Lack Of Androgen Receptor Expression Research Articles

Analysis of B7-H4 Expression Across Salivary Gland Carcinomas Reveals Adenoid Cystic Carcinoma–Specific Prognostic Relevance

B7-H4 (VTCN1), a member of the B7 family, is overexpressed in several types of cancer. Here we investigated the pattern of expression of B7-H4 in salivary gland carcinomas (SGC) and assessed its potential as a prognostic marker and therapeutic target. Immunohistochemistry (IHC) analyses were performed in a cohort of 340 patient tumors, composed of 124 adenoid cystic carcinomas (ACC), 107 salivary duct carcinomas (SDC), 64 acinic cell carcinomas, 36 mucoepidermoid carcinomas (MEC), 9 secretory carcinomas (SC), as well as 20 normal salivary glands (controls). B7-H4 expression was scored and categorized into negative (<5% expression of any intensity), low (5%–70% expression of any intensity or >70% with weak intensity), or high (>70% moderate or strong diffuse intensity). The associations between B7-H4 expression and clinicopathologic characteristics, as well as overall survival, were assessed. Among all tumors, B7-H4 expression was more prevalent in ACC (94%) compared with those of SC (67%), MEC (44%), SDC (32%), and acinic cell carcinomas (0%). Normal salivary gland tissue did not express B7-H4. High expression of B7-H4 was found exclusively in ACC (27%), SDC (11%), and MEC (8%). In SDC, B7-H4 expression was associated with female gender (P = .002) and lack of androgen receptor expression (P = .012). In ACC, B7-H4 expression was significantly associated with solid histology (P < .0001) and minor salivary gland primary (P = .02). High B7-H4 expression was associated with a poorer prognosis in ACC, regardless of clinical stage and histologic subtype. B7-H4 expression was not prognostic in the non-ACC SGC evaluated. Our comparative study revealed distinct patterns of B7-H4 expression according to SGC histology, which has potential therapeutic implications. B7-H4 expression was particularly high in solid ACC and was an independent prognostic marker in this disease but not in the other SGC assessed.

Abstract LB298: Molecular phenotype classification of metastatic prostate cancer by cell-free DNA methylation analysis

Abstract Introduction: Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease which can be classified into clinically relevant subtypes based on the expression of transcription factors (TF), such as the androgen receptor (AR) and neuroendocrine markers. Neuroendocrine prostate cancer (NEPC), characterized by gain of stem-like and neuroendocrine features and lack of AR expression is a clinically aggressive variant. Due to the absence of adequate biomarkers, NEPC is usually detected at a very advanced stage. There is mounting evidence that molecular subtype changes seen in NEPC are enforced by widespread epigenetic alterations, in particular DNA methylation changes. In this study, we aim to devise a novel DNA methylation-based assay for molecular subtyping and disease monitoring from cell-free DNA (cfDNA). Methods: We analyzed genome wide methylation patterns in 60 prostate cancer patient-derived xenograft (PDX) and 133 mCRPC tumors using array- and sequencing-based assays. We integrated DNA methylation with TF cistrome data to determine the landscape of methylation alterations at key lineage TF binding sites (TFBS). A linear regression model was trained on low-pass Enzymatic Methyl-Seq (EM-seq) cfDNA data derived from PDXs to identify molecular subtype specific DNA methylation changes at these TFBS. The model performance was optimized with in silico admixture experiments. This model was then used to discern tumor molecular phenotypes from cfDNA in three independent cohorts of mCRPC patients using low-pass whole genome bisulfite sequencing and EM-seq. Results: We observed a strong association between TFBS methylation and TF expression. For lineage specific TFs such as AR and ASCL1, we identified core sets of TFBSs whose differential methylation allowed for accurate assay-independent molecular subtype classification in tumor tissues. Applying an optimized quantitative model to mCRPC patients who underwent comprehensive tissue sampling by rapid autopsy we observed perfect subtype prediction from both tissue samples and cfDNA (AUC=1). A similar analytical performance was observed in additional clinical mCRPC cohorts with cfDNA. Conclusions: We show that methylation patterns at TFBSs can determine TF activity and can be used to classify molecular subtypes from both tumor tissue and cfDNA. For prostate cancer, we demonstrate that this approach can accurately detect NEPC by cost-effective low-pass EM-seq. More broadly, this study provides a novel analysis framework for robustly assessing molecular tumor phenotypes in cfDNA with applications in solid and liquid tumor diagnostics. Citation Format: Mohamed Adil, Brian Hanratty, Pallabi Mustafi, Ilsa Coleman, Radhika Patel, Anna-Lisa Doebley, Robert Patton, Eden Cruikshank, Patricia Galipeau, Ruth Dumpit, Martine Roudier, Jin-Yih Low, Navonil De Sarkar, Robert Montgomery, Eva Corey, Colm Morrissey, Peter Nelson, Gavin Ha, Michael Haffner. Molecular phenotype classification of metastatic prostate cancer by cell-free DNA methylation analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB298.

