Importance Of Androgen Receptor Research Articles

Harnessing androgen receptor: Revolutionizing diabetes treatment in men with selective androgen receptor modulators

Diabetes is a chronic metabolic disorder. Among different types, type 2 diabetes mellitus (T2DM) is the most prevalent accounting for 90 % to 95 % of all reported cases. T2DM is more prevalent in men compared to women. According to several literature reports, men with low testosterone levels and/or decreased androgen receptor (AR) expression are prone to T2DM. Consistently, androgen therapy was effective in treating T2DM in several preclinical and clinical studies. Activation of AR has been shown to induce the gene expression of NGN3 and NEUROD1 which helps in pancreatic β-cell differentiation, regeneration, and proliferation. AR activation can also increase the gene expression of glucose transporter (GLUT4) in the hepatocytes, skeletal muscle fibers, and adipocytes. An increase in GLUT4 expression could augment the activity of insulin and could help to reduce the insulin dose. However, testosterone therapy even for the short term was found to produce significant adverse effects. The rate-limiting being the increase in the hematocrit value. Hence, to minimize or avoid the serious adverse effects associated with androgen therapy, selective androgen receptor modulators (SARMs) could be employed to harness the goodness of the AR. A preliminary study based on the network analysis of the genes associated with T2DM has also pointed towards the importance of AR in T2DM.

The importance of androgen receptors in breast cancer.

Breast cancer (BC) is the most common malignancy among women worldwide, and one of the leading causes of cancer-related deaths in females. For the breast malignant tumors there are numerous targeted therapies, depending on the receptors expressed. Regulating the process of epithelial-mesenchyme transcription, the steroid nuclear receptors are important in invasion and progression of BC cells. Till now, it is known that androgen receptor (AR) is present in about 60-80% of BC cells but, unfortunately, there is no targeted therapy available yet. We revised the recent literature that included the AR mechanism of action in patients diagnosed with breast cancer, the preclinical, retrospective and clinical studies and the aspects related to the prognosis of these patients, depending on the molecular subtype. A total of 12 articles were eligible for this review. AR positivity was assessed using immunohistochemistry. Herein, neither 1 nor 10% cut-points were robustly prognostic. AR was an independent prognostic marker of BC outcome, especially in triple negative BC group. AR is a potential targeted pathway which can improve the prognostic of AR positive patients with BC. Further preclinical and clinical studies are necessary to clarify the mechanism of action and to establish the drugs which can be used, either alone or in combination.

Dissociation of disease onset, progression and sex differences from androgen receptor levels in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder caused by loss of motor neurons. ALS incidence is skewed towards males with typically earlier age of onset and limb site of onset. The androgen receptor (AR) is the major mediator of androgen effects in the body and is present extensively throughout the central nervous system, including motor neurons. Mutations in the AR gene lead to selective lower motor neuron degeneration in male spinal bulbar muscular atrophy (SBMA) patients, emphasising the importance of AR in maintaining motor neuron health and survival. To evaluate a potential role of AR in onset and progression of ALS, we generated SOD1G93A mice with either neural AR deletion or global human AR overexpression. Using a Cre-LoxP conditional gene knockout strategy, we report that neural deletion of AR has minimal impact on the disease course in SOD1G93A male mice. This outcome was potentially confounded by the metabolically disrupted Nestin-Cre phenotype, which likely conferred the profound lifespan extension observed in the SOD1G93A double transgenic male mice. In addition, overexpression of human AR produced no benefit to disease onset and progression in SOD1G93A mice. In conclusion, the disease course of SOD1G93A mice is independent of AR expression levels, implicating other mechanisms involved in mediating the sex differences in ALS. Our findings using Nestin-Cre mice, which show an inherent metabolic phenotype, led us to hypothesise that targeting hypermetabolism associated with ALS may be a more potent modulator of disease, than AR in this mouse model.

