Androgen Receptor Binding Research Articles

Comprehensive genomics in androgen receptor-dependent castration-resistant prostate cancer identifies an adaptation pathway mediated by opioid receptor kappa 1

Castration resistance is a lethal form of treatment failure of prostate cancer (PCa) and is associated with ligand-independent activation of the androgen receptor (AR). It is only partially understood how the AR mediates survival and castration-resistant growth of PCa upon androgen deprivation. We investigated integrative genomics using a patient-derived xenograft model recapitulating acquired, AR-dependent castration-resistant PCa (CRPC). Sequencing of chromatin immunoprecipitation using an anti-AR antibody (AR-ChIP seq) revealed distinct profiles of AR binding site (ARBS) in androgen-dependent and castration-resistant xenograft tumors compared with those previously reported based on human PCa cells or tumor tissues. An integrative genetic analysis identified several AR-target genes associated with CRPC progression including OPRK1, which harbors ARBS and was upregulated upon androgen deprivation. Loss of function of OPRK1 retarded the acquisition of castration resistance and inhibited castration-resistant growth of PCa both in vitro and in vivo. Immunohistochemical analysis showed that expression of OPRK1, a G protein-coupled receptor, was upregulated in human prostate cancer tissues after preoperative androgen derivation or CRPC progression. These data suggest that OPRK1 is involved in post-castration survival and cellular adaptation process toward castration-resistant progression of PCa, accelerating the clinical implementation of ORPK1-targeting therapy in the management of this lethal disease.

Cistrome and transcriptome analysis identifies unique androgen receptor (AR) and AR-V7 splice variant chromatin binding and transcriptional activities

The constitutively active androgen receptor (AR) splice variant, AR-V7, plays an important role in resistance to androgen deprivation therapy in castration resistant prostate cancer (CRPC). Studies seeking to determine whether AR-V7 is a partial mimic of the AR, or also has unique activities, and whether the AR-V7 cistrome contains unique binding sites have yielded conflicting results. One limitation in many studies has been the low level of AR variant compared to AR. Here, LNCaP and VCaP cell lines in which AR-V7 expression can be induced to match the level of AR, were used to compare the activities of AR and AR-V7. The two AR isoforms shared many targets, but overall had distinct transcriptomes. Optimal induction of novel targets sometimes required more receptor isoform than classical targets such as PSA. The isoforms displayed remarkably different cistromes with numerous differential binding sites. Some of the unique AR-V7 sites were located proximal to the transcription start sites (TSS). A de novo binding motif similar to a half ARE was identified in many AR-V7 preferential sites and, in contrast to conventional half ARE sites that bind AR-V7, FOXA1 was not enriched at these sites. This supports the concept that the AR isoforms have unique actions with the potential to serve as biomarkers or novel therapeutic targets.

Abstract PD1-07: Mutant ESR1 receptors antagonize the tumor suppressor function of androgen receptors

