AR Gene Expression Research Articles

Seasonal heterochrony of reproductive development and gene expression in a polymorphic salamander.

Life cycle evolution includes ecological transitions and shifts in the timing of somatic and reproductive development (heterochrony). However, heterochronic changes can be tissue-specific, ultimately leading to the differential diversification of traits. Salamanders exhibit alternative life cycle polymorphisms involving either an aquatic to terrestrial metamorphosis (biphasic) or retention of aquatic larval traits into adulthood (paedomorphic). In this study, we used gene expression and histology to evaluate how life cycle evolution impacts temporal reproductive patterns in males of a polymorphic salamander. We found that heterochrony shifts the distribution of androgen signaling in the integument, which is correlated with significant differences in seasonal reproductive gland development and pheromone gene expression. In the testes, androgen receptor (ar) expression does not significantly vary between morphs or across seasons. We found significant differences in the onset of spermatogenesis, but by peak breeding season the testes were the same with respect to both histology and gene expression. This study provides an example of how seasonal heterochronic shifts in tissue-specific ar gene expression can disparately impact seasonal development and expression patterns across tissues, providing a potential mechanism for differential diversification of reproductive traits.

NR5A1/SF-1 Collaborates with Inhibin α and the Androgen Receptor.

Steroidogenic factor 1 (SF-1) is a nuclear receptor that regulates steroidogenesis and reproductive development. NR5A1/SF-1 variants are associated with a broad spectrum of phenotypes across individuals with disorders of sex development (DSDs). Oligogenic inheritance has been suggested as an explanation. SF-1 interacts with numerous partners. Here, we investigated a constellation of gene variants identified in a 46,XY severely undervirilized individual carrying an ACMG-categorized 'pathogenic' NR5A1/SF-1 variant in comparison to the healthy carrier father. Candidate genes were revealed by whole exome sequencing, and pathogenicity was predicted by different in silico tools. We found variants in NR1H2 and INHA associated with steroidogenesis, sex development, and reproduction. The identified variants were tested in cell models. Novel SF-1 and NR1H2 binding sites in the AR and INHA gene promoters were found. Transactivation studies showed that wild-type NR5A1/SF-1 regulates INHA and AR gene expression, while the NR5A1/SF-1 variant had decreased transcriptional activity. NR1H2 was found to regulate AR gene transcription; however, the NR1H2 variant showed normal activity. This study expands the NR5A1/SF-1 network of interacting partners, while not solving the exact interplay of different variants that might be involved in revealing the observed DSD phenotype. It also illustrates that understanding complex genetics in DSDs is challenging.

The possible role of epigenetics in the etiology of hypospadias

Hypospadias is a common malformation of the genitourinary system and is thought with a complex interplay between genetics and environmental factors likely contributing to its pathogenesis. This study aimed to investigate the receptor gene expressions of sex hormones, FGFR2, FGF8 and BMP7 and DNA methylations in these genes as an epigenetic mark, which may play a role in the etiology of hypospadias. The samples from the foreskin of 20 patients with hypospadias and 20 healthy children who underwent circumcision operations were collected. AR, ESR1, FGF8, FGFR2 and BMP7 gene expressions and DNA methylation rates of these genes were investigated in tissues. While ESR1, FGFR2 and BMP7 gene expressions were found to be significantly higher in the hypospadias group, AR gene expression was found to be lower. In the hypospadias group, DNA methylation rates were found to be significantly higher in the ESR1, FGF8 and FGFR2 genes, but lower in the AR gene (Table). Recent clinical studies suggest that epigenetic modifications may play a significant role in genital development, potentially contributing to the etiology of hypospadias. Our recent study demonstrated significant differences in foreskin AR, ESR1, and FGFR2 gene expression between patients with hypospadias and controls. To address this, the present study investigated DNA methylation levels of these same genes in hypospadias patients, hypothesizing that epigenetic modifications might be responsible for the observed gene expression changes. We again observed abnormalities in AR, ESR1, and FGFR2 gene expression in hypospadias patients. Furthermore, we found that DNA methylation patterns associated with these genes differed significantly between hypospadias and control groups. Our study demonstrates significant alterations in DNA methylation of sex hormone receptor genes (ESR1 and AR), FGFR2, and FGF8, which correlate with abnormal expression of these genes in hypospadias cases. These findings suggest a potential role for epigenetic modifications in hypospadias etiology.

Genomic profiling of metastatic castration-resistant prostate cancer samples resistant to androgen-receptor pathway inhibitors.

The androgen receptor axis inhibitors (ARPI) (e.g, enzalutamide, abiraterone acetate) are administered in daily practice for men with metastatic castration-resistant prostate cancer (mCRPC). However, not all patients respond, and mechanisms of both primary and acquired resistance remain largely unknown Experimental Design: In a prospective trial MATCH-R (NCT02517892), 59 mCRPC patients underwent whole exome sequencing (WES) and/or RNA-sequencing (RNA-seq) of samples collected before starting ARPI. Also, 18 mCRPC patients underwent biopsy at time of resistance. The objectives were to identify genomic alterations associated with resistance to ARPIs as well as to describe clonal evolution. Associations of genomic and transcriptomic alterations with primary resistance were determined using Wilcoxon and Fisher's exact tests. WES analysis indicated that no single-gene genomic alterations was strongly associated with primary resistance. RNA-seq analysis showed that AR gene alterations and expression levels were similar between responders and non-responders. RNA-based pathway analysis found that patients with primary resistance had a higher Hedgehog pathway score, a lower AR pathway score and a lower NOTCH pathway score than patients with a response. Subclonal evolution and acquisition of new alterations in AR-related genes or neuroendocrine differentiation are associated with acquired resistance. ARPIs do not induce significant changes in the tumor transcriptome of most patients; however, programs associated with cell proliferation are enriched in resistant samples. Low AR activity, activation of stemness programs, and Hedgehog pathway were associated with primary ARPIs' resistance, whereas most acquired resistance was associated with subclonal evolution, AR-related events, and neuroendocrine differentiation.