Abstract 1017: Novel inhibitors of AR-v7 nuclear import: new therapeutic opportunities for CRPC

Abstract Reactivation of androgen receptor (AR) signaling by active splice variants (AR-Vs) is one of key drivers of castration resistant prostate cancer (CRPC). AR-v7 is the most prevalent AR-V and its expression has been clinically associated with poor overall survival, resistance to the AR inhibitors enzalutamide and abiraterone, as well as taxane resistance. Given that treatment with AR inhibitors and taxanes are the only effective therapeutic modalities in CRPC, development of specific AR-v7 inhibitors is urgently needed. Mechanistically, AR-v7 re-activates AR signaling by being constitutively active in the nucleus. While taxane chemotherapy inhibits the nuclear import of AR which is significantly associated with clinical outcomes in CRPC, it has no effect on AR-v7 nuclear localization and activity. Mechanistically, AR-v7 lacks the microtubule-binding domain and—unlike AR—does not utilize the canonical importin-α/β pathway, or RanGTP for nuclear import. Using wheat germ agglutinin to block active protein nuclear uptake resulted in AR-v7 cytoplasmic sequestration, indicating a requirement for an alternative transport receptor. Further, mutation of AR-v7 dimerization domain (D-box) led to its cytoplasmic sequestration, indicating that the D-box is also required for nuclear import. As inhibition of AR nuclear import is a clinically validated therapeutic strategy, we developed a novel drug discovery platform to identify compounds that specifically inhibit AR-v7 nuclear import. Using cells stably expressing inducible AR-v7 in conjunction with an enzyme complementation assay we tested 166,000 compounds by high throughput screening (HTS). The robust HTS performance (Z>0.8) together with subsequent counter screens including confirmation and compound titration, cell toxicity, a tertiary imaging based screen, led to identification of lead compounds that inhibit AR-v7 nuclear import. The lead compounds share structural features, across two main chemotypes, which are amenable to structure-activity relationship studies to identify the most desirable compound for in vivo studies. Using newly synthesized compounds from each of the two chemotypes, we showed specific dose-dependent inhibition of AR-v7 nuclear import. Currently, we are testing these compounds, on inhibition of AR-v7 transcriptional activity across several cell models including enzalutamide-resistant cells as well as inhibition of tumor growth in AR-v7 xenograft models. In parallel we are testing the lead compound for potential direct binding to the AR-v7 dimerization domain or to candidate alternative nuclear transport receptors. Further development of our lead small molecules will yield novel chemotypes, with desirable pharmacological properties that target the unique AR-v7 nuclear import pathway and can be clinically combined with existing AR therapies. Note: This abstract was not presented at the meeting. Citation Format: Seaho Kim, Mohd Azrin Jamalruddin, Eiman Mukhtar, Michael Miller, Leigh Baxt, Stacia Kargman, Andrew Stamford, Peter Meinke, Paraskevi Giannakakou. Novel inhibitors of AR-v7 nuclear import: new therapeutic opportunities for CRPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1017.

Transcriptional networks in the human epididymis.

The epithelial lining of the human epididymis is critical for sperm maturation. This process requires distinct specialized functions in the head, body, and tail of the duct. These region-specific properties are maintained by distinct gene expression profiles which are governed by transcription factor networks, non-coding RNAs, and other factors. We used genome-wide protocols including DNase-seq, RNA-seq and ChIP-seq to characterize open (active) chromatin, the transcriptome and occupancy of specific transcription factors (TFs) respectively, in caput, corpus, and cauda segments of adult human epididymis tissue and primary human epididymis epithelial (HEE) cell cultures derived from them. RNA-seq following TF depletion or activation, combined with gene ontology analysis also determined TF targets. Among regional differentially expressed transcripts were epithelial-selective transcription factors (TFs), microRNAs, and antiviral response genes. Caput-enriched TFs included hepatocyte nuclear factor 1 (HNF1) and the androgen receptor (AR), both of which were also predicted to occupy cis-regulatory elements identified as open chromatin in HEE cells. HNF1 targets were identified genome-wide using ChIP-seq, in HEE cells. Next, siRNA-mediated depletion of HNF1 revealed a pivotal role for this TF in coordinating epithelial water and solute transport in caput epithelium. The importance of AR in HEE cells was shown by AR ChIP-seq, and by RNA-seq after synthetic androgen (R1881) treatment. AR has a distinct transcriptional program in the HEE cells and likely recruits different co-factors (RUNX1 and CEBPβ) in comparison to those used in prostate epithelium. Our data identify many transcription factors that regulate the development and differentiation of HEE cells. Moreover, a comparison between immature and adult HEE cells showed key TFs in the transition to fully differentiated function of this epithelium. These data may help identify new targets to treat male infertility and have the potential to open new avenues for male contraception.

