Androgen Receptor Gene Expression Research Articles

Investigation of the regulatory effects of synthesized antisense oligonucleotides on androgen receptor (AR) exon 3 splicing in prostate cancer cells

The Androgen Receptor (AR) gene plays a key role in castration-resistant prostate cancer (CRPC). Controlling the progression of CRPC by inhibiting AR gene expression is one of the core directions for prostate cancer (Pca) drug development. A 23-amino acids retention, named exon 3a, into the DNA binding domain of the splice variant AR23 has been shown to prevent AR from entering the nucleus and restore the sensitivity of cancer cells to related therapies. In this study, we conducted a preliminary investigation of the splicing modulation of the AR gene in order to develop a splice-switching therapy for Pca by promoting exon 3a inclusion. Using mutagenesis-coupled RT-PCR with AR minigene and over-expression of certain splicing factors, we found that serine/arginine-rich (SR) proteins are key factors facilitating the recognition of the 3′ splice site of exon 3a (L-3′ SS), while the deletion or blocking of the polypyrimidine tract (PPT) region of the original 3′ splice site of exon 3 (S-3′ SS) could strongly enhance exon 3a splicing without affecting the function of any SR protein. Furthermore, we designed a series of antisense oligonucleotides (ASOs) to screen drug candidates, and ASOs targeting S-3′ SS and its PPT region or the exonic region of exon 3 turned out to be most effective in rescuing exon 3a splicing. A dose–response test indicated ASO12 as the lead candidate drug significantly promoting the inclusion of exon 3a to more than 85%. MTT assay confirmed that the cell proliferation was significantly inhibited after ASO treatment. Our results provide the first glance to AR splicing regulation. With several promising therapeutic ASO candidates obtained here, further development of ASO drugs to treat CRPC is strongly encouraged.

Vitamin E modulates androgen receptor gene expression to attenuate ovarian dysfunctions in a rat model of dehydroepiandrosterone-induced polycystic ovary

Objective: To investigate the protective effect of vitamin E in dehydroepiandrosterone (DHEA)-induced polycystic ovary in rats. Methods: Premature female Wistar rats were randomly allocated into four groups, with 7 rats in each group. Group I received corn oil (vehicle) and served as the control group; group II received 0.2 mL of 0.06 mg/g DHEA in corn oil; group III received 200 mg/kg vitamin E; group IV received DHEA plus vitamin E. All treatments lasted for 15 days, with DHEA administered subcutaneously, while vitamin E and corn oil were administered orally. After the experiment, serum samples and ovaries were harvested for biochemical, immunohistochemical, hormonal, and histological analysis. The ovarian mRNA expression of androgen receptor was analyzed by reverse transcriptase quantitative polymerase chain reaction (qPCR). Results: The antioxidant and metabolic enzyme activity significantly decreased in the DHEA-treated rats compared to the control rats (P<0.05). Administration of vitamin E to DHEA-treated rats significantly decreased cytokines and malondialdehyde compared to the DHEA-treated rats. The histological analysis showed reduced atretic and cystic ovaries, increased E-cadherin and Bcl-2 expression, and reduced expression of Bax in the DHEA-treated rats co-treated with vitamin E. The mRNA expression of androgen receptor was upregulated in the DHEA-treated rats compared to the control rats. Conclusions: Vitamin E ameliorates the hyperandrogenic effect of DHEA-induced polycystic ovaries via metabolic, antioxidant, and anti-apoptotic pathways.

FT-6876, a Potent and Selective Inhibitor of CBP/p300, is Active in Preclinical Models of Androgen Receptor-Positive Breast Cancer.

Patients with triple-negative breast cancer (TNBC) expressing the androgen receptor (AR) respond poorly to neoadjuvant chemotherapy, although AR antagonists have shown promising clinical activity, suggesting these tumors are AR-dependent. cAMP responsive element binding protein (CREB)-binding protein (CBP) and p300 are transcriptional co-activators for the AR, a key driver of AR+ breast and prostate cancer, and may provide a novel therapeutic target in AR+ TNBC. The aim of this study was to determine the therapeutic potential of FT-6876, a new CBP/p300 bromodomain inhibitor, in breast cancer models with a range of AR levels in vitro and in vivo. Effects of FT-6876 on the CBP/p300 pathway were determined by combining chromatin immunoprecipitation (ChIP) with precision run-on sequencing (PRO-seq) complemented with H3K27 acetylation (Ac) and transcriptional profiling. The antiproliferative effect of FT-6876 was also measured in vitro and in vivo. We describe the discovery of FT-6876, a potent and selective CBP/p300 bromodomain inhibitor. The combination of ChIP and PRO-seq confirmed the reduction in H3K27Ac at specific promoter sites concurrent with a decrease in CBP/p300 on the chromatin and a reduction in nascent RNA and enhancer RNA. This was associated with a time- and concentration-dependent reduction in H3K37Ac associated with a decrease in AR and estrogen receptor (ER) target gene expression. This led to a time-dependent growth inhibition in AR+ models, correlated with AR expression. Tumor growth inhibition was also observed in AR+ tumor models of TNBC and ER+ breast cancer subtypes with consistent pharmacokinetics and pharmacodynamics. Our findings demonstrate FT-6876 as a promising new CBP/p300 bromodomain inhibitor, with efficacy in preclinical models of AR+ breast cancer.