Lack of Androgen Receptor Expression Selects for Basal-Like Phenotype and Is a Predictor of Poor Clinical Outcome in Non-Metastatic Triple Negative Breast Cancer.

Background: Androgen receptor (AR) has emerged as a significant favorable prognostic indicator in estrogen receptor expressing (ER+) breast cancer (BCa); however, its clinical and biological relevance in triple negative breast cancer (TNBC) and association with cancer stem cell (CSC) markers remain ambiguous.Methods: We examined the immunohistochemical expression of AR in a cohort of stage I–III TNBC cases (n = 197) with a long-term clinical follow-up data (mean follow-up = 53.6 months). Significance of AR expression was correlated with prognostic biomarkers including cancer stem cell markers (CD44, CD24, and ALDH1), basal markers (CK5, CK14, and nestin), proliferation marker (ki-67), apoptotic marker (Bcl-2), and COX-2. Expression of CK5 and nestin was used for the categorization of TNBC into basal (TN, CK5+, and/or nestin+) and non-basal (TN, CK5−, and/or nestin−) phenotypes, and Kaplan–Meier curves were used for estimation of overall survival and breast cancer-specific survival (BCSS).Results: AR expression was observed in 18.8% of non-metastatic TNBC tumors. Expression of AR correlated with lower grade (P < 0.001) and conferred a favorable prognostic significance in patients with axillary lymph node metastasis (P = 0.005). Lack of AR expression correlated with expression of CSC phenotype (CD44+/CD24−) (P < 0.001), COX-2 (P = 0.02), basal markers (CK5: P = 0.03), and nestin (P = 0.01). Basal-like phenotype (TN, CK5+, and/or nestin+) correlated with quadruple-negative breast cancer (QNBC) and showed a significant association with adverse prognostic markers including high proliferation index (P < 0.001), expression of COX-2 (P = 0.009), and CSC phenotype (CD44+/CD24−: P = 0.01). Expression of AR remained an independent prognostic indicator for improved overall survival (P = 0.003), whereas basal-like phenotype was associated with an adverse BCSS (P = 0.013).Conclusions: Assessment of AR and basal markers identified biologically and clinically distinct subgroups of TNBC. Expression of AR defined a low-risk TNBC subgroup associated with improved overall survival, whereas expression of basal markers (CK5 and nestin) identified a high-risk subgroup associated with adverse BCSS. Integration of immunohistochemical analysis of AR and basal biomarkers to the assessment of TNBC tumors is expected to improve the prognostication of an otherwise heterogeneous disease.

Abstract P3-05-02: Upregulation of canonical Wnt signaling may underlie an aggressive disease course in androgen receptor-negative triple negative breast cancer

