Androgen Receptor Knockdown Research Articles

Genetic comprehension of organophosphate flame retardants, an emerging threat to prostate cancer

In recent years, organophosphate ester flame retardants (OPFRs), which have been regarded as alternatives for brominated flame retardants (BFRs), have become widely used in building materials, textiles, and electric equipment. Elucidating the relationship between OPFRs and tumors holds great significance for the treatment and prevention of diseases. In this work, we found a new method for predicting the correlation between the interactive genes of OPFRs and tumors. Transcriptome profiles and OPFR information were obtained from The Cancer Genome Atlas and the Genotype–Tissue Expression, Comparative Toxicogenomics, and PharmMapper databases. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis showed that interactive genes were mainly enriched in prostate cancer, steroid metabolic process, and steroid hormone regulation. Furthermore, protein–protein interaction network analysis revealed 33 biological hub genes. The operating characteristic curves and survival analysis showed the role of key genes in predicting the prognosis of prostate cancer. Gene target prediction and gene set variation analysis proved that OPFRs and their metabolites exert potential effects on prostate cancer. Colony formation assay showed that the cells with AR, mTOR and DDIT3 knockdown could remarkably mitigate the cell proliferation ability in both PC-3 and LNCap cells. Transwell assay demonstrated that the silencing of AR, mTOR and DDIT3 could significantly inhibit the cell invasion capacity of prostate cells. Triphenyl phosphate (TPP) significantly increase the cell proliferation ability and promote cell invasion capacity. AR, mTOR and DDIT3 in the PC-3 and LNCap cells were significantly upregulated with 10–6 M TPP treated.

Abstract 2572: γ-Tocotrienol inhibition of androgen receptor (AR) expression and activation in triple negative breast cancer (TNBC) MBA-MB-231 and MDA-MB-453 cells is associated with a reduction in proliferation, migration and epithelial-to-mesenchymal transition (EMT)

Abstract Triple negative breast cancer (TNBC) is an aggressive invasive malignancy with the lowest 5-years survival rate. Gene expression profiling indicates that TNBC is a heterogeneous disease and at present, there is no targeted therapy available for treatment. Approximately one third of TNBC expressed androgen receptor (AR) and evaluation of AR positive TNBC primary tumors shows nuclear localization of AR, an indication of transcriptionally active receptors. AR levels are most abundant in the luminal AR (LAR) molecular subtype of TNBC, but other non-LAR molecular TNBC subtypes also display high levels of AR expression and activity. Previous studies have shown that AR inhibition or AR knockdown significantly reduces baseline proliferation, anchorage-independent growth, migration and invasion and increase apoptosis in different TNBC cell lines. γ-Tocotrienol is a natural isoform of vitamin E that displays potent anticancer activity. Studies were conducted to determine if γ-tocotrienol effects on AR levels and activity play a role in mediating γ-tocotrienol induced inhibition of TNBC cell proliferation, migration and epithelial-to-mesenchymal transition (EMT). In vitro studies showed that treatment with 0-12 μM γ-tocotrienol induced a dose-responsive significant decrease in TNBC MDA-MB-231 and MDA-MB-453 cell proliferation and viability. Treatment with 35 nM dihydrotestosterone (DHT) resulted in an increase of AR expression and corresponding increase in the growth of both TNBC cell lines. Treatment of MDA-MB-231 and MDA MB-453 cells with γ-tocotrienol (5 μM and 7 μM, respectively), significantly inhibited DHT-induced proliferation of both TNBC cell lines. Western blot analysis showed that γ-tocotrienol treatment significantly reduced DHT-induced cytoplasmic and nuclear AR expression in MDA-MB-453 cells, but induced only a slight and insignificant reduction in AR expression in MDA-MB-231 cells. Immunocytochemistry studies confirmed that γ-tocotrienol treatment induced a decrease in cytoplasmic and nuclear AR expression in both TNBC cell lines, but this decrease was only found to be significant in MDA-MB-453 cells. Other studies showed that γ-tocotrienol treatment significantly reduced cancer cell migration and this finding was associated with an increased expression in cytokeratin 8 (an epithelial cell biomarker) and decrease in vimentin (a mesenchymal cell biomarker) in both TNBC cell lines, and is an indication of a reversal in EMT. In summary, these results demonstrated that γ-tocotrienol treatment inhibits AR expression as well as DHT-dependent cell proliferation, migration and EMT in TNBC. These findings also suggest that AR may be a potential therapeutic target for the treatment of both LAR and non-LAR TNBC subtypes. Citation Format: Tasmin A. Sultana, Nayef Aldabaan, Paul W. Sylvester. γ-Tocotrienol inhibition of androgen receptor (AR) expression and activation in triple negative breast cancer (TNBC) MBA-MB-231 and MDA-MB-453 cells is associated with a reduction in proliferation, migration and epithelial-to-mesenchymal transition (EMT) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2572.