Effect of Dietary Supplementation of Either Zinc Oxide or Nano-Zinc Oxide on Growth Performance, some Serum Biochemical Parameters, Androgen Receptor Gene Expression and Microscopical Structure of the Testis in Growing V-Line Male Rabbits

Effect of Dietary Supplementation of Either Zinc Oxide or Nano-Zinc Oxide on Growth Performance, some Serum Biochemical Parameters, Androgen Receptor Gene Expression and Microscopical Structure of the Testis in Growing V-Line Male Rabbits

Cholesterol Triggers Nuclear Co-Association of Androgen Receptor, p160 Steroid Coactivators, and p300/CBP-Associated Factor Leading to Androgenic Axis Transactivation in Castration-Resistant Prostate Cancer.

Cholesterol modulates intratumoral androgenic signaling in prostate cancer; however, the molecular mechanisms underlying these changes in castration-resistant prostate cancer (CRPC) are not fully elucidated. Herein, we investigated the effect of cholesterol on androgen receptor (AR) coactivators expression and tumorigenesis in vitro and in vivo. Herein, we monitored the expression of AR coactivators (SRC-1, 2, 3 and PCAF) genes in PC-3 cells exposed to 2µg/mL of cholesterol for 8 hours by qPCR. We also performed cell migration at 0, 8, 24, 48 and 72h and flow cytometry assays (viability, apoptosis, and cell cycle) after a 24h exposure. Immunofluorescence assay was performed to evaluate the protein expression of the AR coactivators. Additionally, in vivo experiments were conducted using 22 male NOD/SCID mice. Mice were fed a standard (Control) or hypercholesterolemic (HCOL) diet for 21 days and then subcutaneously implanted with PC-3 cells. The tumor volume was calculated every two days, and after four weeks, the tumors were resected, weighed, and the serum lipid profile was measured. We also measured the intratumoral lipid profile and AR coactivators gene and protein expression by qPCR and Western Blot, respectively. Intratumor testosterone and dihydrotestosterone (DHT) concentrations were determined using ELISA. Cholesterol up-regulated the gene expression of coactivators SRC-1, SRC-2, SRC-3 and PCAF, increasing AR expression in PC-3 cells. Next, cholesterol-supplemented PC-3 cells exhibited increased cell migration and altered cell cycle phases, leading to changes in proliferation and reduced apoptosis. We found that SRC-1, SRC-2, SRC-3 and PCAF proteins co-localized in the nucleus of cholesterol-supplemented cells and co-associate with AR. In the in vivo model, the hypercholesterolemic (HCOL) group displayed higher serum total and intratumoral cholesterol levels, increased testosterone and dihydrotestosterone concentrations, and up-regulated AR coactivator expression. The tumor volume of the HCOL group was significantly higher than the control group. Our findings revealed that increased nuclear translocation of the coactivators leads to up-regulated AR gene and protein expression, potentially influencing tumor progression. Studies targeting cholesterol-modulated changes in AR coactivator expression may provide insights into the molecular mechanisms associated with the CRPC phenotype.

RF10 | PMON249 MicroRNA-21 Ablation Exacerbates Androgen-Mediated White Adipose Tissue Dysfunction and Impaired Insulin Signaling in a Mouse Model of Polycystic Ovary Syndrome

Abstract Background Polycystic ovary syndrome (PCOS) is characterized by oligo- or anovulation, polycystic ovaries, and hyperandrogenism. PCOS is associated with white adipose tissue (WAT) expansion and dysfunction, which has been linked to impaired insulin signaling in a number of metabolic diseases. MicroRNAs dysregulation in WAT is involved in the development of obesity and its associated complications. MicroRNA-21 (miR-21), the most abundant circulatory microRNA in women with PCOS, regulates androgen receptor (AR) expression in prostate cancer however, its role in WAT dysfunction in PCOS, and the associated systemic insulin resistance, is unknown. Using a well-established PCOS model of hyperandrogenemia we aim to test the hypothesis that miR-21 regulates AR expression to modulate androgen-mediated WAT dysfunction and the associated local and systemic insulin resistance. Methods Three-week-old miR-21 knockout (miR-21KO) or wild type (WT) female mice were treated with dihydrotestosterone (DHT, 8 mg/Silastic tube) or vehicle for 90 days (n=6/group). Body composition (EchoMRI) and oral glucose tolerance test (OGTT) were assessed. HOMA-IR index was calculated from fasting serum glucose and insulin levels. Retroperitoneal fat (RPF), a WAT depot that is linked to poor plasma glycemic indices and systemic inflammation, was collected to assess adipocyte size and perform molecular determinations. RPF miR-21 levels, as well as gene and protein expression of AR, and insulin signaling markers (IRS-1/2, insulin receptor-β, PI3K, AKT, Glut4, GSK-α, PTEN) were quantified by RT-qPCR and Western blot. Results In WT mice, DHT increased body weight (25.07 ± 0.52 vs 21.79 ± 0.47 g, p<0.05), fat mass (4.60 ± 0.46 vs 1.98 ± 0.12 g, p<0.05), RPF mass (29.68 ± 3.39 vs. 172.64 ± 32.75 g, p<0.05), RPF miR-21 (2.5-fold) and AR expression (2.1-fold). Adipocyte size analysis revealed a hypertrophic response to DHT, which was associated with impaired OGTT (186.10 ± 5.99 vs 250.70 ± 14.76 mg. min/dL, p<0.05) and downregulation of IRS-1/2, insulin receptor-β, and AKT, indicating impaired insulin signaling. MiR-21 ablation had no effect on DHT-mediated total fat or RPF mass increases, but it exacerbated DHT-mediated AR upregulation, RPF hypertrophy, and insulin resistance as measured by the HOMA-IR index. DHT-mediated reduction in GSK-α, which mediates obesity-induced inflammation, was abolished in miR-21KO mice. Glut4 protein was only downregulated in miR-21KO DHT-treated mice, despite DHT downregulating Glut4 mRNA in both strains. Furthermore, DHT only upregulated PTEN, a negative insulin signaling regulator and known target of miR-21 in miR-21KO mice. Conclusion and significance: These findings imply that WAT miR-21 plays a protective role in PCOS by alleviating androgen-mediated adipose tissue structural and molecular derangements, as well as the associated local and systemic insulin resistance via downregulating AR. Modulation of miR-21 levels in adipose tissue could be a novel therapeutic approach for the treatment of PCOS-related metabolic derangements. Presentation: Saturday, June 11, 2022 1:00 p.m. - 1:05 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