Abstract B074: Targeting CBP/p300 and its downstream transcriptional machinery in advanced PCa

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men in the US. There is a largely unmet clinical need to identify and develop novel strategies, that work either alone or in concert with AR-directed therapeutics, to combat CRPC. The highly conserved histone acetyltransferases CBP/p300 are potent co-activators for AR, and high p300 expression is associated with locally advanced disease and castration-resistant AR function. This study shows that CBP and p300 are highly expressed and correlate closely with AR gene expression and AR activity score in primary PCa and CRPC. By employing clinically relevant PCa models, the clinical significance of CBP/p300 expression in PCa patients as well as mechanistic evaluation of CBP/p300 transcriptional reprogramming and DNA damage response pathways have been undertaken. The molecular response to CBP/p300 inhibition will be assessed to discern novel metrics for precision medicine for PCa patients to improve therapeutic efficacy. Previous studies have relied on non-specific compounds and genetic silencing to target CBP/p300. CCS1477 (inobrodib) is a first-in-class bromodomain inhibitor developed by Cell Centric and targeted to inhibit CBP/p300 mediated bromodomain activity, and thus regulate cell survival. Inhibition of the CBP/p300 bromodomain resulted in significant downregulation of AR-FL, AR-V7, and its targets’ mRNA expression, as well as inhibition of associated factors such c-MYC and its downstream targets, in multiple PCa models. Transcriptomic analysis indicated that both CBP and p300 expression correlate with expression of genes involved in double strand break (DSB) DNA repair process including homologous recombination (HR) and non-homologous end joining (NHEJ) in both primary PCa and CRPC models. CCS1477 directly impacted DNA damage response and repair dynamics, as shown via delay in time to resolution of DNA damage foci formation, including RAD51 and γH2AX foci. Inhibition of CBP/p300 activity decreased tumor cell proliferation, blocked CRPC xenograft growth in vivo, and decreased proliferation ex vivo in patient-derived prostate tumor explants. Importantly, CBP/p300 expression correlated with HR genes in human prostate tissue samples in different cohorts. Lastly, inhibition of CBP/p300 activity also decreased HR gene expression in patients further supporting the essential role CBP/p300 plays in DNA repair. In sum, CBP/p300 inhibition mediates HR repair and impacts patient outcome. In conclusion, these studies identify CBP/p300 as a driver of PCa tumorigenesis through coordinated control of critical transcriptional events and lay the groundwork to optimize therapeutic strategies for advanced PCa via CBP/p300 inhibition, potentially in combination with AR-directed therapies. Combined, these studies have the capacity for significant near-term impact in the prevention and/or management of metastatic disease. Citation Format: Sumaira Sardar, Lakshmi Ravindranath, Christopher McNair, Saswati Chand, Wei Yuan, Denisa Bogdan, Jon Welti, Adam Sharp, Matthew Schiewer, Lisa Butler, Johann DeBono, Kris Frese, Nigel Brooks, Neil Pegg, Karen Knudsen, Ayesha A. Shafi. Targeting CBP/p300 and its downstream transcriptional machinery in advanced PCa [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B074.

Abstract 1449: Targeting CBP/p300 and its downstream transcriptional machinery in advanced prostate cancer