Androgen receptor signalling confers clonogenic and migratory advantages in urothelial cell carcinoma of the bladder.

Bladder urothelial cell carcinoma (UCC) incidence is about three times higher in men compared with women. There are several indications for the involvement of hormonal factors in the aetiology of UCC. Here, we provide evidence of androgen signalling in UCC progression. Microarray and qPCR analysis revealed that the androgen receptor (AR) mRNA level is upregulated in a subset of UCC cases. In an AR‐positive UCC‐derived cell line model, UM‐UC‐3‐AR, androgen treatment increased clonogenic capacity inducing the formation of big stem cell‐like holoclones, while AR knockdown or treatment with the AR antagonist enzalutamide abrogated this clonogenic advantage. Additionally, blockage of AR signalling reduced the cell migration potential of androgen‐stimulated UM‐UC‐3‐AR cells. These phenotypic changes were accompanied by a rewiring of the transcriptome with almost 300 genes being differentially regulated by androgens, some of which correlated with AR expression in UCC patients in two independent data sets. Our results demonstrate that AR signals in UCC favouring the development of an aggressive phenotype and highlights its potential as a therapeutic target for bladder cancer.

Histone Lysine Demethylase 1A Is a Master Regulator of Genes Necessary for Trophoblast Cell Proliferation

Histone lysine demethylase 1A is a master regulator of genes necessary for trophoblast cell proliferation.A proper functioning placenta is critical for pregnancy, fetal growth and development and postnatal health. Trophoblast cell proliferation and differentiation is critical for placental development and function. Recently we demonstrated that the histone lysine demethylase KDM1A binds to androgen receptor (AR) in human and sheep trophoblast cells, and targets the same promoter region of vascular endothelial growth factor A (VEGFA), suggesting a role for KDM1A and AR in early placental angiogenesis. The goal of this study was to determine the function of KDM1A during early placental development. We hypothesized that KDM1A regulates genes that are necessary for trophoblast cell proliferation, and early placental development. To this end, both in vitro and in vivo approaches were used in this study. ACH-3P cells (human first trimester trophoblast cells (CT and EVT) fused with the choriocarcinoma cell line AC1-1) were used, and a KDM1A knock out (KO) cell line was generated using CRISPR-Cas 9 based genome editing. KDM1A KO in ACH-3P cells led to significant (P<0.05) reduction in AR and VEGFA. Furthermore, factors important for cell proliferation and trophoblast cell development high mobility group AT-hook 1 (HMGA1), LIN28, and MYC protooncogene (cMYC) were significantly (P<0.05) lower in KDM1A KO ACH-3P cells. Cell proliferation assays revealed a significant (P<0.05) reduction in KDM1A KO ACH-3P cells compared to scramble controls. An in vivo experiment was conducted to demonstrate a role for KDM1A in placental development, using the sheep as a model. Day 9 hatched blastocysts were flushed and infected with a Lenti-CRISPRv2 KDM1A target construct (n=4) to knockout KDM1A specifically in the trophectoderm, or with SC (n=5). Infected embryos were transferred to recipient ewes and embryos were collected at gestational day 16. Data suggests that KDM1A KO in trophoblast cells is necessary for conceptus elongation. Current experiments are ongoing to determine the effects of KDM1A and AR knockdown using shRNA lentiviral target vectors on conceptus elongation and pregnancy. Collectively these results indicate that KDM1A plays a central role in regulating genes necessary for trophoblast cell proliferation.This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2019-67015-29000 from the USDA National Institute of Food and Agriculture.

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

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

Crosstalk between Androgen-ZIP9 Signaling and Notch Pathway in Rodent Sertoli Cells.

Our recent study demonstrated altered expression of Notch ligands, receptors, and effector genes in testes of pubertal rats following reduced androgen production or signaling. Herein we aimed to explore the role of nuclear androgen receptor (AR) and membrane androgen receptor (Zrt- and Irt-like protein 9; ZIP9) in the regulation of Notch pathway activation in rodent Sertoli cells. Experiments were performed using TM4 and 15P-1 Sertoli cell lines and rat primary Sertoli cells (PSC). We found that testosterone (10−8 M–10−6 M) increased the expression of Notch1 receptor, its active form Notch1 intracellular domain (N1ICD) (p < 0.05, p < 0.01, p < 0.001), and the effector genes Hey1 (p < 0.05, p < 0.01, p < 0.001) and Hes1 (p < 0.05, p < 0.001) in Sertoli cells. Knockdown of AR or ZIP9 as well as antiandrogen exposure experiments revealed that (i) action of androgens via both AR and ZIP9 controls Notch1/N1ICD expression and transcriptional activity of recombination signal binding protein (RBP-J), (ii) AR-dependent signaling regulates Hey1 expression, (iii) ZIP9-dependent pathway regulates Hes1 expression. Our findings indicate a crosstalk between androgen and Notch signaling in Sertoli cells and point to cooperation of classical and non-classical androgen signaling pathways in controlling Sertoli cell function.