Importance of androgen receptor (AR) to estrogen receptor ratio in breast cancer patients during endocrine therapy determined on circulating epithelial tumor cells (CETCs) in breast cancer patients.

e24031Background: The androgen receptor (AR) is expressed in the majority of breast cancers and across the three main breast cancer subtypes. Despite the high frequency of AR expression in breast c...

15P - Estrogen-dependent breast cancer: The importance of androgen receptor in exemestane treatment

15P - Estrogen-dependent breast cancer: The importance of androgen receptor in exemestane treatment

Glutamine Transporters Are Targets of Multiple Oncogenic Signaling Pathways in Prostate Cancer.

Despite the known importance of androgen receptor (AR) signaling in prostate cancer, the processes downstream of AR that drive disease development and progression remain poorly understood. This knowledge gap has thus limited the ability to treat cancer. Here, it is demonstrated that androgens increase the metabolism of glutamine in prostate cancer cells. This metabolism was required for maximal cell growth under conditions of serum starvation. Mechanistically, AR signaling promoted glutamine metabolism by increasing the expression of the glutamine transporters SLC1A4 and SLC1A5, genes commonly overexpressed in prostate cancer. Correspondingly, gene expression signatures of AR activity correlated with SLC1A4 and SLC1A5 mRNA levels in clinical cohorts. Interestingly, MYC, a canonical oncogene in prostate cancer and previously described master regulator of glutamine metabolism, was only a context-dependent regulator of SLC1A4 and SLC1A5 levels, being unable to regulate either transporter in PTEN wild-type cells. In contrast, rapamycin was able to decrease the androgen-mediated expression of SLC1A4 and SLC1A5 independent of PTEN status, indicating that mTOR complex 1 (mTORC1) was needed for maximal AR-mediated glutamine uptake and prostate cancer cell growth. Taken together, these data indicate that three well-established oncogenic drivers (AR, MYC, and mTOR) function by converging to collectively increase the expression of glutamine transporters, thereby promoting glutamine uptake and subsequent prostate cancer cell growth.Implications: AR, MYC, and mTOR converge to increase glutamine uptake and metabolism in prostate cancer through increasing the levels of glutamine transporters. Mol Cancer Res; 15(8); 1017-28. ©2017 AACR.

Abstract 3822: Expression of androgen receptor and CD24 correlates with improved 5-year survival in Pakistani patients with invasive breast cancer

Abstract Introduction: Androgen receptor (AR) expression has been shown to correlate with improved survival in luminal A, B and triple negative (TN) breast cancer (BCa). Cancer stem cells (CSC) with CD44+/CD24- and ALDH1+ phenotype is associated with pro-invasive gene signature in BCa. Aim of our study was to evaluate prognostic significance of AR expression in relation to CD44, CD24 and ALDH1 in invasive BCa. Methodology: Immunohistochemical expression of AR, CD44, CD24 and ALDH1 was evaluated in 180 FFPE blocks of BCa cases from one institution. Clinico-pathological details and follow-up data were collected from medical charts. Data was analyzed on SPSS 19. Five-year survival was estimated by Kaplan Meier. Results: Median age of patients was 55 years (range: 28-87). Fifteen percent of patients were diagnosed with stage I, 52.2% with stage II, 25% with stage III and 7.8% with stage IV disease. Mean duration of follow-up was 4.5 years (SD+ 2.7) and 29% mortalities were attributed to BCa. AR was expressed in 63.9%, ER in 61%, PR in 51% & HER-2/neu in 30% of tumors. CD44, CD24 and ALDH1 were expressed in 60%, 43.3% & 28.3% of tumors respectively. AR expression was associated (p-value<0.001) with grade II tumors expressing ER, PR and CD24 but lacking ALDH1. Multivariable analysis confirmed AR (adjusted HR 0.43, 95% CI 0.19-0.95) and PR (adjusted HR: 0.20, 95% CI: 0.07-0.60) to be independently associated with better outcome. Five year survival of patients with AR+ versus AR- expressing tumors, showed that patients with AR+ tumors had significantly better outcome (p-value=0.03) with survival advantage of 39.6 months. Survival advantage decreased by 9.6 months in tumors expressing CD44+/CD24-/AR- (p-value=0.01) phenotype and by 12 months in patients whose tumors were phenotypically ALDHI+ /AR- (p-value=0.12). We stratified TN tumors into 2 groups: A. TN/CD24+/AR+; B. TN/CD24-/AR- and found that group A tumors had a trend towards better 5 survival (p-value=0.09). Conclusions: Three individual markers (CD44, CD24, AR) may afford prognostic information. Combinations of CD44, CD24 and AR permitted patient stratification into 3 risk categories: poor risk CD24-/AR-; intermediate risk CD44+/CD24-/AR- and good risk CD24+/AR+. Stratification of TN group showed that loss of AR and CD24 was associated with poorest outcome. These results underscore the relevance and importance of AR and CD24 as prognostic markers in invasive BCa. Citation Format: Nazia Riaz, Romana Idress, Sadia Habib, Azhar Hussain, El-Nasir Lalani. Expression of androgen receptor and CD24 correlates with improved 5-year survival in Pakistani patients with invasive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3822. doi:10.1158/1538-7445.AM2017-3822