Abstract Background: Based on the expression of androgen receptor (AR), triple-negative breast cancer (TNBC) is subdivided into AR-positive and AR-negative subtypes. Although AR is a potential therapeutic target for AR-positive TNBC, its prognostic role in TNBC remains controversial. Lack of AR expression is associated with a more aggressive disease course. Whereas AR-positive TNBC responds well to AR antagonists, there are, beyond chemotherapy, no targeted drugs available for AR-negative patients. Thus, we aimed to examine the impact of AR loss on the prognosis for TNBC and to uncover molecular pathways/drivers that can be therapeutically targeted in AR-negative TNBC. Methods: We evaluated AR expression immunohistochemically in annotated, formalin-fixed paraffin-embedded samples from 420 TNBC patients obtained from various institutions in the US. Samples with ≥1% of AR-positive nuclei were deemed AR-positive. We next performed genome-wide copy number profiling (array-CGH analysis) to determine whether losses and/or deletions of the AR gene contributed to tumor aggressiveness of AR-negative TNBC. Finally, using a combination of in silico analysis, in vitro assays, and RNA-sequencing, we found molecular drivers/pathways that were upregulated in AR-negative TNBC. Results: AR was expressed in approximately 25% of the total cases, and loss of AR was associated with poor overall survival of TNBC patients (p=0.03; n=316 for AR-negative, n=104 for AR-positive) among all treated patients as well as among the subset of adjuvant chemotherapy-treated patients (p=0.06; n=230 for AR-negative, n=75 for AR-positive). Further, the array-CGH analysis showed that AR-negative TNBC presented higher levels of copy number alterations compared to AR-positive TNBC (28.5±4.40 and 13.79±3.48, P&amp;lt;0.01, respectively). These alterations affected mainly the cytobands 1p36.33-p36.32, 1q21.1-q44, 9p24.3-p13.1, 10p15.3-p11.21, 11p15.5-p11.2, 12p13.33-p11.21, 19p13.2-p12, 20q11-q13.33, and 22q11.1-q12.3 cytobands, which were present in higher numbers of AR-negative TNBC. Next-generation RNA-sequencing of TNBC cases and gene set enrichment analysis revealed upregulation of the Wnt/β-catenin axis in AR-negative cases. Proteomic data from TCGA showed upregulation of β-catenin and Dvl3 expression-evidence of overexpression of Wnt signaling in AR-negative TNBC. Furthermore, protein levels of β-catenin were higher in AR-negative TNBCs compared to AR-positives (p&amp;lt;0.05), suggesting aberrant activation of Wnt/β-catenin signaling in AR-negative TNBC. Using reporter gene assays in cultured cells, we confirmed the activation of Wnt signaling and elevated levels of Wnt target genes (Axin2, CD44, and Cyclin D1) in AR-negative TNBC cells relative to AR-positives. Enhanced nuclear accumulation of β-catenin in AR-negative TNBC indicated reduced β-catenin phosphorylation. Knockdown of AR in AR-positive TNBC cells showed features of AR-negative phenotype in AR-positive cells, in that we observed reduced expression of E-cadherin (suggesting enhanced epithelial-mesenchymal transition) and higher β-catenin expression. Concomitantly, β-catenin target genes were also upregulated resulting in enhanced proliferation, migration and invasion capacities (p&amp;lt;0.05). Conclusion: Our results suggest an association of AR loss with poor clinical outcome in TNBC. The data support the dysregulation of the oncogenic Wnt/β-catenin pathway in AR-negative cancer cells. Finally, we elucidated a previously unknown link of AR loss to a more aggressive disease course and discovered actionable targets in AR-negative TNBCs. Citation Format: Shristi Bhattarai, Chakravarthy Garlapati, Komal Arora, Karuna Mittal, Sergey Klimov, Uma Krishnamurti, Xiaoxian B Li, Deepika Wali, Meenakshi Gupta, Upender Manne, Shobhna Kapoor, Luicane Cavalli, Ritu Aneja. Upregulation of canonical Wnt signaling may underlie an aggressive disease course in androgen receptor-negative triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-05-02.

Role of androgen receptor expression in non-muscle-invasive bladder cancer: a systematic review and meta-analysis.

In order to evaluate the potential prognostic/predictive role of androgen receptor (AR) expression in non-muscle-invasive bladder cancer (NMIBC), and whether it may represent a therapeutic target, we conducted a systematic search of the literature using 'androgen receptor or AR', 'testosterone', 'bladder cancer' and 'non-muscle invasive bladder cancer or NMIBC' as keywords. Eleven studies met the inclusion/exclusion criteria. No significant association was found between AR status and patients' gender (p=0.232), tumor size (p=0.975), tumor stage (p=0.237), tumor grade (p=0.444), tumor multicentricity (p=0.397), concomitant CIS (p=0.316) and progression of disease (p=0.397). On the other hand, relative lack of AR expression was significantly correlated to recurrent disease (p=0.001). Evidence for a direct correlation between AR expression and recurrence-free survival of patients with NMIBC indicate ARs as potential markers of BC behavior; moreover, the finding of a role of androgen blockade therapy in improving survival highlights the potential clinical application of this pathway, which deserves to be further explored.

Abstract P3-05-11: Immune and basal signature correlation to targeted therapies and race in QNBC patients