Inhibition of Androgen Receptor Signaling Promotes Prostate Cancer Cell Migration via Upregulation of Annexin A1 Expression

Recent studies indicate that androgen deprivation therapy (ADT), the main therapeutic approach for metastatic prostate cancer (PCa), accelerates PCa invasion and metastasis. Annexin A1 (ANXA1) is a Ca2+-regulated phospholipid-binding protein that can promote PCa migration and invasion. The aim of this study is to determine whether ANXA1 is regulated by ADT and participates in PCa progression after ADT, and to explore the possible mechanism of ANXA1-mediated PCa migration. Expression of ANXA1 and androgen receptor (AR) in PCa cell lines and tissues was detected, and the association between these two proteins were analyzed. Expression of ANXA1 was evaluated after AR knockdown or AR inhibition in PCa cell lines. Cell migration of PCa cell liness after ANXA1 knockdown or overexpression was determined by invitro migration assay. Transcriptome analysis was used to explore the possible mechanism of ANXA1-mediated PCa migration. ANXA1 expression in PCa cell lines and tissues was reversely associated with AR. Invitro studies revealed an increase in ANXA1 expression after AR knockdown or treatment with AR antagonist. Moreover, functional assays indicated that ANXA1 knockdown in PCa cells significantly inhibited cell migration, while ANXA1 overexpression in PCa cells significantly accelerated cell migration. Transcriptome analysis showed that ANXA1 regulated multiple genes involved in cell junction organization, such as CADM1, LIMCH1 and PPM1F. Our results indicate that ADT might accelerate PCa metastasis via ANXA1 expression and PCa cell migration.

Androgen Receptor Signaling Reduces the Efficacy of Bacillus Calmette-Guérin Therapy for Bladder Cancer via Modulating Rab27b-Induced Exocytosis.

Although intravesical bacillus Calmette-Guérin (BCG) immunotherapy has been the gold standard for nonsurgical management of non-muscle-invasive bladder cancer, a considerable number of patients exhibit resistance to the adjuvant treatment with unexplained mechanisms. This study aimed to investigate whether and how androgen receptor (AR) signals modulate BCG cytotoxicity in bladder cancer. AR knockdown or overexpression in bladder cancer lines resulted in induction or reduction, respectively, in intracellular BCG quantity and its cytotoxic activity. Microarray screening identified Rab27b, a small GTPase known to mediate bacterial exocytosis, which was upregulated in BCG-resistant cells and downregulated in AR-shRNA cells. Knockdown of Rab27b, or its effector SYTL3, or overexpression of Rab27b also induced or reduced, respectively, BCG quantity and cytotoxicity. In addition, treatment with GW4869, which was previously shown to inhibit Rab27b-dependent secretion, induced them and reduced Rab27b expression in bladder cancer cells. Meanwhile, AR expression was upregulated in BCG-resistant lines, compared with respective controls. In a mouse orthotopic xenograft model, Rab27b/SYTL3 knockdown or GW4869 treatment enhanced the amount of BCG within tumors and its suppressive effect on tumor growth. Moreover, in non-muscle-invasive bladder cancer specimens from patients subsequently undergoing BCG therapy, positivity of AR/Rab27b expression was associated with significantly higher risks of tumor recurrence. AR activation thus correlates with resistance to BCG treatment, presumably via upregulating Rab27b expression. Mechanistically, it is suggested that BCG elimination from urothelial cells is induced by Rab27b/SYTL3-mediated exocytosis. Accordingly, Rab27b inactivation, potentially via antiandrogenic drugs and/or exocytosis inhibition are anticipated to sensitize the efficacy of BCG therapy, especially in patients with BCG-refractory AR/Rab27b-positive bladder cancer.