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men in the US. There is a largely unmet clinical need to identify and develop novel strategies, that work either alone or in concert with AR-directed therapeutics, to combat CRPC. The highly conserved histone acetyltransferases CBP/p300 are potent co-activators for AR, and high p300 expression is associated with locally advanced disease and castration-resistant AR function. This study shows that CBP and p300 are highly expressed and correlate closely with AR gene expression and AR activity score in primary PCa and CRPC. By employing clinically relevant PCa models, the clinical significance of CBP/p300 expression in PCa patients as well as mechanistic evaluation of CBP/p300 transcriptional reprogramming and DNA damage response pathways have been undertaken. The molecular response to CBP/p300 inhibition will be assessed to discern novel metrics for precision medicine for PCa patients to improve therapeutic efficacy. Previous studies have relied on non-specific compounds and genetic silencing to target CBP/p300. CCS1477 (inobrodib) is a first-in-class bromodomain inhibitor developed by Cell Centric and targeted to inhibit CBP/p300 mediated bromodomain activity, and thus regulate cell survival. Inhibition of the CBP/p300 bromodomain resulted in significant downregulation of AR-FL, AR-V7, and its targets’ mRNA expression, as well as inhibition of associated factors such c-MYC and its downstream targets, in multiple PCa models. Transcriptomic analysis indicated that both CBP and p300 expression correlate with expression of genes involved in double strand break (DSB) DNA repair process including homologous recombination (HR) and non-homologous end joining (NHEJ) in both primary PCa and CRPC models. CCS1477 directly impacted DNA damage response and repair dynamics, as shown via delay in time to resolution of DNA damage foci formation, including RAD51 and γH2AX foci. Inhibition of CBP/p300 activity decreased tumor cell proliferation, blocked CRPC xenograft growth in vivo, and decreased proliferation ex vivo in patient-derived prostate tumor explants. Importantly, CBP/p300 expression correlated with HR genes in human prostate tissue samples in different cohorts. Lastly, inhibition of CBP/p300 activity also decreased HR gene expression in patients further supporting the essential role CBP/p300 plays in DNA repair. In sum, CBP/p300 inhibition mediates HR repair and impacts patient outcome. In conclusion, these studies identify CBP/p300 as a driver of PCa tumorigenesis through coordinated control of critical transcriptional events and lay the groundwork to optimize therapeutic strategies for advanced PCa via CBP/p300 inhibition, potentially in combination with AR-directed therapies. Combined, these studies have the capacity for significant near-term impact in the prevention and/or management of metastatic disease. Citation Format: Sumaira Sardar, Lakshmi Ravindranath, Christopher McNair, Saswati Chand, Wei Yuan, Denisa Bogdan, Jon Welti, Adam Sharp, Matthew Schiewer, Lisa Butler, Johann de Bono, Kris Frese, Nigel Brooks, Neil Pegg, Karen Knudsen, Ayesha Shafi. Targeting CBP/p300 and its downstream transcriptional machinery in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1449.

PD16-02 CONCURRENT TARGETING AR/AR VARIANTS AND AKR1C3 FOR ADVANCED PROSTATE CANCER THERAPY

You have accessJournal of UrologyCME1 Apr 2023PD16-02 CONCURRENT TARGETING AR/AR VARIANTS AND AKR1C3 FOR ADVANCED PROSTATE CANCER THERAPY Cameron Armstrong, Shu Ning, Wei Lou, Christopher Evans, Pui-Kai Li, and Allen Gao Cameron ArmstrongCameron Armstrong More articles by this author , Shu NingShu Ning More articles by this author , Wei LouWei Lou More articles by this author , Christopher EvansChristopher Evans More articles by this author , Pui-Kai LiPui-Kai Li More articles by this author , and Allen GaoAllen Gao More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003271.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: AR/AR-variants and AKR1C3 play critical roles in prostate cancer progression and driving resistance to antiandrogens, and are attractive targets for therapeutic intervention for advanced prostate cancer. However, there are currently no clinically available therapies that simutanously target both AR/AR variants and AKR1C3. We have developed a number of novel small molecules (LX) that can concurrently inhibit both AR/AR-variants and AKR1C3. METHODS: A library of novel compounds (LX) was designed and synthesized according to structure based computer modeling. Cell growth assays were used to assess their efficacy at inhibiting CRPC growth. The effects of the LXs on AR/AR-variants and AKR1C3 expression were evaluated by Western blot. PSA-luciferase assays were used to determine effects on AR signaling activity. RNA-seq was performed on lead LXs (LX-1 and LX-5). Resistant cell sublines generated from C4-2B cells resistant to enzalutamide (MDVR), apalutamide (ApalR), darolutamide (DaroR), or abiraterone (AbiR) were treated with LX-1 or their respective antiandrogen and cell number was determined. Mice bearing VCaP xenograft tumors and LuCaP35CR PDX tumors were treated with LX-1 and effects on tumor growth were assessed. RESULTS: The lead compound, LX-1, showed the greatest effect at reducing cell number, AR/AR variant expression, and AKR1C3 activity. PSA-luciferase activity was greatly reduced by LX-1. RNA-seq analysis showed a robust reduction in AR and AR-V7 signaling gene expression by LX-1. LX-1 treatment significantly inhibited the growth of resistant cells including enzalutamide resistant MDVR, apalutamide resistant ApalR, darolutamide resistant DaroR, and abiraterone resistant AbiR. Co-treatment of LX-1 and anti-androgens in resistant lines improved treatment response. LX-1 inhibited conversion of the testosterone precursor androstenedione into testosterone in AKR1C3 overexpressing C4-2B and LNCaP cells. Additionally, LX-1 treatment reduced testosterone production by LUCaP35CR tumor cells which express high levels of AKR1C3 in the presence of androstenedione in a dose-dependent manner ex vivo. Furthermore, treatment with LX-1 reduced tumor growth in both VCaP and LuCaP35CR PDX models and reduced intratumoral testosterone. CONCLUSIONS: We generated novel small molecule inhibitors that concurrent target AR/AR-variant and AKR1C3. These compounds, specifically LX-1, effectively reduce CRPC growth and synergize with antiandrogens including enzalutamide in vitroand in vivo, suggesting potential for treating advanced prostate cancer. Source of Funding: NIH CA253605, CA 225836, CA271327 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e489 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Cameron Armstrong More articles by this author Shu Ning More articles by this author Wei Lou More articles by this author Christopher Evans More articles by this author Pui-Kai Li More articles by this author Allen Gao More articles by this author Expand All Advertisement PDF downloadLoading ...