Abstract Background: African American women have worse prognosis and poor survival rates due to lack of Androgen Receptor expression. These subtypes, the Quadruple Negative Breast Cancers (QNBC), currently have no treatments. Previously, we found that AA TNBC have 28% more chance of being QNBC and have 25% higher rate of progression vs the Caucasian Americans (CA). Also, these QNBC patients have a unique immune and basal signature. Here, we explore individual genes and their correlation to race and immune therapies. Methods: We analyzed AR mRNA expression in 925 tumors from CA or AA women from The Cancer Genome Atlas and independently validated AR protein levels by immunohistochemistry in 197 primary tumors. Samples were dichotomized as AR positive or negative using mean gene expression cutoff. Gene expression comparisons among groups were determined using standard t-tests, ANOVA, and odds ratios with a significance threshold alpha of 0.05. a of 0.05. Therapy targets were selected from the list of significantly differentiated genes. Boxplots and heatmaps were made using R packages. Cumulative Incidence plots for time to progression were constructed using XLStats and Grays test for significance. Results: QNBC CA have higher expression of PDCD4 (p &amp;lt;.001), CD46 (p &amp;lt;.001), CD3EAP (p =.04) and CD84 (p =.07) compared to QNBC AA. CD47 (p = .63) and PD1 (p =.96) express no significant differences in race[CY1]. QNBC AA show higher expression of NFKB (p &amp;lt;.001), CCL2 (p &amp;lt;.001), and IL12 (p &amp;lt;.001) compared to QNBC CAs. Interestingly, QNBCs with high expressions of CD47 (p = .04) have poor prognosis[L2]. Thus, QNBC patients with low expression of CD46 (p =.025), PDCD4 (p &amp;lt; .001), and CD3EAP (p &amp;lt; .001) have poor prognosis and overall survival. Conclusions: Current immune targeted therapies like CD47 and PD1 looks favorable for treatment in some QNBC patients. Race appears to effect other major therapy target gene expressions in the QNBC immune basal signature as well. This may imply that QNBC AA and CA have different immune response which may explain racial progression differences. Assessment of immune markers may contribute to more accurate prognosis. Citation Format: Hughley RW, Colomb WD, Shweta T, Yates C. Immune and basal signature correlation to targeted therapies and race in QNBC patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-11.

Abstract A67: Lack of androgen receptor expression correlates with high expression of ALDH1 and poor overall and 5-year disease free survival in patients with invasive breast cancer

Abstract Introduction: A sub-population of cells referred to as cancer stem cells has been identified utilizing specific markers such as aldehyde dehydrogenase 1 (ALDH1) amongst others in solid tumors including breast cancers. Evidence from in vitro studies has shown that high ALDH1 expression identifies tumorigenic cell population which is capable of self-renewal and generating tumors which recapitulate the heterogeneity of the primary tumors. Expression of androgen receptor is present in 70-90% cases of invasive breast cancers at a frequency that is higher than the expression of estrogen or progesterone receptors. However, prognostic relevance of androgen receptor expression in relation to the expression of ALDH1 has not been investigated. Methodology: Immunohistochemical expression of ALDH1 and AR was evaluated in formalin fixed paraffin embedded blocks from 177 invasive breast cancer cases at a single institution in Pakistan from 2006-2010. Clinico-pathological details and follow-up data were collected from patients' medical charts. Data analysis was performed on SPSS version 19. Over-all survival and 5-year disease free survival were estimated by Kaplan Meier. Results: The mean age of the patients was 53.9 (+ 12.40) years. Fifteen percent patients were diagnosed with stage I disease, 52% with stage II, 25% and 8% presented with stage III and stage IV disease, respectively. Twelve percent patients presented with T1 tumors, 49.7% patients presented with T2 tumors whereas 23.7% and 14.6 % patients presented with T3 and T4 tumors respectively. Fifty-seven percent patients were node positive. Estrogen and progesterone receptor expression was present in 62% and 52% of tumors respectively. HER-2/neu was overexpressed /amplified in 30% tumors. Immunohistochemical expression of androgen receptor (AR) was present in 63.8% tumors whereas ALDH1 expression was present in 28.8% tumors. Lack of androgen receptor expression was associated with high expression of ALDH1 ( p= 0.009). Cases were divided into two groups: Group1: ALDH1-/AR+ and Group 2: ALDH1+/ AR-. No significant differences were observed between these groups with respect to age, tumor size, stage of disease, HER-2 neu over-expression/amplification or nodal status. Group 1 phenotype correlated with nuclear grade I and II tumors (p &amp;lt;0.05), estrogen receptor (p &amp;lt;0.05) and progesterone receptor (p &amp;lt;0.05) expression. Group 2 phenotype was associated with nuclear grade III tumors (p &amp;lt;0.05) and estrogen and progesterone receptor negativity (p &amp;lt;0.05). Although not statistically significant, but a trend towards a better over-all survival and 5-year disease free survival was observed in group 1 as compared to group 2. Conclusions: 1. Group 1 phenotype correlated with better prognostic features such as well to moderately differentiated and endocrine responsive tumors. 2. Group 2 phenotype correlated with poor prognostic features such as higher nuclear grade and lack of expression of hormone receptors. 3. Expression of stem cell marker, ALDH1, in group 2 phenotype correlated with poor over-all and 5-year disease free survival. Citation Format: Nazia Riaz, Romana Idress, Sadia Habib, Syed Zulfiqar Naqvi, Shaista Masood Khan, Anwar Ali Siddiqui, El-Nasir Lalani. Lack of androgen receptor expression correlates with high expression of ALDH1 and poor overall and 5-year disease free survival in patients with invasive breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A67.