Abstract 4706: Transcriptional repression of mitochondrial deacetylase SIRT3 promotes prostate cancer metastasis

Abstract Prostate cancer (PCa) is the most common cancer and the second leading cause of cancer death among men in America. Aggressive prostate tumors that rapidly metastasize to other organs such as bones account for high morbidity and mortality. Metastatic PCa is resistant to androgen depletion therapy, and increased expression of androgen receptor (AR) coregulator Steroid Receptor Coregulator-2 (SRC-2) remains a major underlying driver of lethal PCa progression. Increased expression of SRC-2 positively correlates with PCa recurrence and poor disease outcome. SRC-2 directly binds to AR and regulates its transcriptional activity even in castrate-levels of androgen. Although the role of SRC-2 as a coactivator of AR is well established, we recently discovered that SRC-2 can also function as a co-repressor of AR on certain gene promoters such as mitochondrial deacetylase Sirtuin 3 (SIRT3). Analysis of human PCa databases revealed that expression of SIRT3 is reduced in prostate tumors as the disease progresses to metastatic PCa, and there exists an inverse correlation with SRC-2 expression. To confirm that SRC-2 is functioning as a co-repressor on SIRT3 promoter we performed chromatin immunoprecipitation (ChIP) and identified strong occupancy of SRC-2 on the SIRT3 promoter along with AR, whereas SRC-2 knockdown reduced AR enrichment. Consistently with this observation SRC-2 or AR knockdown significantly increased SIRT3 expression in PCa lines C4-2 and 22RV1, validating that SRC-2 functions as a transcriptional co-repressor of AR on SIRT3 promoter. This suggests that AR/SRC-2 can transcriptionally silence potential tumor suppressors. Next, we investigated the role of SIRT3 in PCa, and adenovirus-mediated ectopic expression of SIRT3 significantly impaired cell cycle progression arresting TRAMP-C2 and C4-2 cells in G0/G1 phase with reduced cell growth and viability, suggesting SIRT3 may function as a tumor suppressor in PCa. Since SRC-2 represses SIRT3 gene expression we analyzed mRNA expression of SRC-2 and SIRT3 in metastatic lesions from human prostate cancer patients using an advanced prostate cancer genomics dataset. We found that increased levels of SRC-2 with a concomitant reduction in SIRT3 expression is prevalent in metastatic lesions of PCa patients that spread to bones and lymph nodes. We used intra-iliac artery (IIA) injection to selectively deliver C4-2 cells expressing stable knockdown of SRC-2 (shSRC-2) or NTshRNA into the hind limb tissues. After 12 weeks we found SRC-2 ablation almost completely abolished PCa colonization in the bone whereas all animals transplanted with C4-2-NTshRNA developed bone metastasis. In conclusion, our study indicates that transcriptional repression of tumor suppressor SIRT3 by SRC2 may function as an oncogenic driver of lethal PCa progression to the bone. This work is supported by DoD grant: W81XWH-16-1-0297, NCI grant: K22CA207578 and Roswell Park Alliance Foundation. Citation Format: Mayrel Palestino Dominguez, Abhisha Sawant Dessai, Eriko Katsuta, Tao Dai, Subhamoy Dasgupta. Transcriptional repression of mitochondrial deacetylase SIRT3 promotes prostate cancer metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4706.

Effects of environmental steroid mixtures are regulated by individual steroid receptor signaling

Fish are exposed to steroids of different classes in contaminated waters, but their effects are not sufficiently understood. Here we employed an anti-sense technique using morpholino oligonucleotides to knockdown the glucocorticoid receptors (GRs, GRα and GRβ) and androgen receptor (AR) to investigate their role in physiological and transcriptional responses. To this end, zebrafish embryos were exposed to clobetasol propionate (CLO), androstenedione (A4) and mixtures containing different classes of steroids. CLO caused a decrease of spontaneous muscle contraction and increase of heart rate, as well as transcriptional induction of pepck1, fkbp5, sult2st3 and vitellogenin (vtg1) at 24 and/or 48 h post fertilization (hpf). Knockdown of GRs eliminated these effects, while knockdown of AR decreased the ar transcript but caused no expressional changes, except induction of sult2st3 after exposure to A4 at 24 hpf. Exposure to a mixture of 6 steroids comprising progesterone (P4) and three progestins, cyproterone acetate, dienogest, drospirenone, 17β-estradiol (E2) and CLO caused a significant induction of pepck1, sult2st3, vtg1 and per1a. Knockdown of GRs eliminated the physiological effects and the up-regulation of vtg1, sult2st3, pepck1, fkbp5 and per1a. Thus, as with CLO, responses in mixtures were regulated by GRs independently from the presence of other steroids. Exposure to a mixture comprising A4, CLO, E2 and P4 caused induction of vtg1, cyp19b, sult2st3 and fkbp5. Knockdown of AR had no effect, indicating that regulation of these genes occurred by the GRs and estrogen receptor (ER). Our findings show that in early embryos GRs cause vtg1 and sult2st3 induction in addition to known glucocorticoid target genes. Each steroid receptor regulated its own target genes in steroid mixtures independently from other steroids. However, enhanced expressional induction occurred for vtg1 and fkbp5 in steroid mixtures, indicating an interaction/cross-talk between GRs and ER. These findings have importance for the understanding of molecular effects of steroid mixtures.