Abstract P2-11-22: Expression of milk whey protein genes in human breast carcinoma cells isolated by LCM impacts prediction of clinical outcomes

Abstract Background: Milk whey protein consumption has been associated with breast cancer prevention, therapy and treatment tolerance, and other studies detected these proteins or their mRNAs in breast cancer models or tissues. Our objectives are to ascertain expression of certain whey protein genes in human breast carcinoma cells isolated by Laser Capture Microdissection (LCM) and to determine if gene expression is related to risk of recurrence and overall survival in lesions with various ER/PR status. Genes selected were: LALBA (alpha-lactalbumin), LPO (lactoperoxidase), LTF (lactotransferrin), PAEP (progestogen associated endometrial protein or glycodelin) and SSP1 (secreted phosphoprotein 1 or osteopontin). This retrospective investigation is unique in that highly quantified ER and PR protein levels and ESR1 and PGR relative expression from pure populations of carcinoma cells were employed to stratify cancers into ER/PR subcategories to assess relationships of expression of candidate genes with clinical outcomes. Methods: This investigation of primary breast carcinomas from 170 postmenopausal and 77 premenopausal patients used a matchless deidentified dataset of quantified estrogen receptor (ER) and progestin receptor (PR) protein results with clinical follow-up. ER/PR protein levels, expressed as fmol/mg cytosol protein, were quantified earlier from each breast carcinoma with FDA-approved kits, either Radioligand-Binding Assay (LBA, NEN/DuPont) and/or enzyme immunoassay (EIA, Abbott Labs). Patients were treated with the standard of care at the time of diagnosis. The PixCell IleTM (ThermoFisher, Arcturus) LCM instrument was used to procure only carcinoma cells from tissue sections of de-identified, frozen biopsies, using protocols we employed earlier. Total RNA of each fixed specimen was extracted, purified and amplified to obtain relative expression levels of approximately 22,000 genes by microarray, providing an assessment of mRNA in carcinoma cells only. Relationships of relative gene expression, quantified ER/PR and other features of primary breast cancers were analyzed with clinical results using R software v4.2.0 (Vigorous Calisthenics) by Univariable Cox Regression, Kaplan Meier plots and various tests (i.e., Wilcoxon, Log Ranked, Spearman and/or Chi Square tests). Bidirectional elimination stepwise regression was also used to derive best predictors of patient outcomes. Results: Violin plots of expression distribution of each milk protein gene of 247 biopsies regardless of patient menopausal status revealed that LALBA and SSP1 were highly elevated (p < 0.001) in ER+ (n = 146) or PR+ (n = 146) breast cancer cells while PAEP was quite diminished (p < 0.001). No difference was observed in LPO expression in ER+ or PR+ cells and LTF was only elevated slightly (p < 0.05) in ER+ cells. Regardless of ER/PR status of breast cancer, LTF, LALBA, SSP1, AR and ESR1 gene expression levels were increased significantly in postmenopausal versus premenopausal patients while PAEP was decreased (p < 0.001). No difference was observed in LPO or PGR expression. Correlation matrices examined gene to gene and receptor protein to gene relationships. Using Kaplan Meier analyses of each gene alone with Log Ranked Tests of outcomes, only SSP1 expression was associated with clinical outcome, i.e., overexpression was associated with lower PFS and OS (p = < 0.05). Conclusions: Application of bidirectional elimination stepwise regression of gene expression of the five milk proteins collectively with ER/PR protein status of each carcinoma revealed that LALBA gene expression enhanced prediction of PFS in ER+ cancer while PAEP expression was associated with improved prediction of OS in ER+ carcinomas. We report that expression of LALBA and PAEP genes, largely associated with milk production in normal breast tissue, are also expressed in certain breast carcinomas and influence prediction of patient survival. Citation Format: James L. Wittliff, Michael W. Daniels. Expression of milk whey protein genes in human breast carcinoma cells isolated by LCM impacts prediction of clinical outcomes [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-22.

The involvement of high succinylation modification in the development of prostate cancer.

Succinylation modification of the lysine site plays an important role in tumorigenesis and development, but it is rarely reported in prostate cancer (PCa), so this study aims to elucidate its expression in and clinical correlation with PCa. A total of 95 tumor, 3 normal and 52 paired adjacent tissue of PCa were involved for succinylation stanning. 498 PCa samples with 20 succinylation modification-related genes from TCGA were downloaded for model construction. Statistical methods were employed to analyze the data, including Non-Negative Matrix Factorization (NMF) algorithm, t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm and Cox regression analysis. The pan-succinyllysine antibody stanning indicated that tumor tissues showed higher succinyllysine level than adjacent tissues (p<0.001). Gleason grade and PDL1 expression levels were significantly different (p<0.001) among the high, medium and low succinylation staining scores. The types of PCa tissue were divided into four clusters using RNA-seq data of 20 succinylation-related genes in TCGA database. Clinical characterize of age, PSA level, and pathological stage showed differences among four clusters. The expression of succinylation-related genes (KAT5, SDHD and GLYATL1) and PCa related genes (PDL1, AR and TP53) were significantly different in 52 matched tumor and adjacent tissues (p<0.001). GLYATL1 and AR gene expression was significantly related to the pathological stage of PCa. Succinylation was significantly increased in PCa tissues and was closely related to Gleason grade and PD-L1 expression. Model construction of 20 genes related to succinylation modification showed that the later the pathological stage of PCa, the higher the level of succinylation modification.

Association of androgen receptor expression with glucose metabolic features in triple-negative breast cancer.