Abstract P2-09-05: The potent anti-androgen enzalutamide demonstrates broad anti-tumour activity across all androgen receptor-positive triple-negative breast cancer subtypes

Abstract Background: Up to 30% of triple-negative breast cancers (TNBC) show expression of the androgen receptor (AR) and may therefore be candidates for AR-targeted therapy. Molecular profiling of TNBC has identified the luminal AR (LAR) subset, which shares many aspects of ER-positive luminal cancers but is resistant to ER-targeted therapy. LAR cancers highly express the AR, are heavily enriched in hormone-regulated pathways and demonstrate tumour growth in response to androgen stimulation that can be inhibited by the mixed AR agonist/antagonist bicalutamide. The objective of this study was to investigate the role of AR signalling in TNBC in more detail and to evaluate the potential of the second generation anti-androgen Enzalutamide in this setting relative to bicalutamide. Methods: A panel of 13 TNBC breast cancer cell lines was selected, representing different molecular subtypes of TNBC (including 4 LAR cell lines). Baseline AR expression was determined using qPCR (Taqman) and Western-Blot (Antibody, Santa Cruz Biotechnology). The effects of enzalutamide and bicalumatide on cell proliferation and survival were determined using MTT assays with and without DHT stimulation. Comprehensive molecular profiling (including Illumina Human HT-12 v4 whole genome mRNA and Illumina Human 450K methylation microarrays) was applied before and after enzalutamide treatment to define the molecular signature associated with sensitivity to enzalutamide therapy. All statistical calculations and IC50 doses were calculated using Prism v6.0 (GraphPad). Results: A wide range of AR expression was confirmed across the cell line panel defining 3 groups with complete lack of AR expression, low/moderate AR expressions levels, or very high expression in the LAR subtype (with up to 100-fold greater AR expression compared to other TNBC subtypes). Enzalutamide demonstrated broad anti-tumour activity across all AR+ TNBC subtypes (but not in AR-negative subtypes), with IC50 values being similar in LAR and non-LAR cell lines. Treatment with enzalutamide resulted in more effective growth inhibition and a broader range of activity compared to bicalutamide. Data on molecular profiles of enzalutamide sensitivity will be presented at the meeting. Conclusion: AR inhibition is a promising therapeutic strategy for both LAR and non-LAR AR-expressing TNBC. The potent anti-androgen enzalutamide shows broad activity across all AR+ TNBC subtypes and warrants further clinical evaluation. A phase 2 study of enzalutamide in AR+ TNBC has been initiated. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-05.

Male Central Precocious Puberty: Serum Profile of Anti-Müllerian Hormone and Inhibin B before, during, and after Treatment with GnRH Analogue

We aimed to describe the functional changes of Sertoli cells, based on the measurement of serum anti-Müllerian hormone (AMH) and inhibin B during treatment with GnRHa and after its withdrawal in boys with central precocious puberty. Six boys aged 0.8 to 5.5 yr were included. AMH was low at diagnosis in patients >1 yr but within the normal range in younger patients. AMH increased to normal prepubertal levels during treatment. After GnRHa withdrawal, AMH declined concomitantly with the rise in serum testosterone. At diagnosis, inhibin B was elevated and decreased throughout therapy, remaining in the upper normal prepubertal range. In patients with testicular volume above 4 mL AMH remained higher in spite of suppressed FSH. After treatment withdrawal, inhibin B rose towards normal pubertal levels. In conclusion, AMH did not decrease in patients <1 yr reflecting the lack of androgen receptor expression in Sertoli cells in early infancy. Serum inhibin B might result from the contribution of two sources: the mass of Sertoli cells and the stimulation exerted by FSH. Sertoli cell markers might provide additional tools for the diagnosis and treatment followup of boys with central precocious puberty.