Abstract 2076: Targeting the FGFR pathway in androgen receptor negative castration resistant prostate cancer

Abstract Metastatic castration-resistant prostate cancer (CRPC) has a poor prognosis and remains a significant therapeutic challenge. The continued importance of androgen receptor (AR) signaling as a growth and survival pathway in most advanced prostate cancer (PC) has prompted the development of therapeutics directed toward further suppressing AR ligands or the AR itself. However, total androgen blockade can give rise to AR-negative PC and currently there are no effective therapies for AR-negative PC. We have developed two novel patient-derived xenograft (PDX) models from a liver metastasis and a rib metastasis of a patient with CRPC: LuCaP 173.1 (Neuroendocrine; NE) and LuCaP 173.2 (Double negative: DN (AR-negative and NE-negative)). Using RNA-Seq we observed that both of these lines express high levels of fibroblast growth factors (FGF’s) and downstream effectors. We hypothesized that FGF signaling promotes survival and proliferation of NEPC and DNPC. To determine if blocking the FGFR survival pathway can attenuate the growth of NEPC/DNPC tumors, to characterize the molecular response and resistance to FGF signaling inhibition, and to support further evaluation in the clinic, we treated both PDX models with the FGFR inhibitor CH5183284. CH5183284 significantly reduced LuCaP 173.2 tumor volume (TV) (p<0.0001). The reduction in TV in CH5183284-treated animals bearing LuCaP 173.1 tumors was not as significant. To address this difference, we analyzed FGFR1 expression at the transcript and protein levels. Analysis of the FGFRs in the LuCaP 173.1 PDX model revealed a loss of FGFR1 expression. FGFR2 and FGFR3 transcripts were expressed at similar levels in both PDXs. The loss of FGFR1 expression may explain the low efficacy of CH5183284 in the LuCaP 173.1 PDX model. We are currently assessing the molecular pathways impacted by FGFR inhibition and mechanisms of resistance to treatment in these studies. We have demonstrated that FGFR inhibition attenuated tumor growth in FGFhigh AR-negative PC. We intend to target the FGFR survival pathway in additional NE and DNPC PDX models to investigate tumor heterogeneity in response to therapy and identify mechanisms of resistance. These studies will provide translational evidence that FGFR inhibitors could represent a new therapy for CRPC patients with AR-negative PC. Citation Format: Colm Morrissey, Eva Corey, Lisha Brown, Ilsa Coleman, Holly Nguyen, Michael Schweizer, Peter Nelson. Targeting the FGFR pathway in androgen receptor negative castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2076. doi:10.1158/1538-7445.AM2017-2076

Reduction in expression of the benign AR transcriptome is a hallmark of localised prostate cancer progression.