OR30-5 Androgen-Regulated Secreted Factors Promote Tumorigenesis and Immune Suppression in Breast Cancer

Abstract Background Breast cancer (BC) is the most diagnosed cancer and the second-leading cause of cancer-associated death in women in the United States. BCs that express the estrogen and/or progesterone receptor (ER/PR) are treated with ER-targeting endocrine therapies that effectively reduce disease burden and extend lifespan. However, up to 50% of patients with ER+BCs will experience recurrence, often at a metastatic site, within 10 years of diagnosis. Novel targeted therapies are needed to treat both metastatic ER+BC resistant to current treatment strategies and chemoresistant ER-BCs.We identified chitinase 3–like-1 (CHI3L1/BRP-39/YKL-40) and prolactin inducible protein (PIP/GCDFP-15) as androgen receptor (AR)-regulated in BC. PIP is reported to be detectable in >90% of BCs, but its function in BC is not well-characterized. CHI3L1 is a glycosyl hydrolase family 18 protein member with reported roles in aging and numerous pathologies. During mammary gland involution, CHI3L1 contributes to a pro-tumorigenic wound-healing-like environment and increases macrophage infiltration. Further, CHI3L1 correlates with tumor-suppressive M2 macrophage infiltration in ER-BC. We previously reported that ER+ cells and patient-derived xenografts with activating mutations in the ESR1 gene encoding ER—a known mechanism of endocrine therapy resistance—had elevated AR and CHI3L1 gene and protein expression. Based on the above and our finding that metastatic BCs biopsies harboring ESR1 mutations exhibited increased AR expression, we hypothesized that AR regulation of CHI3L1 and PIP promote progression to metastatic disease in part through immune suppressive effects. Methods A ConTra(V3) transcription factor binding site analysis was performed on CHI3L1 and PIP promoters. CHI3L1 and PIP were measured via immunohistochemistry in xenograft tumors in mice treated with or without the androgen dihydrotestosterone (DHT). Immunoblotting, immunohistochemistry, and immunofluorescence approaches were also utilized to assess CHI3L1 protein expression in DHT-treated, tamoxifen resistant (TAMR), and long term E2 deprived (LTED) ER+BC cells. CIBERSORT analysis was conducted on the Firehouse Legacy TCGA dataset. Macrophage efferocytosis assays were performed on an IncuCyte Live Cell Imaging system. Results We identified highly conserved AR binding sites in the CHI3L1 and PIP promoters. CHI3L1 protein was elevated in TAMR and LTED BC cells, which was further elevated in BC cells with ESR1 mutations, as well as DHT-treated cells. Further, CHI3L1 and PIP were increased in xenograft tumors after DHT treatment. CIBERSORT analysis revealed that CHI3L1 and PIP high tumors had altered immune profiles, including differential macrophage levels. Finally, CHI3L1 and PIP treatment significantly reduced macrophage efferocytosis of BC cells (p ≤ 0.0001). Conclusions Our work suggests that AR-mediated regulation of CHI3L1 and PIP may alter immune cell infiltration/function in BC and may be important in endocrine therapy resistant ER+BCs. Further characterization and manipulation of these AR-regulated proteins is underway to test the potential to target and reverse their immune suppressive functions. Presentation: Tuesday, June 14, 2022 10:45 a.m. - 11:00 a.m.

Dual-Acting Peptides Target EZH2 and AR: A New Paradigm for Effective Treatment of Castration-Resistant Prostate Cancer.

Prostate cancer starts as a treatable hormone-dependent disease, but often ends in a drug-resistant form called castration-resistant prostate cancer (CRPC). Despite the development of the antiandrogens enzalutamide and abiraterone for CRPC, which target the androgen receptor (AR), drug resistance usually develops within 6 months and metastatic CRPC (mCRPC) leads to lethality. EZH2, found with SUZ12, EED, and RbAP48 in Polycomb repressive complex 2 (PRC2), has emerged as an alternative target for the treatment of deadly mCRPC. Unfortunately, drugs targeting EZH2 have shown limited efficacy in mCRPC. To address these failures, we have developed novel, dual-acting peptide inhibitors of PRC2 that uniquely target the SUZ12 protein component, resulting in the inhibition of both PRC2 canonical and noncanonical functions in prostate cancer. These peptides were found to inhibit not only the EZH2 methylation activity, but also block its positive effect on AR gene expression in prostate cancer cells. Since the peptide effect on AR levels is transcriptional, the inhibitory peptides can block the expression of both full-length AR and its splicing variants including AR-V7, which plays a significant role in the development of drug resistance. This dual-mode action provides the peptides with the capability to kill enzalutamide-resistant CRPC cells. These peptides are also more cytotoxic to prostate cancer cells than the combination of enzalutamide and an EZH2 inhibitory drug, which was recently suggested to be an effective treatment of mCRPC disease. Our data show that such a dual-acting therapeutic approach can be more effective than the existing front-line drug therapies for treating deadly mCRPC.