Abstract Background: Acquired ESR1 mutations are a dominant driver of distant metastasis in metastatic breast cancer, inducing a basal-like phenotype that is relatively resistant to ER antagonists with decreased progression free survival. The majority of ER+ patients also express the androgen receptor (AR), and although AR expression is associated with better outcomes, high AR expression has also been associated with resistance to endocrine therapy (ET). Thus, there is currently a conundrum on how best to target AR, and to define when it is participating as a proliferative/metastatic driver, or when it may be a potential tumor suppressor associated with good prognosis. Methods: We generated ESR1 Y537S or D538G homozygous mutations in MCF-7 cells using CRISPR Cas-9 technology. ChIP-Seq, transcriptome analyses, and quantitative analysis of ER ChIP-Seq profiles from patient biopsies were integrated. Nuclear fractionation and immunoblot analyses were performed. Results: Correlation and gene set enrichment analyses demonstrated that the androgen response pathway was significantly reduced in ESR1 mutant, compared to wild-type (WT) ER cells. A dramatic redistribution in AR binding sites to heterochromatin was observed in ESR1 mutant cells. AR and ER co-occupied DNA binding sites were redistributed only to ERE motifs present in a restricted set of gene enhancer regions. Quantitative analysis of ER DNA binding profiles from ER+ patients showed that the highest recruitment of ER to AR/ER binding sites were in patients with metastatic breast cancer. Integration of AR/ER co-bound sites with the differentially-expressed mutant transcriptome identified a gene signature that predicted poor disease-free and overall survival in ER+ primary breast cancer patients from the METABRIC database. Elevated gene expression of these AR/ER co-regulated genes, including NCOA3, BMP7, N4BP3, and FOXA1, were validated in a cohort of metastatic tumors (N=97), compared to primary tumors (N=276). AR protein levels were elevated in ESR1 mutant tumors. Mechanistic studies demonstrated that elevated AR phosphorylation at specific sites (pS515, pS650) and decreased phosphorylation (pS308) occurs in mutant cells. These sites are known to affect AR transcriptional activity, protein stability, and nuclear export. Decreased K48-AR ubiquitination was observed, suggesting that elevated AR protein levels result from altered post-transcriptional modification of AR in mutant cells. Conclusions: We propose a two-prong genomic activation mechanism in ESR1 mutant tumors with AR redistribution to heterochromatin, and genomic co-activation of AR and ER. We hypothesize that the loss of AR tumor suppressor function may help drive metastasis in ESR1 mutant tumors via AR’s collaboration with ESR1 mutant oncogenic activity. Efficacious targeting of AR may require disruption of this driver AR/ER-regulated transcriptional program. Citation Format: Sandra L. Grimm, Guowei Gu, Sarah K. Herzog, Thomas L. Gonzalez, Hangqing Lin, Amanda R. Beyer, Yassine Rechoum, Tasneem Bawa-Khalfe, Ashfia F. Khan, Lili Du, W. Fraser Symmans, Ralf Kittler, Cristian Coarfa, Suzanne A.W. Fuqua. Mutant ESR1 receptors antagonize the tumor suppressor function of androgen receptors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD1-07.

Exploiting the tumor-suppressive activity of the androgen receptor by CDK4/6 inhibition in castration-resistant prostate cancer

The androgen receptor (AR) plays a pivotal role in driving prostate cancer (PCa) development. However, when stimulated by high levels of androgens, AR can also function as a tumor suppressor in PCa cells. While the high-dose testosterone (high-T) treatment is currently being tested in clinical trials of castration-resistant prostate cancer (CRPC), there is still a pressing need to fully understand the underlying mechanism and thus develop treatment strategies to exploit this tumor-suppressive activity of AR. In this study, we demonstrate that retinoblastoma (Rb) family proteins play a central role in maintaining the global chromatin binding and transcriptional repression program of AR and that Rb inactivation desensitizes CRPC to the high-dose testosterone treatment invitro and invivo. Using a series of patient-derived xenograft (PDX) CRPC models, we further show that the efficacy of high-T treatment can be fully exploited by a CDK4/6 inhibitor, which strengthens the chromatin binding of the Rb-E2F repressor complex by blocking the hyperphosphorylation of Rb proteins. Overall, our study provides strong mechanistic and preclinical evidence on further developing clinical trials to combine high-T with CDK4/6 inhibitors in treating CRPC.

A genome-scale CRISPR screen reveals PRMT1 as a critical regulator of androgen receptor signaling in prostate cancer.

Androgen receptor (AR) signaling is the central driver of prostate cancer across disease states. While androgen deprivation therapy (ADT) is effective in the initial treatment of prostate cancer, resistance to ADT or to next-generation androgen pathway inhibitors invariably arises, most commonly through the re-activation of the AR axis. Thus, orthogonal approaches to inhibit AR signaling in advanced prostate cancer are essential. Here, via genome-scale CRISPR-Cas9 screening, we identify protein arginine methyltransferase 1 (PRMT1) as a critical mediator of AR expression and signaling. PRMT1 regulates the recruitment of AR to genomic target sites and the inhibition of PRMT1 impairs AR binding at lineage-specific enhancers, leading to decreased expression of key oncogenes, including AR itself. In addition, AR-driven prostate cancer cells are uniquely susceptible to combined AR and PRMT1 inhibition. Our findings implicate PRMT1 as a key regulator of AR output and provide a preclinical framework for co-targeting of AR and PRMT1 in advanced prostate cancer.

Up-regulation of androgen receptor by heat shock protein 27 and miR-1 induces pathogenesis of androgenic alopecia.