BackgroundDespite the importance of androgen receptor (AR) signalling to prostate cancer development, little is known about how this signalling pathway changes with increasing grade and stage of the disease.ObjectiveTo explore changes in the normal AR transcriptome in localised prostate cancer, and its relation to adverse pathological features and disease recurrence.DesignPublically accessible human prostate cancer expression arrays as well as RNA sequencing data from the prostate TCGA. Tumour associated PSA and PSAD were calculated for a large cohort of men (n=1108) undergoing prostatectomy.Outcome Measurements and Statistical AnalysisWe performed a meta-analysis of the expression of an androgen-regulated gene set across datasets using Oncomine. Differential expression of selected genes in the prostate TCGA database was probed using the edgeR Bioconductor package. Changes in tumour PSA density with stage and grade were assessed by Student's t-test, and its association with biochemical recurrence explored by Kaplan-Meier curves and Cox regression.ResultsMeta-analysis revealed a systematic decline in the expression of a previously identified benign prostate androgen-regulated gene set with increasing tumour grade, reaching significance in nine of 25 genes tested despite increasing AR expression. These results were confirmed in a large independent dataset from the TCGA. At the protein level, when serum PSA was corrected for tumour volume, significantly lower levels were observed with increasing tumour grade and stage, and predicted disease recurrence.ConclusionsLower PSA secretion-per-tumour-volume is associated with increasing grade and stage of prostate cancer, has prognostic relevance, and reflects a systematic perturbation of androgen signalling.

Abstract B04: Epithelial nuclear androgen receptor (AR) in radical prostatectomy samples as assessed by quantitative immunofluorescence is differentially expressed in African American versus Caucasian cases

Abstract Introduction: Numerous biomarkers are being evaluated as potential predictors of prostate cancer (PCa) recurrence after definitive surgical treatment with radical prostatectomy (RP). In particular, there is a significant literature on the importance of androgen receptor (AR) protein expression in predicting the aggressiveness of PCa as well as the probability of response to secondary therapeutic interventions (e.g. androgen deprivation therapy. The AR axis has been a primary target for researchers seeking treatment options for patients whose prostate cancer is not cured by surgery alone. By utilizing quantitative immunofluorescence (IF) multiplexed biomarkers assessd via spectral imaging, we evaluated whether AR expression is deferentially expressed by race. Methods: We performed multiplex immunoflorescence immunohistochemistry on tissue microarrays (TMA) made from representative cores of tumor tissue from formalin-fixed, paraffin-embedded prostatectomy samples from cases treated for PCa by RP at the Henry Ford Health System between 2001 and 2005. There were 400 stained cores sampled from 149 African American (AA) cases and 453 cores from 195 Caucasian American (CM) cases. The samples were stained by a multiplex IF assay which contained the nuclear marker DAPI, along with AR and CK18. The co-localization of DAPI, CK18 and AR permitted quantitative analysis of AR expression in only the epithelial nuclei, a known cellular compartment of prognostic expression. Differences in expression were evaluated using the Wilcoxon rank sum test. The association between race and AR expression level was adjusted for potential confounding covariates using non-parametric generalized additive models. Results: There was a statistically significant difference in the expression of AR signal in epithelial nuclei between Caucasian men and African American men, p-value = 0.03. The association between Race and AR remained statistically significant (p=0.02) even after adjusting for established clinical risk factors of PCa (age, grade, stage and PSA). It is also noteworthy that there were no statistically significant differences in age, grade, stage or PSA when comparing Caucasian and African American men in our dataset. Conclusions: AR levels in RP epithelial nuclei have previously been found to be a useful tool in guiding therapeutic response for men who have failed surgical therapy for their prostate cancer. However, our results suggest that even with other clinical risk factors being equal, such molecular metrics may be different across racial subgroups of patients. It is possible that expression of these molecular features may be affected by external exposures, or genetic factors which differ by race, but are not necessarily related to the underlying disease mechanisms we seek to measure. Additional research into such differences is necessary as the African American community would benefit from the development of molecular diagnostic assays specifically tailored to this population. Current practices in biomarker discovery rarely address the possibility of racial variation, thereby leading to a potentially inaccurate or incorrect assessment of disease severity and consequent therapy for African American PCa patients. Citation Format: Russell Bailey McBride, Faisal M. Khan, Benjamin A. Rybicki, Russell Cordon-Cardo. Epithelial nuclear androgen receptor (AR) in radical prostatectomy samples as assessed by quantitative immunofluorescence is differentially expressed in African American versus Caucasian cases. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr B04.