Abstract 957: Aurora kinase and N-myc are involved in neuroendocrine differentiation of prostate cancer and are new drug targets

Abstract Background: Neuroendocrine prostate cancer (NEPC) is an aggressive variant of prostate cancer that can arise de novo or from existing prostate adenocarcinoma (PCA), and is promoted by the use of androgen deprivation therapy. We sought to better understand the molecular transformation of NEPC and identify new drug targets. Methods: Using Next Generation RNA sequencing and oligonucleotide arrays, we profiled NEPC, PCA, and benign prostate (BEN), and validated findings on tumors from a large cohort of patients using IHC and FISH. Functional studies were performed using NCI-H660 (NEPC), VCaP and LnCaP (PCA), RWPE (BEN) cell lines and xenografts. Results: There was a spectrum of NEPC, ranging from pure NEPC to those with mixed features of PCA and NEPC. ERG rearrangement was detected by FISH break-apart in 47% of NEPC, but ERG protein expression by IHC was absent and corresponded directly with lack of androgen receptor expression. We sequenced 7 NEPC, 30 PCA, 5 BEN. There were significant molecular differences between NEPC and PCA, with 936/25932 genes differentially expressed (P&amp;lt;0.001). The cell cycle kinases Aurora kinase A and B (AURKA, AURKB) were overexpressed in NEPC, and notably this was independent of cell proliferation (Ki67 expression). N-myc (MYCN), a gene frequently amplified in neuroblastoma, was overexpressed in NEPC (P&amp;lt;0.001), and both AURKA and MYCN were amplified. Using IHC and FISH, we validated these findings on a large cohort (30 BEN, 118 PCA, 30 NEPC) and found majority (&amp;gt;80%) of NEPC showed Aurora kinase A and B overexpression, and 35% harbored AURKA and co-existent MYCN amplification. A small subset of PCA (5%) and no BEN were positive. Transfection of MYCN induced AURKA expression as well as Aurora kinase activity in RWPE in vitro, and AURKA could also induce MYCN expression. MYCN and AURKA independently induced expression of the neuroendocrine (NE) markers, SYP and NSE. After validating NCI-H660 as model of NEPC, we observed enhanced in vitro and in vivo sensitivity to the Aurora kinase inhibitor PHA-739358 compared to LnCaP and VCaP, with &amp;gt; 50% tumor shrinkage in NEPC xenografts and minimal to no effect in PCA. Phosphorylated histone 3 expression, a downstream marker of Aurora kinase activity, was significantly inhibited in the treated NCI-H660 but not in treated PCA xenografts. Notably, NE marker expression was completely suppressed in the treated NCI-H660 xenografts, again supporting a role of Aurora kinase in modulating the NE phenotype. Conclusions: There is likely clonal origin of NEPC from PCA (with ERG fusion positivity seen in both), but ERG expression is limited to PCA and driven by AR signaling. We discovered significant overexpression and gene amplification of Aurora kinases and N-myc in NEPC and a small subset of PCA, and evidence that that they cooperate and induce a NE phenotype in prostate cells. In vitro and in vivo data confirms that these are novel drug targets for NEPC. 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 957. doi:10.1158/1538-7445.AM2011-957

Granulosa Cell-Specific Androgen Receptors Are Critical Regulators of Ovarian Development and Function.

The physiological significance of androgens in female reproduction was unclear until female mice with global knockout of androgen receptor (AR) expression were found to have reduced fertility with abnormal ovarian function. However, because ARs are expressed in a myriad of reproductive tissues, including the hypothalamus, pituitary, and various ovarian cells, the role of tissue-specific ARs in regulating female fertility remained unknown. To examine the importance of ovarian ARs in female reproduction, we generated granulosa cell (GC)- and oocyte-specific AR-knockout (ARKO) mice by crossing AR-flox mice with MisRIIcre (GC-specific) or growth differentiation factor growth differentiation factor-9cre (oocyte-specific) mice. Relative to heterozygous and wild-type mice, GC-specific ARKO mice had premature ovarian failure and were subfertile, with longer estrous cycles and fewer ovulated oocytes. In addition, ovaries from GC-specific knockout mice contained more preantral and atretic follicles, with fewer antral follicles and corpus lutea. Finally, in vitro growth of follicles from GC-specific AR-null mice was slower than follicles from wild-type animals. In contrast to GC-specific AR-null mice, fertility, estrous cycles, and ovarian morphology of oocyte-specific ARKO mice were normal, although androgens no longer promoted oocyte maturation in these animals. Together, our data indicate that nearly all reproductive phenotypes observed in global ARKO mice can be explained by the lack of AR expression in GCs. These GC-specific ARs appear to promote preantral follicle growth and prevent follicular atresia; thus they are essential for normal follicular development and fertility.