The pathogenesis of androgenetic alopecia (AGA) is related to the level of androgen and its metabolic pathways. The binding of androgen and androgen receptor (AR) depends on the assistance of heat shock protein 27 (HSP27). HSP27 combined with microRNAs (miR)-1 can regulate AR levels. However, it is not clear whether HSP27 and miR-1 jointly participate in the pathogenesis of AGA. This study aims to investigate the role of AR up-regulation in the pathogenesis of AGA and underlying mechanisms. A total of 46 male AGA patients (AGA group), who admitted to the First Affiliated Hospital of Guangzhou Medical University from September 2019 to February 2020, and 52 healthy controls admitted to the same period were enrolled in this study. Serum levels of dihydrotestosterone (DHT) and HSP27 in patients and healthy controls were measured by ELISA. Western blotting was used to detect the protein expression of HSP27 and AR in scalp tissues of patients and the healthy controls. The levels of HSP27, AR, and miR-1 were analyzed using real-time PCR. Human dermal papilla cells were transfected with HSP27 siRNA to inhibit the expression of HSP27. MiR-1 and miR-1 inhibitors were transfected simultaneously or separately into cells and then the changes in AR protein expression were detected. The levels of DHT and HSP27 in the AGA group were (361.4±187.7) pg/mL and (89.4±21.8) ng/mL, respectively, which were higher than those in the control group [(281.8±176.6) pg/mL and (41.2±13.7) ng/mL, both P<0.05]. However, there was no significant difference in serum HSP27 and AR levels among AGA patients with different degrees of hair loss (P>0.05). Correlation analysis showed that there was a positive correlation between HSP27 level and DHT level in the AGA patients (P<0.05). The level of HSP27 mRNA in scalp tissue was negatively correlated with that of miR-1 mRNA (P<0.05). Compared with the control group, the levels of HSP27 protein, AR protein, HSP27 mRNA, and AR mRNA in scalp tissues of AGA group were significantly increased (P<0.05). The up-regulation of HSP27 in scalp tissues of AGA patients was closely related to the increased levels of AR. However, the level of miR-1 in scalp tissues of AGA patients was significantly down-regulated, contrary to the expression of AR (P<0.05). Further in cell studies showed that inhibition of HSP27 or miR-1 expression in human dermal papilla cells could inhibit the expression of AR, and inhibition of both HSP27 and miR-1 expression was found to have an accumulative effect on AR, with statistically significant differences (all P<0.05). HSP27 could combine with miR-1 to up-regulate AR levels, which is closely related to the development of AGA.

Endocrine Disruptors and Prostate Cancer.

The role of endocrine disruptors (EDs) in the human prostate gland is an overlooked issue even though the prostate is essential for male fertility. From experimental models, it is known that EDs can influence several molecular mechanisms involved in prostate homeostasis and diseases, including prostate cancer (PCa), one of the most common cancers in the male, whose onset and progression is characterized by the deregulation of several cellular pathways including androgen receptor (AR) signaling. The prostate gland essentiality relies on its function to produce and secrete the prostatic fluid, a component of the seminal fluid, needed to keep alive and functional sperms upon ejaculation. In physiological condition, in the prostate epithelium the more-active androgen, the 5α-dihydrotestosterone (DHT), formed from testosterone (T) by the 5α-reductase enzyme (SRD5A), binds to AR and, upon homodimerization and nuclear translocation, recognizes the promoter of target genes modulating them. In pathological conditions, AR mutations and/or less specific AR binding by ligands modulate differently targeted genes leading to an altered regulation of cell proliferation and triggering PCa onset and development. EDs acting on the AR-dependent signaling within the prostate gland can contribute to the PCa onset and to exacerbating its development.

(+)-JJ-74-138 is a Novel Noncompetitive Androgen Receptor Antagonist.

Identification of novel androgen receptor (AR) antagonists may lead to urgently needed new treatments for patients with prostate cancer resistant to current AR antagonists. AR is presently the main target for treating prostate cancer. Clinically approved AR antagonists compete with dihydrotestosterone (DHT) for binding to the ligand-binding domain (LBD) of AR, and patients eventually develop resistance to these treatments. One approach to overcoming resistance is to discover compounds that inhibit AR in alternative ways. Our lab previously identified a small molecule, JJ-450, that is capable of inhibiting AR lacking LBD. To optimize the efficacy of this class of inhibitors, we developed structural analogues of JJ-450 and identified (+)-JJ-74-138 as a promising candidate. Here, we show that (+)-JJ-74-138 is more potent than JJ-450 in the inhibition of androgen-independent AR activity in enzalutamide-resistant LN95 cells. Further studies showed (+)-JJ-74-138 inhibition of castration-resistant PSA expression in all tested castration-resistant prostate cancer (CRPC) cells. (+)-JJ-74-138 inhibited mRNA expression of AR and ARv7 target genes and reduced AR level in the nucleus in the absence of androgens. Also, this analogue noncompetitively inhibited androgen-stimulated AR activity in C4-2, LN95, and 22Rv1 CRPC cells. At low dosages, (+)-JJ-74-138 inhibited the proliferation of enzalutamide-resistant AR-positive LN95 and 22Rv1 cells, but not AR-negative PC3 and DU145 cells. A surface plasmon resonance assay detected (+)-JJ-74-138 binding to AR and a chromatin immunoprecipitation assay indicated (+)-JJ-74-138 inhibited AR binding to androgen response elements. In addition, (+)-JJ-74-138 inhibited 22Rv1 xenograft tumor growth. Our observations suggest that (+)-JJ-74-138 is a novel noncompetitive AR antagonist capable of inhibiting enzalutamide-resistant CRPC.