Role of nitric oxide-based immunotherapy in augmenting prostate cancer progression by targeting androgen receptor heterogeneity.

e17537 Background: A significant proportion of men with Prostate Cancer (PCa) develop castration resistant prostate cancer (CRPC) and do not respond to hormonal agents that decrease androgens. In trying to understand the causes of androgen resistance that develop in CRPC, it is considered most relevant to study the role of Androgen receptor (AR) in the development and progression of PCa from androgen dependent to androgen independent state. Recent studies have highlighted the significance of tumor microenvironment (TME) in regulation of PCa progression in addition to AR. A key molecule in the regulation of TME interactions is nitric oxide (NO). We have shown in our recent study, the critical association of NO with the TME in CRPC. However, the effects of NO to modulate the progression of PCa to CRPC with respect to AR still remains largely unexplored. Methods: 22RV1, LNCaP, LNCaPAPIPC(cells expressing no AR), and LNCaPshAR/pATK (cells expressing low AR), cells were used for the study. Cell proliferation was first assessed by MTT assay. The castrated SCID mice were grafted with 22RV1 cells and were treated with GSNO at the dosage of 10mg/kg/day IP. After treatment, animals were humanely sacrificing. Tumor RNA and proteins were analysed for markers that are important for PCa progression using qPCR, western blot and cytokine antibody array. Animal experiments were carried out in compliance with the IACUC of University of Miami. GraphPad Prism (GraphPad Software) was used for statistical analysis. Results: In addition to reducing the tumor burden, the expression of anti-inflammatory (M2) macrophages (CD206 and Arginase1) is decreased and that of the pro-inflammatory (M1) macrophage (iNOS) is increased in mice which received increased NO levels. Furthermore, to study the effects of NO on progression of PCa from androgen dependent to androgen independent stage, we characterized the LNCAP cell models with differential extent of AR knockdown (LNCaP, LNCaPshAR/pATK and LNCaPAPIPC) for the effects of increased NO levels. Results showed that NO had significant impact on cell proliferation on androgen dependent PCa cells however the effects were negligible in cells expressing low or no AR, suggesting that effects of NO on PCa cell proliferation are AR dependent. Conclusions: Our results suggest that during PCa progression, NO suppresses TAMs to target the TME in an AR dependent manner. Further studies are undergoing to establish the impacts of NO in PCa progression.

An androgen-independent mechanism underlying the androgenic effects of 3-methylcholanthrene, a potent aryl hydrocarbon receptor agonist

Aryl hydrocarbon receptor (AhR) and androgen receptor (AR) are ligand-activated transcription factors with profound cross-talk between their signal transduction pathways. Previous studies have shown that AhR agonists activate the transcription of AR-regulated genes in an androgen-independent manner; however, the underlying mechanism remains unclear. To decipher this mechanism, we evaluated the effects of 3-methylcholanthrene (3MC), a potent AhR agonist, on the transcription of AR-regulated genes in three AR-expressing cell lines. 3MC induced the expression of not only three representative AR-regulated chromosomal genes but also the exogenous AR-responsive luciferase reporter gene. No significant difference in the 3MC-induced luciferase activity was detected in the presence of SKF-525A, a non-specific inhibitor of CYP enzymes. The androgenic effects of 3MC were diminished by AhR and AR knockdown. Following 3MC treatment, the amount of nuclear AhR and AR increased synchronously. Co-immunoprecipitation revealed that AhR and AR formed a complex in the nucleus of cells treated with 3MC. AR was recruited to the proximal promoter and distal enhancer regions of the PSA gene upon the addition of 3MC. We propose that AhR activated by 3MC forms a complex with unliganded AR which translocates from the cytoplasm to the nucleus. Nuclear AR now binds the transcriptional regulatory region of AR-regulated genes and activates the transcription.

Berberine Ameliorates Prenatal Dihydrotestosterone Exposure-Induced Autism-Like Behavior by Suppression of Androgen Receptor.