Androgen receptor in human endothelial cells.

Androgen receptor (AR) is a ligand-inducible transcription factor, and a member of the steroid-thyroid-retinoid receptor superfamily, that mediates the biological effects of androgens in a wide range of physiological and pathological processes. AR expression was identified in vascular cells nearly 20 years ago, and recent research has shown that AR mediates a variety of actions of androgens in endothelial and vascular smooth muscle cells. In this mini-review, we review evidence indicating the importance of AR in human endothelial cell (HUVEC) homeostatic and pathogenic processes. Although a role for AR in the modulation of HUVEC biology is evident, the molecular mechanisms by which AR regulates HUVEC homeostasis and disease processes are not fully understood. Understanding these mechanisms could provide critical insights into the processes of pathogenesis of diseases ranging from cardiovascular disease to cancer that are major causes of human morbidity and mortality.

Abstract 3467: Identification of androgen receptor splice variants, ESR1, CYP17, and CYP19 in human breast cancer.

Abstract Many advances have been made in the diagnosis and treatment of breast cancer in recent years, but it still remains one of the leading causes of death in women. It is well known that the growth of breast cells is regulated by the interactions of different steroid hormones, in particular androgens and estrogens that are related due to their connected metabolic pathways. A number of novel inhibitors of steroidogenic enzymes have been developed that target pre-receptor events_those pertaining to the production, transport, and conversion of steroid ligands. Critical pre-receptor steps include the conversion of pregnenolone-like steroids into androgens, mediated largely by the 17α-hydroxylase/lyase (CYP17) enzyme complex, and the conversion of testosterone and androstenedione to estradiol and estrone, mediated by aromatase (CYP19). Both conversions are implicated in the emergence of tumor resistance and thus are targets for intervention. Androgen Receptor (AR) is the sex hormone receptor most frequently found in both primary and secondary breast tumors, which is indicitative of the importance of AR in regulating the growth of breast cancer cells. It is estimated that 90% of human genes undergo alternative splicing and AR is no exception. Alternative splicing of the AR could culminate in a receptor that is capable of translocation, or can bind DNA without ligand, leading current AR therapies to be less efficacious. Using TaqMan qRT-PCR we examined 213 female breast-cancer FFPET samples, 80 ER- PR- Her2- samples, 68 ER- PR- Her2+ samples, and 64 ER+ PR+ Her2- samples, as well as 8 breast-cancer cell lines for the presence of ESR1, CYP17, CYP19, full length AR and AR splice variants ARV1, ARV3/V7, ARV567, and Delta3AR. ARV3/V7 and Delta3AR were the most prevalent variants in the ER+ PR+ Her2- and ER- PR- Her2+ sample sets, with >85% of these samples showing expression of either or both of these variants. On the other hand, ARV1, ARV3/V7, and ARV567 were the most prevalent variants in the ER- PR- Her2- sample set, with >90% of these samples showing expression of one or a combination of these variants. Lower expression values of most of the AR variants were observed in higher grade ER+ PR+ Her2- and ER- PR- Her2+ samples as compared to the lower grade samples. CYP19 was highly prevalent in all sample sets with >75% of all samples showing expression, while CYP17 expression was observed in <30% of all the samples tested. Our findings show the relatively high expression of AR variants and CYP19 in breast cancer tissues, which may indicate their role in regulating the growth of these tumors. Hence increased expression of AR splice variants in breast cancer tumors may be an important biomarker of resistance and targets for AR related therapy. Citation Format: Gabriela Martinez, Dana Gaffney, Katherine Bell, Suso Platero, Deborah Ricci, Weimin Li, Jayaprakash D. Karkera. Identification of androgen receptor splice variants, ESR1, CYP17, and CYP19 in human breast cancer. [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 3467. doi:10.1158/1538-7445.AM2013-3467

Abstract A2: Selective inhibition of mTORC1/mTORC2 sensitizes prostate cancer cells to the effects of AR or IGF-1R/IR antagonists, providing an approach to overcome intrinsic mechanisms of resistance