Impact of Androgen Receptor Activity on Prostate-Specific Membrane Antigen Expression in Prostate Cancer Cells.

Prostate-specific membrane antigen (PSMA) is an essential molecular regulator of prostate cancer (PCa) progression coded by the FOLH1 gene. The PSMA protein has become an important factor in metastatic PCa diagnosis and radioligand therapy. However, low PSMA expression is suggested to be a resistance mechanism to PSMA-based imaging and therapy. Clinical studies revealed that androgen receptor (AR) inhibition increases PSMA expression. The mechanism has not yet been elucidated. Therefore, this study investigated the effect of activation and inhibition of androgen signaling on PSMA expression levels in vitro and compared these findings with PSMA levels in PCa patients receiving systemic therapy. To this end, LAPC4, LNCaP, and C4-2 PCa cells were treated with various concentrations of the synthetic androgen R1881 and antiandrogens. Changes in FOLH1 mRNA were determined using qPCR. Open access databases were used for ChIP-Seq and tissue expression analysis. Changes in PSMA protein were determined using western blot. For PSMA staining in patients’ specimens, immunohistochemistry (IHC) was performed. Results revealed that treatment with the synthetic androgen R1881 led to decreased FOLH1 mRNA and PSMA protein. This effect was partially reversed by antiandrogen treatment. However, AR ChIP-Seq analysis revealed no canonical AR binding sites in the regulatory elements of the FOLH1 gene. IHC analysis indicated that androgen deprivation only resulted in increased PSMA expression in patients with low PSMA levels. The data demonstrate that AR activation and inhibition affects PSMA protein levels via a possible non-canonical mechanism. Moreover, analysis of PCa tissue reveals that low PSMA expression rates may be mandatory to increase PSMA by androgen deprivation.

Elevated expression of the colony-stimulating factor 1 (CSF1) induces prostatic intraepithelial neoplasia dependent of epithelial-Gp130.

Macrophages are increased in human benign prostatic hyperplasia and prostate cancer. We generate a Pb-Csf1 mouse model with prostate-specific overexpression of macrophage colony-stimulating factor (M-Csf/Csf1). Csf1 overexpression promotes immune cell infiltration into the prostate, modulates the macrophage polarity in a lobe-specific manner, and induces senescence and low-grade prostatic intraepithelial neoplasia (PIN). The Pb-Csf1 prostate luminal cells exhibit increased stem cell features and epithelial-to-mesenchymal transition. Human prostate cancer patients with high CSF-1 expression display similar transcriptional alterations with the Pb-Csf1 model. P53 knockout alleviates senescence but fails to progress PIN lesions. Ablating epithelial Gp130 but not Il1r1 substantially blocks PIN lesion formation. The androgen receptor (AR) is downregulated in Pb-Csf1 mice. ChIP-Seq analysis reveals altered AR binding in 2482 genes although there is no significant widespread change in global AR transcriptional activity. Collectively, our study demonstrates that increased macrophage infiltration causes PIN formation but fails to transform prostate cells.

The androgen receptor inhibits transcription of GPER1 by preventing Sp1 and Sp3 from binding to the promoters in prostate cancer cells.