In vivo exploration of the functional activity of the non-coding 8q24 prostate cancer risk locus

In vivo exploration of the functional activity of the non-coding 8q24 prostate cancer risk locus

Granulosa Cell-Specific Androgen Receptors Are Critical Regulators of Ovarian Development and Function

The physiological significance of androgens in female reproduction was unclear until female mice with global knockout of androgen receptor (AR) expression were found to have reduced fertility with abnormal ovarian function. However, because ARs are expressed in a myriad of reproductive tissues, including the hypothalamus, pituitary, and various ovarian cells, the role of tissue-specific ARs in regulating female fertility remained unknown. To examine the importance of ovarian ARs in female reproduction, we generated granulosa cell (GC)- and oocyte-specific AR-knockout (ARKO) mice by crossing AR-flox mice with MisRIIcre (GC-specific) or growth differentiation factor growth differentiation factor-9cre (oocyte-specific) mice. Relative to heterozygous and wild-type mice, GC-specific ARKO mice had premature ovarian failure and were subfertile, with longer estrous cycles and fewer ovulated oocytes. In addition, ovaries from GC-specific knockout mice contained more preantral and atretic follicles, with fewer antral follicles and corpus lutea. Finally, in vitro growth of follicles from GC-specific AR-null mice was slower than follicles from wild-type animals. In contrast to GC-specific AR-null mice, fertility, estrous cycles, and ovarian morphology of oocyte-specific ARKO mice were normal, although androgens no longer promoted oocyte maturation in these animals. Together, our data indicate that nearly all reproductive phenotypes observed in global ARKO mice can be explained by the lack of AR expression in GCs. These GC-specific ARs appear to promote preantral follicle growth and prevent follicular atresia; thus they are essential for normal follicular development and fertility.

Lack of Androgen Receptor Expression in Sertoli Cells accounts for the Absence of Anti-Mullerian Hormone Repression during Early Human Testis Development

Lack of Androgen Receptor Expression in Sertoli Cells accounts for the Absence of Anti-Mullerian Hormone Repression during Early Human Testis Development

Lack of Androgen Receptor Expression in Sertoli Cells Accounts for the Absence of Anti-Mullerian Hormone Repression during Early Human Testis Development

Puberty is associated with increased testicular testosterone (TT) synthesis, which is required to trigger spermatogenesis and to repress anti-Mullerian hormone (AMH) production. However, testicular gonadotropin stimulation during fetal and newborn life neither initiates spermatogenesis nor represses AMH. We postulated that a lack of androgen receptor (AR) expression in Sertoli cells (SC) might explain why these processes do not occur during early human development. Using immunohistochemistry and quantitative PCR, we examined the relationship between AR, AMH, and FSH receptor expression in fetal, newborn, and adult human testis. The ability of testosterone to repress AMH secretion was evaluated in male newborns, neonates, and two adults with androgen insensitivity syndrome and also in vitro using SMAT1 SC. FSH receptor was present in SC at all developmental stages. In fetal and newborn testis, AR was expressed in peritubular and Leydig cells but not in SC. This coincided with the absence of spermatogenesis and with strong SC AMH expression. In adult testis, spermatogenesis was associated with AR expression and with a decrease in SC AMH content. Accordingly, AR mRNA expression was lower and AMH mRNA expression higher in fetal testes than in adult testes. In androgen insensitivity syndrome patients, combined gonadotropin stimulation induced an increase in circulating testosterone and AMH, a finding consistent with a failure of TT to repress AMH in the absence of AR signalling. Finally, direct androgen repression of AMH only occurred in AR-expressing SMAT1 cells. Functional ARs are essential for TT-mediated AMH repression in SC.

Lack of Androgen Receptor Expression in Sertoli Cells Accounts for the Absence of Anti-Mullerian Hormone Repression during Early Human Testis Development

Lack of Androgen Receptor Expression in Sertoli Cells Accounts for the Absence of Anti-Mullerian Hormone Repression during Early Human Testis Development

Physiological Androgen Insensitivity of the Fetal, Neonatal, and Early Infantile Testis Is Explained by the Ontogeny of the Androgen Receptor Expression in Sertoli Cells

Although gonadotropins and testosterone are high in the fetal/early postnatal periods, Sertoli cells remain immature and spermatogenesis does not progress. We hypothesized that Sertoli cells do not respond to testosterone because they do not express the androgen receptor. The objective of the study was to describe the precise ontogeny of androgen receptor expression in the human testis from fetal life through adulthood. This was an immunohistochemical study on testicular biopsies from fetal, neonatal, prepubertal, pubertal, and adult human testes. Quantification of androgen receptor expression in Sertoli cells was measured. Evaluation of androgen receptor expression in peritubular and interstitial cells as well as anti-Müllerian hormone and inhibin-alpha was also performed. Androgen receptor expression was first observed in the nuclei of few Sertoli cells at the age of 5 months. Labeling was weak in 2-15% of Sertoli cells until 4 yr of age and progressively increased thereafter. High levels of androgen receptor expression were observed in more than 90% from the age of 8 yr through adulthood. Androgen receptor was positive in peritubular cells and variable in interstitial cells. Anti-Müllerian hormone immunolabeling was strong in all Sertoli cells from fetal life throughout prepuberty and weakened progressively as spermatogenesis developed. Inhibin-alpha expression was detected in all Sertoli cells from fetal life through adulthood. A lack of androgen receptor expression could explain a physiological Sertoli cell androgen insensitivity during fetal and early postnatal life, which may serve to protect the testis from precocious Sertoli cell maturation, resulting in proliferation arrest and spermatogenic development.