Abstract Prostate cancer is a complex disease primarily characterized by dependence on androgen receptor (AR) signaling. Androgen deprivation therapy is efficacious; however, prostate tumors which initially respond to castration or androgen antagonists eventually progress. These castration-resistant tumors may develop hypersensitivity to low levels of androgens and AR overexpression or may adapt to rely on alternate signaling pathways such as the PI3K/mTOR axis. Deregulation of the PI3K/mTOR axis is a feature of prostate cancer, as evidenced by the fact that 40% of primary and 70% of metastatic prostate tumors exhibit loss of the tumor suppressor PTEN. Recent publications have shown that crosstalk between the AR and PTEN/PI3K/mTOR pathways plays a role in prostate cancer development and progression (1,2). Building upon these data, we evaluated the effects of ASP7486 (OSI-027), a selective inhibitor of mTORC1/mTORC2 as monotherapy and combined with bicalutamide, an androgen receptor antagonist. ASP7486 inhibited proliferation across a panel of prostate cancer cell lines, including those refractory to bicalutamide, supporting the hypothesis that tumors which have acquired androgen independence may rely upon mTOR signaling for survival. ASP7486 but not rapamycin, an allosteric mTORC1 inhibitor, induced apoptosis, implying that mTORC2 signaling is an important regulator of survival in prostate cancer. ASP7486, but not rapamycin, significantly upregulated AR expression which may serve to sensitize cells to the effect of an antiandrogen while limiting sensitivity to mTOR inhibition as a monotherapy. Consistent with this, the combination of ASP7486 and bicalutamide synergistically inhibited proliferation in vitro. In prostate tumors which have acquired resistance to androgen antagonists, activation of alternate RTKs may provide a survival mechanism in the absence of functional AR. PI3K pathway alterations and increased expression of IFG-1R are observed in castrate-resistant tumors (3,4). Treatment of prostate cancer cells with ASP7486 leads to increased phosphorylation of multiple RTKs, including IGF-1R and IR. We reasoned that ASP7486-mediated activation of IGF-1R and IR would sensitize these cells to the effects of OSI-906, a selective IGF-1R/IR inhibitor. The combination of ASP7486 and OSI-906, a selective inhibitor of IGF-1R/IR, synergistically inhibited proliferation and induced apoptosis. The combination of the two drugs provided greater inhibition of key signaling effectors than either monotherapy. These effects are not limited to prostate cancer. We have observed ASP7486-induced upregulation of RTK expression and phosphorylation in multiple tumor types, and the combination of ASP7486 and OSI-906 synergistically inhibited proliferation in the majority of cell lines tested. Together these data demonstrate that combinations of ASP7486 with targeted inhibitors can attenuate crosstalk between signaling networks and provide synergistic efficacy in vitro. The importance of AR and IGF-1R/PI3K/mTOR signaling in prostate cancer provides a rationale for targeting the disease and its intrinsic mechanisms of resistance with inhibitors of these pathways or in combination.

Androgen receptor gene haplotype is associated with male infertility

The purpose of the current study was to evaluate the importance of androgen receptor (AR) gene haplotypes and polymorphic CAG/GGN microsatellites in the aetiology of male infertility. We genotyped six haplotype-tagging single nucleotide polymorphisms and CAG/GGN microsatellites of the AR gene in 112 infertile and 212 control Estonian men. A total of 13 AR haplotypes (HAP1-13) were identified, among which HAP4 was found to confer increased risk for male infertility (OR = 5.15, 95% CI = 1.75-15.15, p = 0.003). However, infertile patients and controls had similar lengths and distributions of both AR CAG (mean +/- SD number of repeats 21.1 +/- 2.5 vs. 21.2 +/- 2.3, respectively) and GGN (mean +/- SD number of repeats 22.5 +/- 1.5 vs. 22.4 +/- 1.9, respectively) repeats. In addition, HAP2 was associated with more CAG repeats (r = 1.17, p = 0.033) and HAP3 with fewer CAG repeats (r = -2.93, p < 0.001) than the major haplotype HAP1. HAP3 and HAP4 were associated with more GGN repeats (r = 1.35, p = 0.001 and r = 1.36, p = 0.002, respectively) than HAP1. In conclusion, our results implicated the AR-HAP4 gene haplotype in increased risk for male infertility, while no association was found between AR CAG/GGN microsatellites and impaired spermatogenesis.