G-1, a GPER1 agonist, was shown to inhibit the growth of castration-resistant mouse xenografts but not their parental androgen-dependent tumors. It is currently unknown how the androgen receptor (AR) represses GPER1 expression. Here, we found that two GPER1 mRNA variants (GPER1v2 and GPER1v4) were transcriptionally repressed, not via transcript destabilization, by the androgen-activated AR. Although no AR binding was found in all active promoters near GPER1, data from promoter assays suggested that both variants’ promoters were inhibited by androgen treatment. Site-directed mutagenesis on Sp1/Sp3 binding sites revealed their role in supporting the basal expression of GPER1. Knockdown of Sp1 and Sp3 together but not separately repressed GPER1 expression whereas overexpression of both Sp1 and Sp3 together was required to alleviate AR repression of GPER1. Based on the chromatin immunoprecipitation data, Sp3 was found to bind to the promoters prior to the binding of Sp1 and RNA polymerase II. However, the binding of all three transcription factors was inhibited by DHT treatment. Concordantly, DHT treatment induced nuclear interactions between AR and Sp1 or Sp3. Taken together, these results indicate that AR represses transcription of GPER1 by binding to Sp1 and Sp3 independently to prevent their transactivation of the GPER1 promoters.

Antiandrogenic activity of Riboflavin 5′-phosphate (FMN) in 22Rv1 and LNCaP human prostate cancer cell lines

The androgen receptor is a hormone activated transcription factor that regulates the development and maintenance of male characteristics and represents one of the most well-established drug targets, being implicated not only in prostate cancer but also in many non-cancerous human diseases including androgenetic alopecia, acne vulgaris, and hirsutism. In this study, the antiandrogenic effects of FMN were investigated in 22Rv1 and LNCaP prostate cancer cells. FMN inhibited dihydrotestosterone (DHT)-induced protein expression of androgen receptor in 22Rv1cells. In another prostate cancer LNCaP cells, FMN decreased the protein level of DHT-induced prostate specific antigen (PSA). In addition, FMN downregulated DHT-induced mRNA expression of androgen regulated genes in both cell lines, showing less prominent inhibition in 22Rv1cells where androgen receptor had been significantly decreased by FMN. FMN was found to bind androgen receptor, demonstrating that it acted as a competitive androgen receptor antagonist. FMN increased the phosphorylation of Akt in 22Rv1 cells and this increment was abrogated by PI3K inhibitor wortmannin, resulting in a rescued androgen receptor protein level which was decreased by FMN. Additionally, FMN was found to increase the mRNA and protein level of E3 ligase mouse double minute 2. Our data suggest that the androgen receptor signaling is regulated through PI3K-Akt-MDM2 pathway in 22Rv1 cells. Together, our results indicate that FMN facilitated the degradation of androgen receptor in 22Rv1 cells and inhibited the expression of androgen regulated genes by competing the binding of DHT to androgen receptor in LNCaP cells, demonstrating its therapeutic potential as an antiandrogen.

Effects of Membrane Androgen Receptor Binding on Synaptic Plasticity in Primary Hippocampal Neurons

Effects of Membrane Androgen Receptor Binding on Synaptic Plasticity in Primary Hippocampal Neurons

AR imposes different effects on ZFHX3 transcription depending on androgen status in prostate cancer cells.

Both androgen receptor (AR) and the ZFHX3 transcription factor modulate prostate development. While AR drives prostatic carcinogenesis, ZFHX3 is a tumour suppressor whose loss activates the PI3K/AKT signalling in advanced prostate cancer (PCa). However, it is unknown whether ZFHX3 and AR are functionally related in PCa cells and, if so, how. Here, we report that in AR‐positive LNCaP and C4‐2B PCa cells, androgen upregulates ZFHX3 transcription via androgen‐induced AR binding to the androgen‐responsive elements (AREs) of the ZFHX3 promoter. Androgen also upregulated ZFHX3 transcription in vivo, as castration dramatically reduced Zfhx3 mRNA and protein levels in mouse prostates, and ZFHX3 mRNA levels correlated with AR activities in human PCa. Interestingly, the binding of AR to one ARE occurred in the absence of androgen, and the binding repressed ZFHX3 transcription as this repressive binding was interrupted by androgen treatment. The enzalutamide antiandrogen prevented androgen from inducing ZFHX3 transcription and caused excess ZFHX3 protein degradation. In human PCa, ZFHX3 was downregulated and the downregulation correlated with worse patient survival. These findings establish a regulatory relationship between AR and ZFHX3, suggest a role of ZFHX3 in AR function and implicate ZFHX3 loss in the antiandrogen therapies of PCa.