Many epidemiology studies have shown that maternal polycystic ovary syndrome (PCOS) results in a greater risk of autism spectrum disorders (ASD) development, although the detailed mechanism remains unclear. In this study, we aimed to investigate the potential mechanism and provide a possible treatment for PCOS-mediated ASD through three experiments: Experiment 1: real-time PCR and western blots were employed to measure gene expression in human neurons, and the luciferase reporter assay and chromatin immunoprecipitation (ChIP) was used to map the responsive elements on related gene promoters. Experiment 2: pregnant dams were prenatally exposed to dihydrotestosterone (DHT), androgen receptor (AR) knockdown (shAR) in the amygdala, or berberine (BBR), and the subsequent male offspring were used for autism-like behavior (ALB) assay followed by biomedical analysis, including gene expression, oxidative stress, and mitochondrial function. Experiment 3: the male offspring from prenatal DHT exposed dams were postnatally treated by either shAR or BBR, and the offspring were used for ALB assay followed by biomedical analysis. Our findings showed that DHT treatment suppresses the expression of estrogen receptor β (ERβ) and superoxide dismutase 2 (SOD2) through AR-mediated hypermethylation on the ERβ promoter, and BBR treatment suppresses AR expression through hypermethylation on the AR promoter. Prenatal DHT treatment induces ERβ suppression, oxidative stress and mitochondria dysfunction in the amygdala with subsequent ALB behavior in male offspring, and AR knockdown partly diminishes this effect. Furthermore, both prenatal and postnatal treatment of BBR partly restores prenatal DHT exposure-mediated ALB. In conclusion, DHT suppresses ERβ expression through the AR signaling pathway by hypermethylation on the ERβ promoter, and BBR restores this effect through AR suppression. Prenatal DHT exposure induces ALB in offspring through AR-mediated ERβ suppression, and both prenatal and postnatal treatment of BBR ameliorates this effect. We conclude that BBR ameliorates prenatal DHT exposure-induced ALB through AR suppression, this study may help elucidate the potential mechanism and identify a potential treatment through using BBR for PCOS-mediated ASD.

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.

Seviteronel, a Novel CYP17 Lyase Inhibitor and Androgen Receptor Antagonist, Radiosensitizes AR-Positive Triple Negative Breast Cancer Cells.

Increased rates of locoregional recurrence (LR) have been observed in triple negative breast cancer (TNBC) despite multimodality therapy, including radiation (RT). Recent data suggest inhibiting the androgen receptor (AR) may be an effective radiosensitizing strategy, and AR is expressed in 15–35% of TNBC tumors. The aim of this study was to determine whether seviteronel (INO-464), a novel CYP17 lyase inhibitor and AR antagonist, is able to radiosensitize AR-positive (AR+) TNBC models. In cell viability assays, seviteronel and enzalutamide exhibited limited effect as a single agent (IC50 > 10 μM). Using clonogenic survival assays, however, AR knockdown and AR inhibition with seviteronel were effective at radiosensitizing cells with radiation enhancement ratios of 1.20–1.89 in models of TNBC with high AR expression. AR-negative (AR−) models, regardless of their estrogen receptor expression, were not radiosensitized with seviteronel treatment at concentrations up to 5 μM. Radiosensitization of AR+ TNBC models was at least partially dependent on impaired dsDNA break repair with significant delays in repair at 6, 16, and 24 h as measured by immunofluorescent staining of γH2AX foci. Similar effects were observed in an in vivo AR+ TNBC xenograft model where there was a significant reduction in tumor volume and a delay to tumor doubling and tripling times in mice treated with seviteronel and radiation. Following combination treatment with seviteronel and radiation, increased binding of AR occurred at DNA damage response genes, including genes involved both in homologous recombination and non-homologous end joining. This trend was not observed with combination treatment of enzalutamide and RT, suggesting that seviteronel may have a different mechanism of radiosensitization compared to other AR inhibitors. Enzalutamide and seviteronel treatment also had different effects on AR and AR target genes as measured by immunoblot and qPCR. These results implicate AR as a mediator of radioresistance in AR+ TNBC models and support the use of seviteronel as a radiosensitizing agent in AR+ TNBC.

The combined effect of epigenetic inhibitors for LSD1 and BRD4 alters prostate cancer growth and invasion.

Epigenetic modifications play an important role in prostate tumor development and progression. Epigenetic drugs are emerging as effective modulators of gene expression that act on pathways potentially important in the control of cancer clinically. We investigated two different epigenetic modulating drugs, SP-2509 and JQ1, that target histone lysine demethylase 1 (LSD1), and bromodomain-containing protein (BRD), respectively and their combined effect in three different prostate cancer (PCa) types: 1) androgen receptor (AR)-positive and androgen-sensitive; 2) AR-positive but castration-resistant; and 3) androgen-nonresponsive. We found combined treatment provided a synergistic growth inhibition in castration-resistant PCa cells but knockdown of AR reduced sensitivity to both inhibitors in these cells. In the androgen-sensitive cell lines, AR knockdown attenuated sensitivity to the LSD1 inhibitor but not the JQ1 inhibitor. Strikingly, treatment with SP-2509 slightly, and JQ1 markedly increased invasion in PCa cells with high AR expression but decreased invasion in PCa cells with low/negative AR expression. Our results suggest that these two epigenetic drugs are novel and promising compounds for the development of PCa therapeutics, particularly for castration-resistant disease. However, due to the potential risks, including metastasis, caution must be exercised in the clinical setting.