BackgroundAndrogen receptor (AR) is a potential therapeutic target in triple-negative breast cancer (TNBC). We aimed to elucidate the association of AR expression with glucose metabolic features in TNBC.MethodsTwo independent datasets were analyzed: FDG PET data of our institution and a public dataset of GSE135565. In PET analysis, patients with TNBC who underwent pretreatment PET between Jan 2013 and Dec 2017 were retrospectively enrolled. Clinicopathologic features and maximum standardized uptake value (SUVmax) of tumors were compared with AR expression. In GSE135565 dataset, glycolysis score was calculated by the pattern of glycolysis-related genes, and of which association with SUVmax and AR gene expression were analyzed.ResultsA total of 608 female patients were included in the PET data of our institution. SUVmax was lower in AR-positive tumors (P < 0.001) and correlated with lower AR expression (rho = –0.26, P < 0.001). In multivariate analysis, AR was a deterministic factor for low SUVmax (P = 0.012), along with other key clinicopathologic features. In the GSE135565 dataset, AR expression also exhibited a negative correlation with SUVmax (r = –0.34, P = 0.001) and the glycolysis score (r = –0.27, P = 0.013).ConclusionsLow glucose metabolism is a signature of AR expression in TNBC. It is suggested that evaluation of AR expression status needs to be considered in clinical practice particularly in TNBC with low glucose metabolism.

Prenatal androgenization causes expression changes of progesterone and androgen receptor mRNAs in the arcuate nucleus of female mice across development.

Prenatal exposure to excess androgens is associated with the development of polycystic ovary syndrome (PCOS). In prenatally androgenised (PNA) mice, a model of PCOS, progesterone receptor (PR) protein expression is reduced in arcuate nucleus (ARC) GABA neurons. This suggests a mechanism for PCOS-related impaired steroid hormone feedback and implicates androgen excess with respect to inducing transcriptional repression of the PR-encoding gene Pgr in the ARC. However, the androgen sensitivity of ARC neurons and the relative gene expression of PRs over development and following prenatal androgen exposure remain unknown. Here, we used a quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) of microdissected ARC to determine the relative androgen receptor (Ar) and progesterone receptor (Pgr) gene expression in PNA and control mice at five developmental timepoints. In a two-way analysis of variance, none of the genes examined showed expression changes with a statistically significant interaction between treatment and age, although PgrA showed a borderline interaction. For all genes, there was a statistically significant main effect of age on expression levels, reflecting a general increase in expression with increasing age, regardless of treatment. For PgrB and Ar, there was a statistically significant main effect of treatment, indicating a change in expression following PNA (increased for PgrB and decreased for Ar), regardless of age. For PgrA, there was a borderline main effect of treatment, suggesting a possible change in expression following PNA, regardless of age. PgrAB gene expression changes showed no significant main effect of treatment. We additionally examined androgen and progesterone responsiveness specifically in P60 ARC GABA neurons using RNAScope® (Advanced Cell Diagnostics, Inc.) in situ hybridization. This analysis revealed that Pgr and Ar were expressed in the majority of ARC GABA neurons in normal adult females. However, our RNAScope® analysis did not show significant changes in Pgr or Ar expression within ARC GABA neurons following PNA. Lastly, because GABA drive to gonadotropin-releasing hormone neurons is increased in PNA, we hypothesised that PNA mice would show increased expression of glutamic acid decarboxylase (GAD), the rate-limiting enzyme in GABA production. However, the RT-qPCR showed that the expression of GAD encoding genes (Gad1 and Gad2) was unchanged in adult PNA mice compared to controls. Our findings indicate that PNA treatment can impact Pgr and Ar mRNA expression in adulthood. This may reflect altered circulating steroid hormones in PNA mice or PNA-induced epigenetic changes in the regulation of Pgr and Ar gene expression in ARC neurons.

PD44-08 NOVEL DUAL TARGETING AKR1C3 AND AR/AR VARIANTS INHIBITS ANDROGEN SIGNALING AND OVERCOMES RESISTANCE TO ANTIANDROGEN TREATMENT