Polymorphisms of estrogen receptor β gene are associated with hypospadias

Hypospadias is a common male congenital urethral malformation, defined as the displacement of the urethral meatus ventrally from the tip of the glans penis. The importance of androgen receptor in male external genitalia development has been well recognized. Recently, the presence of active estrogen receptors (ER) in the developing male external genitalia has also been demonstrated. There are two isoforms of the human estrogen receptor, ESR1 and ESR2, which occur, with distinct tissue and cell patterns of expression. We hypothesized that modifications in these nuclear receptors' genes could lead to hypospadias. We screened 60 boys with hypospadias for mutations in the coding regions of ESR1 and ESR2 genes, by denaturing high-performance liquid chromatography and automated sequence analysis. We also genotyped the CA repeat polymorphism in ESR2 and the TA repeat polymorphism in ESR1. The CA repeat polymorphism in ESR2 is prolonged in hypospadias patients compared to controls (p < 0.05). Prolongation of this CA repeat polymorphism has previously been associated with lower levels of testosterone. Six patients presented the genetic variant 2681-4A > G (rs944050) in the heterozygous form in ESR2, which was a significantly higher frequency than in the control population (p < 0.05). One of these patients also presented a 266_267insC in exon 1 of ESR2, which is also a known single nucleotide polymorphism (SNP; rs3832949). In ESR1, no significant gene alteration was found to be associated with hypospadias. Our results suggest that variations in the ESR2 might influence susceptibility to hypospadias.

NF-κB and TNF-α stimulate androgen receptor expression in Sertoli cells

Germ cell development within the mammalian testis requires testosterone stimulation of somatic Sertoli cells via interaction with intracellular androgen receptors (AR). AR expression levels undergo marked changes during spermatogenesis suggesting that the modulation of AR expression is an important mechanism to regulate Sertoli cell responsiveness to testosterone. An analysis of the AR gene promoter revealed three κB enhancer elements that interacted with Sertoli cell p50 and RelA NF-κB proteins, and the overexpression of these NF-κB subunits in Sertoli cells stimulated AR promoter activity. Moreover, TNF-α, a secretory product of round spermatids, stimulated NF-κB binding to the AR promoter, induced AR promoter activity, and increased endogenous AR expression in primary cultures of Sertoli cells. Given the requirement of testosterone for spermatogenesis and the importance of AR in mediating Sertoli cell responsiveness to testosterone, the stimulation of AR expression by NF-κB and TNF-α may represent an important regulatory mechanism required to maintain efficient spermatogenesis.

Testosterone and 5α-dihydrotestosterone inhibit in vitro growth of human breast cancer cell lines

Androgens are of biological and clinical importance for the growth and development of breast cancer in women, and the androgen receptor (AR) has been shown to be a predictor of tumor differentiation. Inthe present study, we investigated the relationship between AR status and testosterone and 5α-dihydrotestosterone (DHT)-dependent proliferation of the human breast carcinoma cell lines MCF-7,T47-D, MDA-MB 435S and BT-20. AR status was studied by means of immunocytochemistry and Western blot analysis. All four cell lines stained positively for AR. Western blot analysis revealed a strong expressionof AR in MCF-7, in contrast to BT-20 cells. According to proliferation kinetics, we observed a significant (p ≤ 0.05) dose-dependent inhibition of cell growth by testosterone and DHT treatment in allfour cell lines. In the estrogen receptor (ER)-negative cell lines BT-20 and MDA-MB 435S, testosterone was a more potent inhibitor of cell proliferation than DHT (p ≤ 0.05), in contrast to the ER-positivecells lines MCF-7 and T47-D, in which a stronger inhibition of proliferation was achieved by DHT. A partial transformation of testosterone to estrogen in ER-positive cells might be an explanation for thiseffect. Our data favor a possible role of androgens in growth regulation of breast cancer. Clinical studies are needed to analyze the importance of AR as a possible predictor in response to endocrine therapyof breast cancer.