Enzalutamide Induces Apoptotic Insults to Human Drug-Resistant and -Sensitive Glioblastoma Cells via an Intrinsic Bax-Mitochondrion-Cytochrome C Caspase Cascade Activation Pathway.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor. Temozolomide (TMZ) is the first-line chemotherapeutic drug for treating GBM. However, drug resistance is still a challenging issue in GBM therapy. Our preliminary results showed upregulation of androgen receptor (AR) gene expression in human GBM tissues. This study was designed to evaluate the effects of enzalutamide, a specific inhibitor of the AR, on killing drug-resistant and -sensitive glioblastoma cells and the possible mechanisms. Data mining from The Cancer Genome Atlas (TCGA) database revealed upregulation of AR messenger (m)RNA and protein expressions in human GBM tissues, especially in male patients, compared to normal human brains. In addition, expressions of AR mRNA and protein in human TMZ-sensitive U87 MG and -resistant U87 MG-R glioblastoma cells were elevated compared to normal human astrocytes. Exposure of human U87 MG and U87 MG-R cells to enzalutamide concentration- and time-dependently decreased cell viability. As to the mechanism, enzalutamide killed these two types of glioblastoma cells via an apoptotic mechanism. Specifically, exposure to enzalutamide augmented enzyme activities of caspase-9 rather than those of caspase-8. Moreover, enzalutamide successively triggered an elevation in levels of the proapoptotic Bax protein, a reduction in the mitochondrial membrane potential, release of cytochrome c, cascade activation of caspases-3 and -6, DNA fragmentation, and cell apoptosis in human TMZ-sensitive and -resistant glioblastoma cells. Pretreatment with Z-VEID-FMK, an inhibitor of caspase-6, caused significant attenuations in enzalutamide-induced morphological shrinkage, DNA damage, and apoptotic death. Taken together, this study showed that enzalutamide could significantly induce apoptotic insults to human drug-resistant and -sensitive glioblastoma cells via an intrinsic Bax-mitochondrion-cytochrome c-caspase cascade activation pathway. Enzalutamide has the potential to be a drug candidate for treating GBM by targeting the AR signaling axis.

Expression of Sex Hormone Receptors in Canine Osteosarcoma.

Simple SummaryOsteosarcoma is a bone tumor that arises from normal bone cells that become malignant. This is a disease that is relatively common in dogs and also affects humans. Dogs are a useful model for human osteosarcoma as they share many similarities including genetic changes, manner of spread and how they respond to treatment. In dogs, the tumors are frequently seen in large breeds and may be caused by environmental and genetic factors. Sex steroids have been shown to regulate bone metabolism directly and indirectly through receptors on bone. The aim of this study was to investigate the expression levels of sex hormone receptors in canine osteosarcoma tissue and cell lines. Our results demonstrate varying levels of receptors in tissue and a high expression of progesterone receptors in the canine osteosarcoma cell lines. Low oxygen levels further increased progesterone receptor expression. Lastly, estradiol decreased the expression of progesterone receptor in one of the cell lines, and progesterone decreased cell proliferation in the same cell line. Further investigation is recommended to determine whether or not sex steroid receptor levels and sensitivity to hormones are related.Sex steroids regulate bone metabolism directly and indirectly through receptors on bone. Estrogen receptors (ER-∝, ER-β), progesterone receptor (PR), and androgen receptor (AR), have been previously identified on human osteosarcoma (OSA) cells, and are considered to influence tumor growth, but their expression and role in canine OSA is unknown. The aim of this study was to characterize sex hormone receptor expression levels in naturally occurring OSA tissue and in three canine OSA cell lines. The expression of ER-α, ER-β, PR, and AR was investigated using RT-PCR. PR expression levels were also quantified in OSA cells cultured under hypoxic conditions or in the presence of estradiol. The effects of progesterone on cell proliferation were quantified. Results demonstrated varying expression levels of these receptors in five OSA subtypes. OSA cell lines demonstrated high gene expression levels of PR and low gene expression levels of ER-α and ER-β and no gene expression of AR. PR expression was increased in OSA cells cultured under hypoxic conditions in a HIF-∝ independent manner. Interestingly, one cell line expressed very high levels of PR, expression of which decreased in response to estradiol. In addition, progesterone decreased OSA cell proliferation in this particular cell line. Further investigation of the role of sex steroids, particularly PR and its ligands, in regulation of canine OSA is recommended.