IL8 Expression Is Associated with Prostate Cancer Aggressiveness and Androgen Receptor Loss in Primary and Metastatic Prostate Cancer.

Chronic inflammation and African ancestry are implicated in prostate cancer aggressiveness, and inflammation-related genes are more highly expressed in prostate cancer in African American men. IL8 secretion is also implicated in prostate cancer progression and castration resistance. We used RNA in situ hybridization to localize IL1β, IL6, IL8, and IL10 mRNA in low- and high-grade prostate cancer from African American and European American men. IL8 was the most abundantly expressed and the only interleukin detected in tumor cells. We further interrogated IL8 expression in primary and metastatic prostate cancer tissue microarrays and both androgen-dependent and castration-resistant patient-derived xenografts (PDX). IL8 was significantly increased in both tumor and benign regions of higher grade cases (ISUP Grade Group 4-5), but there was no difference between races. We determined that IL8 expression in prostate cancer cell lines, distant metastases, and PDX lines was associated with androgen receptor (AR) loss, but not castration resistance. Reciprocal IL8 and AR expression was also observed in high IL8-expressing atrophy lesions with simultaneous AR downregulation. Finally, we show that IL8 is likely repressed by AR binding to the IL8 promoter and is inducible in prostate cancer cells stimulated with lipopolysaccharide only in cells with AR loss. Likewise, AR knockdown in androgen-dependent cells induced IL8 expression, further demonstrating that AR represses IL8 expression. In conclusion, IL8 expression in the tumor microenvironment is associated with aggressive prostate cancer and with AR loss in metastatic disease. IMPLICATIONS: IL8 expression is repressed by AR and is associated with prostate cancer aggressiveness and AR loss in metastatic disease.

Androgen receptor activation reduces the endothelial cell proliferation through activating the cSrc/AKT/p38/ERK/NFκB-mediated pathway

The effect of androgen on angiogenesis has been documented. However, its underlying molecular mechanisms have not been well illustrated. Here, we show that treatment with an androgen receptor (AR) agonist, metribolone (R1881; 0.05–5 nM), or dihydrotestosterone (DHT; 0.5–2 nM), concentration- and time-dependently inhibited proliferation in human umbilical venous endothelial cells (HUVEC). This inhibitory effect was confirmed in human microvascular endothelial cells (HMEC-1). Flow cytometric analysis demonstrated that R1881 induced G0/G1 phase cell cycle arrest in HUVEC. Blockade of the AR activity by pre-treatment with an AR antagonist, hydroxyflutamide (HF), or knockdown of AR expression using the shRNA technique abolished the R1881-induced HUVEC proliferation inhibition, suggesting that AR activation can inhibit endothelial cell proliferation. We further investigated the signaling pathway contributing to the proliferation inhibition induced by AR activation. Our data suggest that R1881 reduced the proliferation rate of HUVEC through activating the AR/cSrc/AKT/p38/ERK/NFκB pathway, subsequently up-regulating p53 expression, which in turn increased the levels of p21 and p27 protein, hence decreasing the activities of cyclin-dependent kinase 2 (CDK2) and CDK4, and finally reduced the cell proliferation rate. An extra-nuclear pathway involved in the proliferation inhibition induced by AR activation in vascular endothelial cells was confirmed by showing that membrane-impermeable testosterone-bovine serum albumin (BSA) treatment significantly increased the levels of p53, p27 and p21 protein and reduced cell proliferation. These data highlight the underlying molecular mechanisms by which AR activation induced proliferation inhibition in vascular endothelial cells.

Abstract 4397: Gli Activation by Steroid Receptors in Prostate and Breast Cancer Cells

