phosphorylation at Ser473 and Thr308 in LNCaP cells. Moreover, we developed resistance cell lines of LNCaP to Enzalutamide (or MDV3100), an AR inhibitor (named as LNCaP 'MDV-R') and PF-04691502, a PI3K/mTOR inhibitor (named as LNCaP 'PF-R'). MTS analysis showed that LNCaP 'PF-R' was strongly resistant to Enzalutamide treatment, and on the other hand, LNCaP 'MDV-R' was 6-fold resistant to PF-04691502 treatment. Mechanistically, LNCaP 'MDV-R' cells had significantly reduced AR, loss of PSA and increase Akt activity in contrast with LNCaP 'PF-R' cells. Combined inhibition of PI3K/mTOR and AR pathways with a variety of small molecular inhibitors led to a synergistic suppression of cell proliferation and a profound increase of apoptosis and cell cycle arrest in both androgen-dependent LNCaP and independent CRPC 22Rv1 cell lines. In conclusion, this study provides preclinical proof-of-concept that the combination of a PI3K/mTOR inhibitor with an AR inhibitor results in a synergistic anti-tumor response in non-CRPC and CRPC models.

Expression and regulation of androgen receptor in the mouse uterus during early pregnancy and decidualization.

The androgen receptor (AR) is a ligand-activated transcription factor that is important for both the male and female reproductive systems. The expression and regulation of AR in the uterine endometrium during early pregnancy and decidualization remain relatively under-investigated, so we sought to immunohistochemically examine the spatiotemporal expression of AR in mouse uteri during the peri-implantation period as well as in response to specific steroid hormones. AR protein was found in the nuclei of uterine stromal cells starting on pregnancy Days 1 and 2, with its abundance increasing on Days 3 and 4. From pregnancy Days 5 to 9, however, the expression of AR markedly declined in stromal zones of uteri. No signal was detected in the decidualized cells surrounding the site of embryo implantation; moreover, no AR immunostaining was observed in decidualized uterine cells in an artificial oil-induced model of decidualization. Progesterone significantly inhibited AR protein expression, whereas estrogen dramatically elevated AR abundance in the stroma of ovariectomized mouse uteri. Taken together, our results are the first to demonstrate that decidualization and progesterone significantly inhibited the AR protein expression in vivo, whereas estrogen increased AR protein levels in the stromal cells of mouse uteri. These responses might be advantageous for the proliferation and differentiation of uterine stroma and for embryo implantation during early pregnancy.

Effects of resistin on ovarian folliculogenesis and steroidogenesis in the vespertilionid bat, Scotophilus heathi

The bat Scotophilus heathi exhibit prolonged anovulatory condition known as delayed ovulation coinciding with the period of extensive fat accumulation. The present study was undertaken to find out whether extensive accumulation of fat in S. heathi is responsible for suppression of ovarian activity by increasing production of adipokine resistin in the bat. This was achieved by (a) investigating variation in serum resistin level in relation to the changes in the body fat mass and (b) evaluating the effect of resistin treatment on ovarian activity with reference to steroid synthesis. An attempt was also made to determine whether resistin mediate its effects on ovary through signal transducer and activator of transcription 3 (STAT3) signaling mechanism. The results showed significant seasonal variation in serum resistin level with the peak level coinciding with the period of maximum fat accumulation, high circulating androgen level and period of anovulation. The treatment with resistin to the bat caused increase in androstenedione due to stimulatory effects on 3β-hydroxysteroid dehydrogenase, but decrease in estradiol level due to inhibitory effect on aromatase. Resistin treatment increased androgen receptor protein together with increased insulin receptor but not through conventional luteinizing hormone receptor and steroidogenic acute regulatory protein mediated pathways. This study further showed that resistin treatment increases androstenedione synthesis and up-regulates insulin receptor in the ovary through STAT3 mediated pathways. These findings suggest that obese women through increased resistin synthesis may causes development of non-ovulatory antral follicles through insulin receptor signaling cascade.

Methylation of the PMEPA1 gene, a negative regulator of the androgen receptor in prostate cancer

The prostate transmembrane protein androgen induced 1 (PMEPA1) gene is highly expressed in prostate epithelial cells and is a direct transcriptional target for the androgen receptor (AR). AR protein levels are controlled by the AR-PMEPA1 negative feedback loop through NEDD4-E3 ligase. Reduced expression of PMEPA1 observed in prostate tumors, suggests that loss of PMEPA1 may play critical roles in prostate tumorigenesis. This study focuses on epigenetic mechanisms of reduced PMEPA1 expression in the cancer of the prostate (CaP). Benign (n = 77) and matched malignant (n = 77) prostate epithelial cells were laser capture micro-dissected from optimum cutting temperature embedded frozen prostate sections from 42 Caucasian American (CA) and 35 African American (AA) cases. Purified DNA specimens were analyzed for CpG methylation of the PMEPA1 gene. PMEPA1 mRNA expression levels were evaluated by qRT-PCR. Analysis of PMEPA1 methylation and mRNA expression in the same tumor cell populations indicated a significant inverse correlation between mRNA expression and methylation in CaP (P = 0.0115). We noted higher frequency of CpG methylation within the evaluated first intronic region of the PMEPA1 gene in prostate tumors of CA men as compared with AA. In CaP cell lines, PMEPA1 expression was induced and AR protein levels were diminished in response to treatment with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (decitabine). Cell culture-based studies demonstrated that decitabine restores PMEPA1 expression in AR-positive CaP cell lines. This report reveals the potential role of PMEPA1 gene methylation in the regulation of AR stability. Thus, downregulation of PMEPA1 may result in increased AR protein levels and function in CaP cells, contributing to prostate tumorigenesis.