W Chromosome Evolution by Repeated Recycling in the Frog Glandirana rugosa

The Y or W sex chromosome of a heteromorphic pair is usually heterochromatinised and degenerated. However, whether chromosome degeneration constantly proceeds toward an extreme end is not fully understood. Here, we present a case of intermittent evolution of W chromosomes caused by interpopulation hybridisation in the Japanese soil-frog, Glandirana rugosa. This species includes two heteromorphic sex chromosome systems, which are separated into geographic populations, namely the XY and ZW groups. In this study, to uncover the evolutionary mechanisms of the heterogeneous W chromosomes, we genetically investigated the geographic differentiation of the ZW populations along with the closely located XY populations. Analysis of mitochondrial cytochrome b sequences detected three distinct clades, named ZW1, ZW2, and ZW3. High throughput analyses of nuclear genomic DNA showed that autosomal alleles of XY populations were deeply introgressed into the ZW3 sub-group. Based on the genotypes of sex-linked single nucleotide polymorphisms, W-borne androgen receptor gene expression, and WW developmental mortality, we concluded that the X chromosomes were recycled to W chromosomes. Upon inclusion of two cases from another group, Neo-ZW, we observed that the X chromosomes were recycled independently at least four times to the new W chromosomes: a repetition of degeneration and resurrection.

P-567 Recombinant hCG (choriogonadotropin beta, CG beta) exerts a positive dose-dependent effect on human follicular steroidogenesis during ovarian stimulation using a constant rFSH administration

Abstract Study question How does increasing doses of CG beta augment follicular steroidogenesis and is it associated with gene expression in cumulus cells? Summary answer With the exception of progesterone, CG beta exerts dose-dependent effects on intrafollicular steroid hormone concentrations and CYP19a1 expression during constant rFSH administration. What is known already CG beta (FE 999302) is a newly developed rhCG, produced by a human cell line (PER.C6®). This variant contains the same amino acid sequence as the endogenous hCG but presents with a different glycosylation profile when compared to urinary hCG or rhCG derived from a CHO cell line. CG beta has also been shown to have a longer half-life and higher relative potency. It is well known that hCG like LH creates an intracellular response through the LHR expressed on theca cells and mature granulosa cells and hereby augment the production of androgens, progesterone, and oestrogen. Study design, size, duration This study is part of a randomised, double-blind, placebo-controlled trial to investigate the efficacy and safety of CG beta as an add-on treatment to rFSH in women undergoing ovarian stimulation during a long GnRH agonist protocol. The primary endpoint of this study was intrafollicular steroid levels in relation to CG beta administration. Secondary outcomes were gene expression of LHR, FSHR, CYP19a1, and androgen receptor (AR) and single nucleotide polymorphisms (SNPs) in LHR(312) and FSHR(307). Participants/materials, setting, methods 619 women with AMH levels 5-35 pmol/L were randomized to receive either placebo or 1, 2, 4, 8, or 12 µg CG beta from day one of stimulation combined with a constant individualized dose of rFSH. Follicular fluid (FF) (n = 558), granulosa (n = 498) and cumulus cells (n = 368) were collected at oocyte retrieval. Steroid FF hormones were measured using ELISAs, gene expression was analysed in cumulus cells by qRT-PCR, and SNP analysis was performed in granulosa cells. Main results and the role of chance This study found that CG beta has a pronounced positive dose-dependent effect on intrafollicular concentrations of 17-OH-progesterone, androstenedione, testosterone, and oestradiol during constant rFSH administration. As compared to the placebo group without CG beta administration the concentrations of steroids were significantly dose-dependently increased (p-value<0.0001) reaching up to 10 times higher values in the highest dose group without a clear plateauing. However, for progesterone, there was no statistically significant difference between the CG beta dose groups and placebo. The gene expression level of CYP19a1 in the cumulus cells collected at oocyte retrieval increased significantly (p-value=0.0325) for the highest dose of CG beta. However, the gene expression level of FSHR, LHR, and AR in cumulus cells were not affected by CG beta administration, as no dose-response trend with increasing dose of CG beta was observed. Additionally, this study revealed that there was no overall clear difference in the number of oocytes retrieved between the different CG beta dose groups based on either the FSHR(307) or LHR(312) genotypes. Limitations, reasons for caution The gene expression was assessed in cumulus cells, but it would also have been interesting to evaluate the mural granulosa cells. Analysis of the gene expression of 3βHSD and other important steroidogenic enzymes in the mural granulosa cells could reveal further explanation to the effect of CG beta administration. Wider implications of the findings To our knowledge, this is the first study to show a clear dose-dependent effect of rhCG on human steroidogenesis during constant rFSH administration in preovulatory follicles. This reflects the importance of the combined effect of FSH and hCG/LH on granulosa cell activity, follicle health, and potentially oocyte quality. Trial registration number 000289

The sex‐dependent role of the androgen receptor in glioblastoma: results of molecular analyses

We sought to analyse the androgen receptor (AR) in glioblastoma (GBM) due to the location of the AR gene on chromosome X, often reported with shorter survival and higher prevalence of GBM among males. Copy number (CN) and mRNA expression of AR were tested with droplet digital PCR in 91 fresh‐frozen GBM samples and 170 formalin‐fixed, paraffin‐embedded samples collected at Linköping University Hospital. The fresh‐frozen cohort was also subjected to pyrosequencing methylation analysis of 17 CpG sites in the AR promoter. Additionally, the gene expression of AR was analysed in the fresh‐frozen cohort and The Cancer Genome Atlas (TCGA) cohort of isocitrate dehydrogenase wild‐type primary GBM (135 females and 219 males). The association of AR expression and overall survival (OS) was tested with Kaplan–Meier log rank analysis after dichotomisation by maximally selected rank statistics. We found that AR CN alterations were more common in female GBM. AR gene expression correlated with methylation levels of different CpG sites in males and females but there was no difference in expression between sexes. Survival analysis of TCGA cohort revealed the opposite effect of AR overexpression on OS of males and females, with high AR expression correlating with shorter OS in females and longer OS in males. Additional gene set enrichment analysis showed that AR expression correlated with DNA repair response, especially in the male group. In summary, we found that high AR gene expression in GBM exhibits sex‐dependent effects on patient survival, which, for males, is linked to DNA repair response.