Abstract P192: Comprehensive preclinical characterization of the mechanism of action of EPI-7386, an androgen receptor N-terminal domain inhibitor

Abstract Background: Androgen receptor (AR) signaling is a main driver of prostate cancer progression and remains a crucial target for therapeutic intervention even in late stages of the disease. While current anti-androgen therapies targeting directly or indirectly the AR ligand binding domain (LBD) are initially effective, resistance ultimately develops, and new methods of inhibiting the AR pathway are needed. The selective targeting of the N-terminal domain (NTD) of the AR represents a novel method of blocking AR signaling to by-pass LBD-related resistance. EPI-7386 is a potent and metabolically stable NTD inhibitor (aniten) currently in a phase 1 dose-escalation study in mCRPC patients (NCT04421222). Here we further characterized the binding to AR NTD and the mechanism of action of EPI-7386. Methods: Target engagement was measured by Cellular Thermal Shift Assay (CETSA) and two-dimensional Nuclear Magnetic Resonance (2D NMR) spectroscopy. The potency and selectivity of EPI-7386 was determined in cellular models expressing different forms of AR using reporter and cell viability assays. qPCR, NanoString, and RNA sequencing were used to explore the activity of EPI-7386 on the AR transcriptome. To determine the effect of EPI-7386 on AR genomic occupancy, Chromatin immunoprecipitation sequencing (ChIP-seq) was performed. Results: We confirmed target engagement of EPI-7386 with an LBD truncated AR variant by CETSA using a cell line which expresses only AR-V567es, suggesting the interaction of EPI-7386 with AR NTD. In the same cell line, AR antagonist enzalutamide that binds to AR LBD showed no target engagement with AR-V567es. Furthermore, 2D NMR study results demonstrate an interaction of EPI-7386 with amino acid residues located in the transcription activation unit 5 (Tau-5) region of the AR NTD, a region which has been described to be involved in interactions with transcriptional cofactors such as CBP/p300. EPI-7386 strongly impaired the transcriptional activity and gene expression driven exclusively by LBD truncated AR variants including AR-V567es and AR-V7 and decreased cell viability. EPI-7386 has been shown to suppress the AR regulated transcriptome and the combination of EPI-7386 with lutamides resulted in broader and deeper inhibition of AR-regulated gene expression. The analysis of the AR cistrome by ChIP-seq showed that EPI-7386 displaces genome-wide androgen induced AR binding and the combination with enzalutamide completely abrogated AR binding. Conclusion: EPI-7386 is a potent AR NTD inhibitor that has the capacity to by-pass AR LBD resistance mechanisms to current anti-androgen therapies by uniquely inhibiting AR-mediated signaling. The agent has the potential for providing clinical benefit as a single agent in patients whose tumors are progressing on anti-androgens or in combination with current anti-androgens in earlier line patients. Citation Format: Nan Hyung Hong, Shihua Sun, Peter Virsik, Alessandra Cesano, Elahe A. Mostaghel, Stephen R. Plymate, Berenger Biannic, Han-Jie Zhou, Ronan Le Moigne. Comprehensive preclinical characterization of the mechanism of action of EPI-7386, an androgen receptor N-terminal domain inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P192.

Development of 2-(5,6,7-Trifluoro-1H-Indol-3-yl)-quinoline-5-carboxamide as a Potent, Selective, and Orally Available Inhibitor of Human Androgen Receptor Targeting Its Binding Function-3 for the Treatment of Castration-Resistant Prostate Cancer.

Prostate cancer (PCa) patients undergoing androgen deprivation therapy almost invariably develop castration-resistant prostate cancer (CRPC). Targeting the androgen receptor (AR) Binding Function-3 (BF3) site offers a promising option to treat CRPC. However, BF3 inhibitors have been limited by poor potency or inadequate metabolic stability. Through extensive medicinal chemistry, molecular modeling, and biochemistry, we identified 2-(5,6,7-trifluoro-1H-Indol-3-yl)-quinoline-5-carboxamide (VPC-13789), a potent AR BF3 antagonist with markedly improved pharmacokinetic properties. We demonstrate that VPC-13789 suppresses AR-mediated transcription, chromatin binding, and recruitment of coregulatory proteins. This novel AR antagonist selectively reduces the growth of both androgen-dependent and enzalutamide-resistant PCa cell lines. Having demonstrated in vitro efficacy, we developed an orally bioavailable prodrug that reduced PSA production and tumor volume in animal models of CRPC with no observed toxicity. VPC-13789 is a potent, selective, and orally bioavailable antiandrogen with a distinct mode of action that has a potential as novel CRPC therapeutics.

Inhibition of GATA2 in prostate cancer by a clinically available small molecule.