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II (PD44)1 Sep 2021PD44-08 NOVEL DUAL TARGETING AKR1C3 AND AR/AR VARIANTS INHIBITS ANDROGEN SIGNALING AND OVERCOMES RESISTANCE TO ANTIANDROGEN TREATMENT Cameron Armstrong, Shu Ning, Wei Lou, Jinge Zhao, Christopher Evans, Pui-Kai Li, and Allen Gao Cameron ArmstrongCameron Armstrong More articles by this author , Shu NingShu Ning More articles by this author , Wei LouWei Lou More articles by this author , Jinge ZhaoJinge Zhao More articles by this author , Christopher EvansChristopher Evans More articles by this author , Pui-Kai LiPui-Kai Li More articles by this author , and Allen GaoAllen Gao More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002058.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: AR/AR-V7 and AKR1C3 play key roles in prostate cancer progression and drive resistance to current therapies. Thus far, there are no clinically available therapies to specifically target either AR-V7 or AKR1C3, nor are there dual inhibitors for AR/AR-V7 and AKR1C3. We have developed a number of novel small molecules (LX) that are dual inhibitors of AR/AR-V7 and AKR1C3. METHODS: We designed and synthesized a library of novel compounds according to the structure based computer modeling. Fourteen were selected for initial study. Cell growth assays were used to assess their efficacy at inhibiting CRPC growth. The effects of the LXs on AR/AR variants and AKR1C3 expression were determined by Western blot. PSA-luciferase assays were used to determine effects on AR signaling activity. RNA-seq was performed on selected LXs (LX-1 and LX-5) based on their improved efficacy over the other LX compounds. Resistant cell sublines generated from C4-2B cells resistant to enzalutamide (MDVR), apalutamide (ApalR), darolutamide (DaroR), or abiraterone (AbiR) were treated with LX-1 or their respective antiandrogen and cell number was determined. Mice bearing VCaP xenograft tumors and LuCaP35CR PDX tumors were treated with LX-1 and the effects on tumor growth were determined. RESULTS: Of the 14 LX compounds, LX-1 had the greatest effect at reducing cell number, AR/AR variant expression, and AKR1C3 activity. LX-5 was the next most effective compound. PSA-luciferase activity was greatly reduced by both LX-1 and LX-5. RNA-seq analysis demonstrated a robust reduction in AR and AR-V7 signaling gene expression by both LX-1 and LX-5. MDVR, ApalR, DaroR, and AbiR cells all showed a reduction in cell number when treated with LX-1. LX-1 inhibited conversion of the testosterone precursor androstenedione into testosterone in AKR1C3 overexpressing C4-2B and LNCaP cells. Additionally, LX-1 treatment reduced testosterone production by LUCaP35CR tumor cells which express high levels of AKR1C3 in the presence of androstenedione in a dose-dependent manner ex vivo. In addition, treatment with LX-1 reduced tumor growth in both VCaP and LuCaP35CR PDX models. Furthermore, LX-1 treatment reduced intratumoral testosterone. CONCLUSIONS: We generated novel small molecule inhibitors that dual target AKR1C3 and AR/AR variants. These compounds, specifically LX-1, effectively reduce CRPC growth in vitro and in vivo, suggesting a clinical potential for treating advanced prostate cancer. Source of Funding: NIH/NCI CA 225836 © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e740-e740 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Cameron Armstrong More articles by this author Shu Ning More articles by this author Wei Lou More articles by this author Jinge Zhao More articles by this author Christopher Evans More articles by this author Pui-Kai Li More articles by this author Allen Gao More articles by this author Expand All Advertisement Loading ...

Androgen and Estrogen Receptor Activation Impacts Cardiac Function in a PCOS-Prone Rodent Model

Abstract Introduction: The global risk and incidence of cardiovascular disease (CVD) is increased in polycystic ovary syndrome (PCOS). Early risk factors and subclinical CVD includes atherogenic dyslipidemia, obesity, insulin resistance, blood pressure, atherosclerosis and impaired cardiac function. Androgen exposure is associated with onset of adiposity, impaired insulin-glucose and lipid metabolism, and cardiac dysfunction. The mechanisms of increased risk of CVD and cardiac dysfunction in PCOS related to hyperandrogenemia, AR and estrogen receptor (ER) activation remain unclear. Aim: The aim of this study was to investigate the effect of androgen treatment on cardiac AR and ER activation, fatty acid metabolism and cardiac function in a PCOS-prone rodent model. Methods: A PCOS-prone rodent model at 6 wks of age with obesity, apoB-remnant lipemia and insulin resistance, and controls were treated with testosterone for 12 weeks. Cardiac function was assessed using transthoracic doppler echocardiography (M-Mode 2D-imaging), lipogenic, AR, ER and other metabolic gene and protein expression were assessed using RTPCR and SDS-PAGE western blot. Results: PCOS-prone animals exhibited left ventricular (LV) hypertrophy, with increased LV mass to body weight (551.6 ± 38.85 mg vs 999 ± 96.17 mg, p&amp;lt;0.05), LV posterior wall diastolic diameter and LV internal diastolic diameter compared to controls. Isovolumetric relaxation time (IVRT) was prolonged (15.91 ± 1.591 msec vs 23.75 ± 0.722 msec, p&amp;lt;0.05). Mild systolic dysfunction was evidenced by increased isovolumetric contraction time (IVCT; 22.5 ± 1.348 msec vs 28.96 ± 1.248 msec, p&amp;lt;0.05) and decreased % ejection fraction and % fractional shortening in PCOS-prone compared to controls. T treatment increased LV mass, IVCT and IVRT in controls but did not exacerbate cardiac function in PCOS-prone animals. T treatment increased cardiac protein expression of PPAR-⍺ in PCOS-prone and controls, and T increased ACC in controls. AR protein expression tended to be reduced, and ER-⍺ was reduced in both T treated control and PCOS-prone animals. Conclusions: The PCOS-prone rodent model demonstrates early cardiac LV hypertrophy and diastolic-systolic dysfunction and T treatment alters fatty acid metabolism, and AR and ER activation are associated with altered cardiac morphology and function in the PCOS-prone and control conditions.