Lack of expression of androgen receptor may play a critical role in transformation from in situ to invasive basal subtype of high-grade ductal carcinoma of the breast

Androgen receptor has been implicated in the pathogenesis of breast carcinoma. In this study, we explored the potential role of androgen receptor in breast cancer by analyzing its expression using immunohistochemistry and its relationship with tumor progress (ductal carcinoma in situ [DCIS] versus invasive ductal carcinoma [IDC]); nuclear grades (high grade [HG] versus non-high grade); expression of estrogen receptor (ER), progesterone receptor (PR), HER-2; and 3 molecular classifications: cytokeratin classification, triple (ER/PR/HER-2) negative classification, and ER/HER-2 classification in 184 breast carcinomas. We found that (1) lack of androgen receptor expression was associated with HG-IDC and with basal subtypes of HG-IDC, suggesting androgen receptor may play an important role in preventing the invasive transformation in this subgroup of breast carcinoma. (2) HG-IDC and HG-DCIS more frequently expressed androgen receptor than ER (55%-93% for androgen receptor and 18%-30% for ER) and were frequently androgen receptor+/ER- (63% for HG-DCIS and 39% for HG-IDC), which made androgen receptor a possible therapeutic target. (3) One third of HG-IDC was negative for androgen receptor, ER, PR, and HER-2, suggesting that further studies are needed to identify other key molecules for targeted therapy. We purpose that androgen receptor should be routinely measured for breast cancer.

Androgen Receptor Expression Helps to Differentiate BCC From Trichoblastoma and Trichoepithelioma

Histological differentiation between basal cell carcinoma and benign trichoblastic neoplasms such as trichoepithelioma and trichoblastoma can be difficult on small biopsies. Therefore, several attempts have been made to identify immunohistochemical differences between these entities. Recent studies showed androgen receptor expression in a number of mature epithelial structures in the skin and in epithelial neoplasms including basal cell carcinoma. In contrast, androgen receptor expression was absent in mature hair follicles or the few trichogenic neoplasms studied to date. These findings suggested that androgen receptor expression might be a useful adjunct in the histological differential diagnosis between basal cell carcinoma and benign trichoblastic neoplasms. Therefore, we performed immunohistochemical analysis of androgen receptor expression in 32 basal cell carcinomas and 10 benign trichoblastic tumors (6 trichoepitheliomas and 4 trichoblastomas). In our study, expression of androgen receptor was detected in 78% of basal cell carcinomas. None of the trichoblastic tumors showed any androgen receptor immunoreactivity. These results confirm the lack of androgen receptor expression in benign trichoblastic neoplasms and indicate that finding of androgen receptor expression points to basal cell carcinoma as the most likely diagnosis.

Secretion of endogenous kallikreins 2 and 3 by androgen receptor-transfected PC-3 prostate cancer cells

Androgen independent PC-3 cells lack androgen receptor (AR) expression and do not produce kallikrein 2 (hK2) or 3 (prostate-specific antigen, PSA). In this paper, we examined the ability of androgens to stimulate PSA and hK2 production in AR transfected PC-3 cells (PC-3(AR)) and compared this to LNCaP cells. PSA and hK2 were measured in the culture medium and cell lysates using an ELISA-based immunofluorometric assay. Only androgens were able to induce PSA and hK2 secretion in PC-3(AR) cells in a dose- and time-dependent manner depending on the level of AR present. The level of androgen-induced PSA and hK2 secretion in PC-3(AR) cells was approximately 1.5 and 0.9% that induced in LNCaP cells, respectively. Insulin-like growth factor-I (IGF-I), which has been shown to activate AR in the absence of ligand, did not activate PSA secretion in the absence of androgen, but further increased the dihydrotestosterone-induced PSA secretion in PC-3(AR) cells. The lack of PSA and hK2 production in parental PC-3 cells is thus a result of their lack of AR expression. PSA and/or hK2 production in PC-3(AR) cells can thus serve as an endogenous reporter system to investigate AR action or to screen putative endocrine disrupters.