Testosterone regulates keratin 33B expression in rat penis growth through androgen receptor signaling.

Androgen therapy is the mainstay of treatment for the hypogonadotropic hypogonadal micropenis because it obviously enhances penis growth in prepubescent microphallic patients. However, the molecular mechanisms of androgen treatment leading to penis growth are still largely unknown. To clarify this well-known phenomenon, we successfully generated a castrated male Sprague Dawley rat model at puberty followed by testosterone administration. Interestingly, compared with the control group, testosterone treatment stimulated a dose-dependent increase of penis weight, length, and width in castrated rats accompanied with a dramatic recovery of the pathological changes of the penis. Mechanistically, testosterone administration substantially increased the expression of androgen receptor (AR) protein. Increased AR protein in the penis could subsequently initiate transcription of its target genes, including keratin 33B (Krt33b). Importantly, we demonstrated that KRT33B is generally expressed in the rat penis and that most KRT33B expression is cytoplasmic. Furthermore, AR could directly modulate its expression by binding to a putative androgen response element sequence of the Krt33b promoter. Overall, this study reveals a novel mechanism facilitating penis growth after testosterone treatment in precastrated prepubescent animals, in which androgen enhances the expression of AR protein as well as its target genes, such as Krt33b.

Abstract 3576: USP22 modulates AR activity to critically regulate prostate cancer progression.

Abstract Recently, the deubiquitylase Ubiquitin-specific protease 22 (USP22) was identified as a member of the 11 gene ‘death-from-cancer’ signature, which can predict poor response to therapy and/or propensity toward metastasis of multiple tumor types, including breast and prostate. However, the mechanism for how USP22 impacts cancer is currently unknown. Our new data implicate a specific role for USP22 in hormone-dependent cancers, especially prostate cancer (PCa) where USP22 expression is elevated in primary and metastatic PCa. USP22 has an established role in regulating oncogenic c-Myc activity as it is recruited by c-Myc for transcriptional activation through deubiquitylation of histones, and is necessary for c-Myc-mediated cell transformation. Similarly, our data in PCa cells demonstrates that USP22 controls c-Myc activity but does not affect expression levels. Additional data has also identified USP22 as a critical effector of androgen receptor (AR) levels and output. This is of great clinical relevance since PCa is intricately dependent on AR signaling for disease initiation and progression to castrate resistant disease (CRPC). First, USP22 overexpression in hormone-dependent and CRPC cell lines increased AR protein expression and activity, but did not perturb transcript. Second, in the absence of ligand, USP22 upregulation resulted in enhanced AR recruitment to target genes, as determined by chromatin immunoprecipitation, which corresponded with significant enhancement of cell proliferation and BrdU incorporation. Third, USP22 depletion resulted in attenuated AR activity and caused a significant reduction in AR protein levels that could be attributed to protein stability. Importantly, in the CRPC setting, USP22 depletion dramatically inhibited in vivo tumor growth while expression correlated with decreased time for tumor regression. In sum, the data suggest that USP22 upregulation is sufficient for aberrant c-Myc activity, altered AR expression and activity, and establishment of a CRPC phenotype. We propose that modulation of UPS22 expression and/or activity may present a novel platform to achieve combinatorial suppression of AR and c-Myc function, and therefore could be potentially developed as a novel approach for treatment of PCa. Citation Format: Randy Schrecengost, Jeffry Dean, Tim Stanek, Ruth Birbe, Jessica Hicks, Tapio Visakorpi, Angelo DeMarzo, Steve McMahon, Karen Knudsen. USP22 modulates AR activity to critically regulate 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 3576. doi:10.1158/1538-7445.AM2013-3576

Estrogen receptor β expression and androgen receptor phosphorylation correlate with a poor clinical outcome in hormone-naïve prostate cancer and are elevated in castration-resistant disease

Patients with advanced prostate cancer (PC) are usually treated with androgen withdrawal. While this therapy is initially effective, nearly all PCs become refractory to it. As hormone receptors play a crucial role in this process, we constructed a tissue microarray consisting of PC samples from 107 hormone-naïve (HN) and 101 castration-resistant (CR) PC patients and analyzed the androgen receptor (AR) gene copy number and the protein expression profiles of AR, Serin210-phosphorylated AR (pAR(210)), estrogen receptor (ER)β, ERα and the proliferation marker Ki67. The amplification of the AR gene was virtually restricted to CR PC and was significantly associated with increased AR protein expression (P<0.0001) and higher tumor cell proliferation (P=0.001). Strong AR expression was observed in a subgroup of HN PC patients with an adverse prognosis. In contrast, the absence of AR expression in CR PC was significantly associated with a poor overall survival. While pAR(210) was predominantly found in CR PC patients (P<0.0001), pAR(210) positivity was observed in a subgroup of HN PC patients with a poor survival (P<0.05). Epithelial ERα expression was restricted to CR PC cells (9%). ERβ protein expression was found in 38% of both HN and CR PCs, but was elevated in matched CR PC specimens. Similar to pAR(210), the presence of ERβ in HN patients was significantly associated with an adverse prognosis (P<0.005). Our results strongly suggest a major role for pAR(210) and ERβ in HN PC. The expression of these markers might be directly involved in CR tumor growth.