Abstract PS5-33: Androgen receptor gene expression and prediction of clinical outcomes of breast cancers exhibiting triple negative or triple positive breast carcinoma cells procured by LCM

Abstract Background: Clinical utility of AR, assessed by IHC of protein or by RT-qPCR of gene expression of intact tissue biopsies, has been the focus of many studies without uniform agreement. Due to cellular heterogeneity of most breast cancer biopsies, it is often unclear if measurements of AR and certain clinically relevant biomarkers accurately reflect their content in isolated cells. Using Laser Capture Microdissection (LCM) to procure only populations of breast carcinoma cells from tissue biopsies provides the inimitable opportunity to examine relationships of AR to a variety of parameters. Methods: We performed retrospective studies using a unique, de-identified dataset of ER/ESR1, PR/PGR and HER2/ERBB2 results with AR gene expression determined from LCM-procured carcinoma cells of biopsies from 247 breast cancer patients with associated clinical follow-up. ER/PR protein levels, expressed as fmol/mg cytosol protein, were quantified from each carcinoma biopsy with FDA-approved kits using enzyme immunoassay (EIA, Abbott Labs) or radio-ligand binding assay (NEN/DuPont). HER2 protein content of biopsies was quantified by EIA (Oncogene Sciences). Microarray analyses of ~22,000 genes were performed on RNA isolated, purified and amplified from LCM-procured carcinoma cells to assess relative gene expression. Biomarker results and de-identified clinical outcomes were examined using REMARK criteria in a CLIA licensed laboratory. Gene expression, quantified biomarkers, features of primary breast cancers and clinical outcomes were analyzed by univariable and multivariable Cox regressions, Fisher’s Exact Test, Kaplan Meier plots and with R software v4.0.0. Clinical relevance of gene expression was externally validated with SurvExpress. (Aguirre-Gamboa et al. PLoS One e742502013). Results: Using the median value of relative AR expression in LCM-procured carcinoma cells as a cutoff, elevated AR mRNA, when considered independently, was associated with the following (given as the means): older patients (61.1 vs 55.8 yo), relative expression of either ESR1 (0.2 vs -3.1), PGR (0.4 vs 0.0) or ERBB2 (0.7 vs -0.1) as well as longer PFS (63.7 vs 52.6 mos) and OS (72.4 vs 61.8 mos). Also, carcinoma cells with increased AR mRNA exhibited higher levels of ER protein (242.4 vs 106.5 fmol/mcp) or PR protein (313.8 vs 90.2 fmol/mcp). Whereas, HER2 protein status was not significantly different between AR median bifurcated groups). Neither patient race nor tissue pathology was examined in these analyses. Significantly, Kaplan Meier analyses clearly indicated that addition of AR gene expression to TNBC status (so called QNBC, Quadruple Negative Breast Cancer) did not alter either PFS (5 yr = 0.78 vs 0.79 % probability) nor OS (5 yr = 0.57 vs 0.56 %) of patients. Similarly, Kaplan Meier analyses also indicated that addition of AR gene expression to TPBC status (so called QPBC, Quadruple Positive Breast Cancer) did not alter PFS (5 yr = 0.87 vs 0.87 % probability) nor OS (5 yr = 0.67 vs 0.66 %) of patients. Also, no differences were ascertained for PFS nor for OS of patients at 10 yrs of outcomes between TNBC and QNBC breast cancers clearly indicating that AR gene expression does not contribute to the assessment of a patient’s clinical course. Conclusions: Collectively, when cancers were compared as TNBC versus TPBC status, expression results of ESR1, PGR and ERBB2 genes using LCM-procured carcinoma cells indicated poorer prognosis and overall survival of breast cancer patients with TNBC. However, inclusion of AR gene expression in either of these bifurcated groups ascertained from LCM-procured cells, did not alter patient PFS nor OS suggesting AR utility in clinical management appears to be unwarranted. Supported in part by grants to JLW from Phi Beta Psi Sorority Charity Trust. Citation Format: James L Wittliff, Michael W. Daniels. Androgen receptor gene expression and prediction of clinical outcomes of breast cancers exhibiting triple negative or triple positive breast carcinoma cells procured by LCM [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-33.

The Important Role of TMPRSS2 Gene in Covid-19 and Prostate Cancer: In Silico Approach

HIGHLIGHTS The main characteristic of SARS-CoV, MERS-CoV and SARS-CoV-2 is that air passes through the upper respiratory tract The tissue of the upper respiratory tract can express both ACE2 and TMPRSS2. AR is abundantly expressed in lungs. TMPRSS2, ACE2, and AR connection explains why high expression of AR gene is present in the male lung.

Impact of Androgen Receptor Gene Expression in Gastric Cancer: a Meta-Analysis Based on the GEO Database, TCGA Database, and Literature

To evaluate androgen receptor and AR gene expression impact of gastric cancer (GC). Meta-analysis of microarray data obtained from the Gene Expression Omnibus (GEO) database, TCGA (The Cancer Genome Atlas) database, and data by systematic search of PubMed, China National Knowledge Internet and Web of Science. This meta-analysis included 30 studies involving 4487 patients. From our results, we can know that the positive rate of AR expression in gastric cancer is 40%. The expression of AR was significantly higher in men (SWD = 1.00, 95% CI 0.03 ~ 1.96, P = 0.042). The higher AR expression was linked with poor over survival (OS) (HR = 1.30, 95% CI 1.13 ~ 1.49, P < 0.0001). AR gene may be an important factor affecting the poor prognosis of GC patients of different genders.

Prolonged use of finasteride-induced gonadal sex steroids alterations, DNA damage and menstrual bleeding in women.