PTCH1 and AXIN2 modulation of AR copy number effects on AR gene overexpression in metastatic castration resistant prostate cancer (mCRPC).

e17026 Background: Androgen receptor (AR) signaling is a critical pathway in prostate cancer progression. Reactivation of the AR pathway is a key driver in mCRPC. Patients with mCRPC often develop resistance to novel AR signaling inhibitors (ARSIs) such as enzalutamide. AR inhibitor resistance leads to shorter overall and progression-free survival and can be attributed to AR gene amplification with subsequent AR overexpression and androgen-sensitization in prostate tumors. Identifying novel modulators of AR amplification in patients progressing on ARSIs may lead towards potential targets in drug discovery. Methods: In this cross-sectional study, an in silico patient model was developed on prescribed drug therapies and failed therapies patient information, PSA measures, ̃100 mutation variables including somatic, germline, and gain/loss mutations, and ̃1K gene expressions data from 275 mCRPC patients obtained from the Tempus database using GNS Healthcare’s Causal artificial intelligence technology, REFS. A causal network model was reconstructed based on patient data with inferred effect estimates. The network model was topologically surveyed for biological interactions. Results: Causal analysis of this model uncovered two germline mutations, located in PTCH1 and AXIN2, which modify the effect of AR copy number gain ( ARgain+ : copy number 6, ARgain- : copy number < 6) on AR gene expression. For PTCH1-mutant group, there was a 2-fold increase in AR gene expression in ARgain+ patients compared to ARgain- patients (p = 2.1e-12), while only 1.4 fold increase in AR gene expression from the PTCH1 non-mutant group (p = 6.3e-08). Similarly, there was a 2-fold increase in AR gene expression in ARgain+ patients compared to ARgain- patients (p = 7.2e-16) for the AXIN2-mutant group, while only one-fold increase in AR gene expression with the AXIN2 non-mutant group (p = 0.001). The C to T missense mutation in codon 3746 in PTCH1 and codon 148 in AXIN2 have higher prevalences, 55% and 72%, respectively in this mCRPC cohort compared to their population allele frequencies, 38% and 47%, respectively. Conclusions: PTCH1 and AXIN2 mutations from our analyses is consistent with a literature reported role for Hedgehog signaling and Wnt/-catenin signaling pathways, respectively, influencing the disease progression in castration-resistant prostate cancer. Our findings suggest that the missense germline mutations in PTCH1 and AXIN2 predicts response to enzalutamide and other ARSIs in CRPC setting, and propose potential targets for combination therapy to overcome ARSI drug resistance in hormone-refractory prostate cancer. [Table: see text]

PD07-02 TARGETING WNT5A/FZD2 SIGNALING OVERCOMES RESISTANCE TO ENZALUTAMIDE IN ADVANCED PROSTATE CANCER

You have accessJournal of UrologyCME1 May 2022PD07-02 TARGETING WNT5A/FZD2 SIGNALING OVERCOMES RESISTANCE TO ENZALUTAMIDE IN ADVANCED PROSTATE CANCER Shu Ning, Chengfei Liu, Wei Lou, Alan Lombard, Leandro D'Abronzo, Aiming Yu, Christopher Evans, and Allen Gao Shu NingShu Ning More articles by this author , Chengfei LiuChengfei Liu More articles by this author , Wei LouWei Lou More articles by this author , Alan LombardAlan Lombard More articles by this author , Leandro D'AbronzoLeandro D'Abronzo More articles by this author , Aiming YuAiming Yu More articles by this author , Christopher EvansChristopher Evans More articles by this author , and Allen GaoAllen Gao More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002526.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Androgen receptor (AR) blockade using antiandrogens is a mainstay for the treatment of castration resistant prostate cancer (CRPC). Unfortunately, drug resistance occurs frequently due to mechanisms that are not completely understood. Wnt5a, a representative ligand of non-canonical Wnt signaling, is expressed in circulating tumor cells from CRPC patients treated with enzalutamide. FZD2, the cognate frizzled receptor for Wnt5a, is the most commonly co-upregulated non-canonical Wnt receptor in prostate cancer. Here we determine the contribution of non-canonical Wnt5a/FZD2 to enzalutamide treatment resistance, and explore the potential of targeting Wnt5a/FZD2 to overcome antiandrogen resistance in castration resistant prostate cancer. METHODS: Wnt5a/FZD2 expression was examined in enzalutamide resistant C4-2B MDVR cells. Wnt5a and FZD2 expression were modulated using specific siRNAs. Cell growth, colony formation, migration and invasion were determined in vitro. RNA sequencing was analyzed on C4-2B MDVR cells with WNT5a/FZD2 knocked down; gene expression of non-canonical Wnt signaling, AR and AR-V7 signature genes were analyzed. A novel RNA bioengineered Wnt5a siRNA (tRNA-siWnt5A) was developed to target Wnt5a/FZD2 signaling. The effect of tRNA-siWnt5a on tumor growth and sensitivity to enzalutamide was evaluated in vitro and in vivo. RESULTS: Wnt5a and FZD2 are highly co-upregulated in CRPC patients and enzalutamide resistant C4-2B MDVR cells compared to parental C4-2B cells. Knocking down Wnt5a and FZD2 abrogates the increase of full-length AR and AR variant expression and diminishes the enrichment of genes involved in the non-canonical Wnt signaling pathway. Blocking Wnt5a and FZD2 using specific siRNAs suppresses prostate cancer cell growth, colony formation, migration and invasion. Wnt5a and FZD2 knockdown with siRNA resensitized C4-2B MDVR cells to enzalutamide treatment. Targeting Wnt5a using the bioengineered tRNA-siWnt5A inhibited the growth of enzalutamide resistant prostate cancer cells and resensitized cells to enzalutamide in vitro, and resistant CRPC LuCaP35CR PDX tumor growth in vivo. CONCLUSIONS: Our studies suggest that Wnt5a/FZD2 confers enzalutamide resistance and prostate cancer survival and proliferation. Targeting the non-canonical Wnt5a/FZD2 pathway suppresses tumors expressing high level of Wnt5a and FZD2, not only overcoming resistance but potentiating anti-tumor effects of enzalutamide in CRPC patients. Source of Funding: CA250082, CA 225836, DOD PC180180 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e98 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shu Ning More articles by this author Chengfei Liu More articles by this author Wei Lou More articles by this author Alan Lombard More articles by this author Leandro D'Abronzo More articles by this author Aiming Yu More articles by this author Christopher Evans More articles by this author Allen Gao More articles by this author Expand All Advertisement PDF DownloadLoading ...