Castration-resistant prostate cancer (CRPC) remains highly lethal and in need of novel, actionable therapeutic targets. The pioneer factor GATA2 is a significant prostate cancer (PC) driver and is linked to poor prognosis. GATA2 directly promotes androgen receptor (AR) gene expression (both full-length and splice-variant) and facilitates AR binding to chromatin, recruitment of coregulators, and target gene transcription. Unfortunately, there is no clinically applicable GATA2 inhibitor available at the moment. Using a bioinformatics algorithm, we screened in silico 2650 clinically relevant drugs for a potential GATA2 inhibitor. Validation studies used cytotoxicity and proliferation assays, global gene expression analysis, RT-qPCR, reporter assay, reverse phase protein array analysis (RPPA), and immunoblotting. We examined target engagement via cellular thermal shift assay (CETSA), ChIP-qPCR, and GATA2 DNA-binding assay. We identified the vasodilator dilazep as a potential GATA2 inhibitor and confirmed on-target activity via CETSA. Dilazep exerted anticancer activity across a broad panel of GATA2-dependent PC cell lines in vitro and in a PDX model in vivo. Dilazep inhibited GATA2 recruitment to chromatin and suppressed the cell-cycle program, transcriptional programs driven by GATA2, AR, and c-MYC, and the expression of several oncogenic drivers, including AR, c-MYC, FOXM1, CENPF, EZH2, UBE2C, and RRM2, as well as of several mediators of metastasis, DNA damage repair, and stemness. In conclusion, we provide, via an extensive compendium of methodologies, proof-of-principle that a small molecule can inhibit GATA2 function and suppress its downstream AR, c-MYC, and other PC-driving effectors. We propose GATA2 as a therapeutic target in CRPC.

Safety effect of fermented oyster extract on the endocrine disruptor assay in vitro and in vivo

Oyster (Crassostrea gigas) is a marine bivalve mollusk widely distributed in coastal areas, and have been long widely used in industrial resources. Several studies demonstrated that fermented oyster (FO) extract attribute to bone health, but whether administration of FO play as an endocrine disruptor has not been studied. Therefore, in the present study, we investigated the effect of FO on the endocrine system in vitro and in vivo. As the results of the competitive estrogen receptor (ER) and androgen receptor (AR) binding affinities, FO was not combined with ER-α, ER-β, and AR. However, 17β-estradiol and testosterone, used as positive control, were interacted with ER and AR, respectively. Meanwhile, oral administration of 100 mg/kg and 200 mg/kg of FO doesn’t have any harmful effect on the body weight, androgen-dependent sex accessory organs, estrogen-dependent-sex accessory organs, kidney, and liver in immature rats. In addition, FO supplementation has no effect on the serum levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone, and 17β-estradiol. However, the relative weight of androgen- and estrogen-dependent organs were significantly increased by subcutaneously injection of 4.0 mg/kg of testosterone propionate (TP) and by orally administration of 1.0 μg of 17α-ethynyl estradiol (EE) in immature male and female rats, respectively. Furthermore, TP and EE administration markedly decreased the serum LH and FSH levels, which are similar those of mature Sprague-Dawley (SD) rat. Furthermore, the testosterone and 17β-estradiol levels were significantly enhanced in TP and EE-treated immature rats. Taken together, our findings showed that FO does not interact with ER and AR, suggesting consequentially FO does not play as a ligand for ER and AR. Furthermore, oral administration of FO did not act as an endocrine disruptor including androgenic activity, estrogenic activity, and abnormal levels of sex hormone, indicating FO may ensure the safety on endocrine system to develop dietary supplement for bone health.