The aim of the present study was to examine the effect of prolonged use of finasteride on serum levels of dihydrotestosterone (DHT), estradiol (E2), progesterone, testosterone and androstenedione in women during the menstrual period. Further, to screen and compare the 5α-reductase activities through the expression of SRD5A1, SRD5A2 and AR gene and to determine the level of VEGF, VKOR and SAA gene expression and DNA damage. A total of 30 Saudi women aged between 25 and 35 years were enrolled in the study. The selected women were divided into two groups. The first group (n = 15) received 5 mg finasteride/day for prolonged period of one year and second group (n = 15) was taken as a healthy control. ELISA technique was used for measuring the serum levels of the targeted hormones, and Comet assay was used for checking the DNA integrity. Our findings revealed significant decrement of DHT, E2, progesterone and androstenedione levels and elevated levels of testosterone in group treated with daily oral doses of 5 mg finasteride/day compared with the control subjects. mRNA expression suggested that finasteride has concrete effects on the gene expression of the selected genes from the treated group in comparison with the control group. In addition, finasteride induced DNA damage, and heavy menstrual bleeding was noted in women treated with finasteride. In conclusion, the present findings revealed that finasteride has adverse health effects in women associated with gonadal sex steroids alterations, DNA damage and heavy menstrual bleeding with no consensus in the treatment of androgenetic alopecia in women.

Enantioselective Elimination and Gonadal Disruption of Lambda-Cyhalothrin on Lizards ( Eremias argus).

In this study, the different metabolic pathways of lambda-cyhalothrin (LCT) enantiomers in Eremias argus feces and enantioselective disruption on hypothalamus-pituitary-gonad (HPG) system were investigated. After 7 days oral exposure to LCT enantiomers, the concentration of 3-phenoxybenzoic acid (PBA), hydroxylated and sulfated LCT were higher in the (+)-LCT exposure group than that in the (-)-LCT exposure group, which indicated a higher metabolic rate of (+)-LCT than (-)-LCT. Although no significant differences were seen on lizard body weight after enantiomers' exposure, the gonadosomatic index was dramatically decreased. The testicular impacts such as increased seminiferous tubule diameters were only observed in the (+)-LCT exposure group. Consistent with this result, the expression of ar gene in the (+)-LCT exposure was significantly higher than that in the (-)-LCT exposure group. In addition, the stronger binding affinity of AR with (+)-LCT further demonstrated the more serious disruption of (+)-LCT on lizard HPG axis than (-)-LCT. This study first elucidated the metabolic pathway and endocrine effects of LCT in lizards at enantiomeric level and provided some evidence for lizard population decline.

Lactational exposure of polychlorinated biphenyls impair Leydig cellular steroidogenesis in F1 progeny rats

The present study was aimed to determine the effects of lactational exposure of PCBs (Aroclor 1254) on Leydig cellular steroidogenesis in F1 progeny rats. Lactating dams were orally treated by gavage with different doses of PCBs (1, 2 and 5mg/kg b.wt./day). Male progenies were sacrificed on PND60. Our results demonstrated that exposure to PCBs decreased the body weight, testis weight and anogenital distance (AGD) index in the F1 progeny rats. Importantly, PCBs exposure reduced the serum levels of LH, testosterone and estradiol. Interestingly, PCBs caused a decrease in the Leydig cell population along with decreased activities of steroidogenic enzymes 3β- and 17β-HSD. Additionally, we observed a significant decrease in LHR, SR-B1, StAR protein, Cyp11a1, 3β-HSD, Cyp17a1, 17β-HSD, 5α-reductase, Cyp19a1 and AR gene expression in the Leydig cells of progeny rats. In conclusion, our study demonstrates that lactational exposure of PCBs alters Leydig cellular steroidogenesis in the F1 progeny rats.

Isorhapontigenin induced cell growth inhibition and apoptosis by targeting EGFR-related pathways in prostate cancer.

Isorhapontigenin (ISO), a naturally phytopolyphenol compound existing in Chinese herb, apples, and various vegetables, has attracted extensive interest in recent years for its diverse pharmacological characteristics. Increasing evidences reveal that ISO can inhibit cancer cell growth by induced apoptosis, however, the molecular mechanisms is not fully understood. In this study, we found for the first time that ISO apparently induced cell growth inhibition and apoptosis by targeting EGFR and its downstream signal pathways in prostate cancer (PCa) cells both in vitro and in vivo, whereas no obviously effect on normal prostate cells. From the results, we found that ISO competitively targeted EGFR with EGF and inhibited EGFR auto-phosphorylation, and then decreased the levels of p-Erk1/2, p-PI3 K, and p-AKT, and further induced down-regulation of p-FOXO1 and promoted FOXO1 nuclear translocation; and finally resulted in a significantly up-regulation of Bim/p21/27/Bax/cleaved Caspase-3/cleaved PARP-1 and a markedly down-regulation of Sp1/Bcl-2/XIAP/Cyclin D1. Moreover, our experimental data demonstrated that treatment of ISO decreased protein level of AR via both inhibiting the expression of AR gene and promoting the ubiquitination/degradation of AR proteins in proteasome. In vivo, we also found that ISO inhibited the growth of subcutaneous xenotransplanted tumor in nude mice by inducing PCa cell growth inhibition and apoptosis. Taken together, all findings here clearly implicated that EGFR-related signal pathways, including EGFR-PI3K-Akt and EGFR-Erk1/2 pathways, were involved in ISO-induced cell growth inhibition and apoptosis in PCa cells, providing a more solid theoretical basis for the application of ISO to treat patients with prostate cancer in clinic.