Hypoxic Signaling Is Modulated by Calcium Channel, CaV1.3, in Androgen-Resistant Prostate Cancer.

Androgen deprivation therapy (ADT) remains a key treatment for advance prostate cancer (PCa), but resistance leads to terminal castrate-resistant prostate cancer (CRPC). Its development is linked to the emergence of a hypoxic tumor microenvironment and associated hypoxia inducible factor (HIF) signaling, which is known to be modulated by intracellular calcium. ADT is also known to upregulate store-operated calcium entry (SOCE) through voltage-gated calcium channel, CaV1.3. Consequently, the role of CaV1.3 in supporting hypoxic signaling and CRPC biology was explored. Androgen-sensitive PCa LNCaP cells were cultured with and without ADT bicalutamide, alongside ADT-resistant CRPC cells (LNCaP-ABL), either in normal or low oxygen (O2) (1%) conditions. HIF-1α, CaV1.3, and androgen receptor (AR) gene expression was measured by qPCR and protein expression with Western blot in the presence or absence of siCaV1.3. SOCE was determined through Fura-2AM fluorescence measurement. Cell proliferation was quantified by WST-1 assay and survival by colony formation. CaV1.3 expression was increased during ADT but not hypoxia, correlating with an associated increase in SOCE. HIF-1α expression was upregulated by ADT under normal O2 conditions and increased during hypoxia across all cells but with a higher fold change in early ADT-resistant and CRPC cells. Under hypoxic conditions CaV1.3 small interfering RNA resulted in a significant reduction in HIF-1α expression for ADT-sensitive cells but increased in CRPC. A similar pattern was also observed for AR expression. Cell survival was found significantly reduced by siCaV1.3 under hypoxic conditions for all cells, with and without ADT. Whereas cell proliferation under the same conditions was reduced in CRPC only. This study highlights that CaV1.3 can modulated HIF signaling and impact on PCa tumor biology under hypoxia, but further investigation is required to ascertain if this mediated through SOCE or a noncanonical mechanism.

The Influence of Androgen Receptors on Muscle Repair and Hypertrophy

The mechanisms underlying inter‐individual variation in adaptation to exercise have yet to be fully elucidated. Recently, high responders to resistance training (RT) have demonstrated greater levels of androgen receptor (AR) content, and therefore AR may be a factor underlying the variation in the adaptive response to resistance exercise. Moreover, there has been inconclusive findings surrounding the impact of AR content in females as well as following damage. In the current study, twenty‐six healthy young men (n=13) and women (n=13) underwent an acute bout of damaging exercise consisting of 300 eccentric kicks, followed by 10 weeks of full‐body RT to investigate changes in AR content following acute muscle damage and following chronic resistance exercise. This was assessed in the context of the satellite cell response and during muscle repair and hypertrophy. Skeletal muscle biopsies from the vastus lateralis were taken at baseline, 48 hours post damage and 48 hours following the last bout of RT. RT‐qPCR and western blots were performed to quantify AR gene expression and protein content, respectively. An immunofluorescence staining protocol was optimized for the visualization of AR protein abundance. Results were considered in the context of the SC response and muscle hypertrophy data from the same cohort. AR content increased from baseline (30457 +/‐ 4399 a.u.) to post‐damage (66448 +/‐ 8083 a.u.) by 218% (p=0.005) and post RT (60889 +/‐ 6636 a.u.) by 199% (p=0.003). Those with the greatest increase in SC content post‐damage also had the greatest increase in AR content (p=0.01). Individuals with the greatest increase in leg‐lean mass following RT also had the greatest increase in AR content (p=0.05). Preliminary data from western blot analysis revealed that AR protein content post‐damage in males was correlated with activated SC number (MyoD+ cells) (p=0.01). We then aimed to investigate sex differences in the context of AR content since there is a paucity of work in females. Males (61550 +/‐ 10961 a.u.) had a 198% greater change in AR content than females (31104 +/‐ 7499 a.u) from baseline to post‐damage (p=0.03). Males also had a greater change in AR content (3.186 +/‐ 0.5443) than females (2.003 +/‐ 0.2625) post‐RT, relative to baseline. In males, those with greater relative gains in muscle fibre CSA (high responders), post‐RT, had a 373% greater change in AR content (64936 +/‐ 11783 a.u.) from baseline to post‐RT than low responders (17388 +/‐ 15438) (p = 0.03). Interestingly in females, those with greater relative gain in CSA post‐RT (1.681 +/‐ 0.4120) had a trend towards lower AR content post‐RT compared to low responders (2.502 +/‐ 0.1946) (p = 0.06). This study is the first to immunofluorescently stain AR content for visualization and quantification in skeletal muscle. Collectively, these findings suggest that AR content influences SC activity and exhibits sex differences following damage and chronic resistance exercise.