PD37-06 LATROPHILIN-3 AS A KEY DOWNSTREAM EFFECTOR OF THE ANDROGEN RECEPTOR INDUCES UROTHELIAL TUMORIGENESIS

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (PD37)1 Sep 2021PD37-06 LATROPHILIN-3 AS A KEY DOWNSTREAM EFFECTOR OF THE ANDROGEN RECEPTOR INDUCES UROTHELIAL TUMORIGENESIS Takuro Goto, Yuki Teramoto, Yujiro Nagata, Guiyang Jiang, Taichi Mizushima, Satoshi Inoue, Hiroki Ide, George Netto, and Hiroshi Miyamoto Takuro GotoTakuro Goto More articles by this author , Yuki TeramotoYuki Teramoto More articles by this author , Yujiro NagataYujiro Nagata More articles by this author , Guiyang JiangGuiyang Jiang More articles by this author , Taichi MizushimaTaichi Mizushima More articles by this author , Satoshi InoueSatoshi Inoue More articles by this author , Hiroki IdeHiroki Ide More articles by this author , George NettoGeorge Netto More articles by this author , and Hiroshi MiyamotoHiroshi Miyamoto More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002047.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: We and others have indicated that androgen receptor (AR) signaling plays a critical role in urothelial cancer outgrowth. Meanwhile, our DNA microarray data suggest that latrophilin-3 (LPHN3), one of latrophilins that are a group of the G-protein-coupled receptor where a spider venom latrotoxin (LTX) is known to bind, represents a downstream target of AR in bladder cancer cells. The present study aims to determine the functional role of LPHN3 in urothelial carcinogenesis. METHODS: We determined the expression of LPHN3 in 145 bladder tumors and paired normal bladder tissues (by immunohistochemistry) as well as in human normal urothelial SVHUC sublines stably expressing wild-type AR or vector only (by RT-PCR and western blot). We then assessed the effects of ligand (e.g. LTX, FLRT3) treatment on neoplastic/malignant transformation of SVHUC-derived cells, as well as bladder tumor development in C57BL/6 mice, induced by chemical carcinogens (e.g. MCA, BBN). RESULTS: Immunohistochemistry showed that LPHN3 was positive in 52% (47% 1+, 5% 2+) of tumors, which was significantly (P=0.049) higher than in non-neoplastic urothelial tissues [37% (all 1+)], and that LPHN3 positivity in non-muscle-invasive tumors was associated with the risk of tumor recurrence after transurethral surgery (P=0.051). The level of LPHN3 expression was higher in SVHUC-AR cells than in AR-negative SVHUC-vector cells. In addition, significant induction in the LPHN3 expression by LTX or FLRT3 treatment was seen in SVHUC, whereas LPHN3 knockdown via shRNA virus infection in SVHUC-AR resulted in considerable induction or reduction of the expression of the proteins positively (e.g. cleaved caspase-3) or negatively (e.g. Bcl-2, Bcl-xL), respectively, associated with apoptosis. Chromatin immunoprecipitation assay further revealed the binding of AR to the promoter region of LPHN3 in SVHUC-derived cells. The in vitro transformation assay showed that LTX and FLRT3 induced the MCA-mediated oncogenic activity in both SVHUC-AR and SVHUC-vector cells, while the inhibitory effect of LPHN3 knockdown on the neoplastic transformation was observed in MCA-SVHUC-AR cells. LTX/FLRT3 also significantly accelerated the development of bladder tumors in BBN-treated female mice. CONCLUSIONS: These findings suggest that LPHN3, which is activated in bladder tumors as a key downstream effector of the AR, promotes urothelial tumorigenesis. Accordingly, LPHN3 inhibition, along with AR inactivation, has the potential of being an effective chemopreventive approach for urothelial cancer. Source of Funding: Ferring Research Institute © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e657-e657 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takuro Goto More articles by this author Yuki Teramoto More articles by this author Yujiro Nagata More articles by this author Guiyang Jiang More articles by this author Taichi Mizushima More articles by this author Satoshi Inoue More articles by this author Hiroki Ide More articles by this author George Netto More articles by this author Hiroshi Miyamoto More articles by this author Expand All Advertisement Loading ...

Reshaping of the androgen-driven chromatin landscape in normal prostate cells by early cancer drivers and effect on therapeutic sensitivity.

SUMMARYThe normal androgen receptor (AR) cistrome and transcriptional program are fundamentally altered in prostate cancer (PCa). Here, we profile the chromatin landscape and AR-directed transcriptional program in normal prostate cells and show the impact of SPOP mutations, an early event in prostate tumorigenesis. In genetically normal mouse prostate organoids, SPOP mutation results in accessibility and AR binding patterns similar to that of human PCa. Consistent with dependence on AR signaling, castration of SPOP mutant mouse models results in the loss of neoplastic phenotypes, and human SPOP mutant PCa shows a favorable response to AR-targeted therapies. Together, these data validate mouse prostate organoids as a robust model for studying epigenomic and transcriptional alterations in normal prostate, provide valuable datasets for further studies, and show that a single genomic alteration may be sufficient to reprogram the chromatin of normal prostate cells toward oncogenic phenotypes, with potential therapeutic implications for AR-targeting therapies.