Abstract Background: The nuclear steroid receptor superfamily encompasses a group of proteins best known for their functions as primary transcription factors that are conditionally active when bound to a ligand. Here, we show that prominent members of this family (androgen receptor [AR], estrogen receptor [ER-α] and glucocorticoid receptor [GR]) have a secondary function of coordinately activating the Gli family of transcription factors. Previously, we found that liganded AR recognizes and binds to Gli2/Gli3 proteins at their Protein Processing Domains. This binding prevents their degradation and stabilizes them in their high molecular weight, transcriptionally-active forms. This interaction bypasses the need for signaling through Hedgehog/Smoothened to stabilize Gli. To determine whether other steroid receptors have this activity, we tested the ability of human ER-α and GR to bind to Gli3 and whether they increased Gli activity in exogenous (293FT) and endogenous breast and prostate cancer cell systems. Methods: Human AR, ER-α or GR expression vectors were co-transfected into 293FT cells along with a myc-tagged Gli3. Protein extracts were tested for co-immunoprecipitation of AR-/ER-/GR-Gli3 complexes. 293FT cells were co-transfected with AR, ER, or GR along with a Gli-reporter vector. Luciferase activity was measured in transfected cells upon treatment with vehicle, R1881 (AR ligand), estradiol (E2-ER ligand); or dexamethasone (dex-GR ligand). LNCaP (express AR), MCF7 (express ER) or LNCaP-AI cells (express GR) were transfected with reporter in the presence or absence of R1881, E2, dex or vehicle and luciferase activity was measured. AR-/ER-Gli3 complexes were detected by in situ proximity ligation assays in prostate cancer or breast cancer cells. The effect of AR or ER-α siRNA knockdown in LNCaP or MCF7 cells on Gli3 protein stability was measured on western blot. Results: Pulldowns of AR, ER or GR co-immunoprecipitates with Gli3. Transfection with AR or ER increased Gli reporter activity in the presence of vehicle but was further increased by R1881 or E2 treatment. Gli reporter activity was unchanged by transfection with GR in the presence of vehicle but the presence of 5 or 10nM of dex tripled this activity. Gli-luciferase activity was significantly increased in R1881-treated LNCaP cells, E2-treated MCF7 cells and in dex-treated LNCaP-AI cells. PLA detected the presence of AR-Gli3 and ER-Gli3 complexes in nuclei of LNCaP and MCF7 cells. AR and ER-α knockdown destabilized Gli3 protein in LNCaP and MCF7 cells. Conclusion: Collectively our results established a secondary function (Gli activation) shared by an important evolutionary spectrum of human steroid receptors (AR, ER, and GR). As Gli is oncogenic and regulates the expression of many growth-related genes, our observations may explain the pro-oncogenic effects of steroid receptors in steroid-dependent tumour systems. Citation Format: Shabnam Massah, Na Li, Sarah Truong, Jane Foo, Gail Prins, Ralph Buttyan. Gli Activation by Steroid Receptors in Prostate and Breast Cancer Cells [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 4397.

Abstract 5273: Multi-omics approaches reveal potential role for corticosterone in prostate cancer

Abstract Prostate cancer (PCa) is the most commonly diagnosed cancer in American men. In 2018, 164,690 new cancer cases and 29,430 cancer deaths are projected to occur in the United States. Currently, there is no efficient diagnostic and safe treatment option for the management of PCa. Using Ultra-High Performance Liquid Chromatography coupled High Resolution Mass Spectrometry (UHPLC-HRMS), we globally profiled more than 1400 metabolites across 79 urine samples related to PCa (19 control, 23 benign prostate hyperplasia (BPH), 11 prostatitis, and 26 PCa samples). Among all, corticosterone was identified as a top differential metabolite. Corticosterone levels were markedly elevated in PCa urine samples and modest but significant elevation of the corticosterone in BPH and prostatitis urine samples compared to controls, with an excellent area under the receiver operating characteristic curve of 0.95, 0.94, and 0.93, respectively. In addition, the expression of corticosterone biosynthetic gene, Steroid 11-beta-hydroxylase (CYP11B1), was elevated in PCa tissues, cell lines, and urine samples, while Corticosteroid 11-beta-dehydrogenase isozyme 2 (HSD11B2), which metabolize corticosterone, were markedly reduced in prostate tumors. With the understanding that androgen signaling is the key factor for prostate cancer, we investigated the role of androgen in regulating CYP11B1 and HSD11B2. We analyzed several androgen treated PCa cell lines derived global gene expression datasets and revealed that treatment with androgen increase in CYP11B1 expression and a concomitant decrease in HSD11B2 expression levels. Interestingly, we uncover that genetic knockdown of Androgen Receptor (AR) diminished CYP11B1 expression and alternatively, increased HSD11B2. Moreover, AR based chromatin occupancy (ChIP-seq) data also suggest direct binding of AR to the promoter of CYP11B1 in multiple PCa cells. The role of corticosterone in PCa is previously unknown. Our multi-omics approaches thus indicate that corticosterone might be potential prognostic marker for PCa. The tumor biology and mechanism through which corticosterone associates with PCa is being investigated in our laboratory. Citation Format: Iqbal Mahmud, Bongyong Lee, Ranjan Perera, Timothy J. Garrett. Multi-omics approaches reveal potential role for corticosterone in prostate cancer [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 5273.