Fenofibrate mitigates testosterone induced benign prostatic hyperplasia via regulation of Akt/FOXO3a pathway and modulation of apoptosis and proliferation in rats

Benign prostatic hyperplasia (BPH) is one of the most age-related health problems that commonly affect men. Regrettably, many individuals may not respond to current medical therapies or develop resistance to them. Accordingly, this study aimed to uncover how potentially fenofibrate, a lipid lowering agent, can ameliorate the induced BPH in rats. Forty rats were categorized randomly into four groups; the control group was given the vehicle (olive oil); the BPH model received testosterone propionate (20 mg/kg daily; s.c.) for 4 weeks; BPH-induced group received finasteride (10 mg/kg daily; p.o.) and BPH-induced group received fenofibrate (80 mg/kg daily; p.o.). After testosterone administration, both weight and relative weight of the prostate increased. Additionally, testosterone upregulated androgen receptor (AR), 5α-reductase gene expression and increased prostate proliferation. Histopathological examination confirmed that testosterone disrupted the histo-architecture of the prostate and caused marked hyperplasia of glands and stroma. On the other hand, fenofibrate administration reverted most hyperplastic changes of testosterone, it significantly reduced weight, relative weight of the prostate and dihydrotestosterone (DHT) level compared to BPH group. Also fenofibrate significantly decreased AR and 5α-reductase gene expression. Fenofibrate significantly suppressed ps473 Akt expression causing FOXO3a nuclear inclusion, which triggered induction of apoptosis. As well, Bax/Bcl2 ratio and caspase 3 content were significantly enhanced. Fenofibrate significantly diminished cyclin D1 immunoexpression and restored normal histo-architecture. In conclusion, this study emphasizes the preventive effect of fenofibrate in BPH rat model. This can be accredited, at least partly, to inhibiting AR and 5α-reductase expressions, the anti-proliferative, and pro-apoptotic activity of fenofibrate via modulation of Akt/FOXO3a pathway.

The Role of Androgen Receptor and microRNA Interactions in Androgen-Dependent Diseases.

The androgen receptor (AR) is a member of the steroid hormone receptor family of nuclear transcription factors. It is present in the primary/secondary sexual organs, kidneys, skeletal muscles, adrenal glands, skin, nervous system, and breast. Abnormal AR functioning has been identified in numerous diseases, specifically in prostate cancer (PCa). Interestingly, recent studies have indicated a relationship between the AR and microRNA (miRNA) crosstalk and cancer progression. MiRNAs are small, endogenous, non-coding molecules that are involved in crucial cellular processes, such as proliferation, apoptosis, or differentiation. On the one hand, AR may be responsible for the downregulation or upregulation of specific miRNA, while on the other hand, AR is often a target of miRNAs due to their regulatory function on AR gene expression. A deeper understanding of the AR–miRNA interactions may contribute to the development of better diagnostic tools as well as to providing new therapeutic approaches. While most studies usually focus on the role of miRNAs and AR in PCa, in this review, we go beyond PCa and provide insight into the most recent discoveries about the interplay between AR and miRNAs, as well as about other AR-associated and AR-independent diseases.

Targeting a splicing-mediated drug resistance mechanism in prostate cancer by inhibiting transcriptional regulation by PKC\u03b21

The androgen receptor (AR) is a central driver of aggressive prostate cancer. After initial treatment with androgen receptor signaling inhibitors (ARSi), reactivation of AR signaling leads to resistance. Alternative splicing of AR mRNA yields the AR-V7 splice variant, which is currently an undruggable mechanism of ARSi resistance: AR-V7 lacks a ligand binding domain, where hormones and anti-androgen antagonists act, but still activates AR signaling. We reveal PKCβ as a druggable regulator of transcription and splicing at the AR genomic locus. We identify a clinical PKCβ inhibitor in combination with an FDA-approved anti-androgen as an approach for repressing AR genomic locus expression, including expression of AR-V7, while antagonizing full-length AR. PKCβ inhibition reduces total AR gene expression, thus reducing AR-V7 protein levels and sensitizing prostate cancer cells to current anti-androgen therapies. We demonstrate that this combination may be a viable therapeutic strategy for AR-V7-positive prostate cancer.