Interaction Of Androgen Receptor Research Articles

Androgen Receptor and Non-Coding RNAs' Interaction in Renal Cell Carcinoma.

Renal cell carcinoma (RCC), the most prevalent among the urogenital cancers, accounts for around 3% of new cancer cases worldwide. Significantly, the incidence of RCC has doubled in developed world countries, ranking it as the sixth most common cancer in males, who represent two-thirds of RCC cases. Males with RCC exhibit a higher mortality rate and tend to develop a more aggressive form of the disease than females. Sex-related risk factors, including lifestyle and biological variations, explain this difference. The androgen receptor (AR) oncogenic signaling pathway has been extensively studied among the biological factors that affect RCC. Recent advancements in high-throughput RNA sequencing techniques have underscored the significant roles played by noncoding-RNAs (ncRNAs), previously dismissed as "junk". The oncogenic potential of AR is manifested through its dysregulation of the ncRNAs' availability and function, promoting RCC tumorigenesis. This review offers a summary of the most recent findings on the role and molecular mechanisms of the AR in dysregulating the ncRNAs that play a role in the progression of RCC and the possibility of utilizing ncRNAs to target AR as a potential therapeutic strategy.

Phase Separation Mediated Sub-Nuclear Compartmentalization of Androgen Receptors.

The androgen receptor (AR), a member of the nuclear steroid hormone receptor family of transcription factors, plays a crucial role not only in the development of the male phenotype but also in the development and growth of prostate cancer. While AR structure and AR interactions with coregulators and chromatin have been studied in detail, improving our understanding of AR function in gene transcription regulation, the spatio-temporal organization and the role of microscopically discernible AR foci in the nucleus are still underexplored. This review delves into the molecular mechanisms underlying AR foci formation, focusing on liquid-liquid phase separation and its role in spatially organizing ARs and their binding partners within the nucleus at transcription sites, as well as the influence of 3D-genome organization on AR-mediated gene transcription.

Effects of 5α-reductase inhibition by dutasteride on reproductive gene expression and hormonal responses in zebrafish embryos

Steroid 5α-reductase (SRD5A) is a crucial enzyme involved in steroid metabolism, primarily converting testosterone to dihydrotestosterone (DHT). Dutasteride, an inhibitor of SRD5A types 1 and 2, is widely used for treating benign prostatic hyperplasia. An adverse outcome pathway (AOP) has been documented wherein SRD5A inhibition decreases DHT synthesis, leading to reduced levels of 17β-estradiol (E2) and vitellogenin (VTG), subsequently impairing fecundity in fish (AOP 289). However, the molecular and cellular mechanisms underlying these effects remain poorly understood. In this study, we assessed the impact of SRD5A inhibition on zebrafish embryos (Danio rerio). Exposure to dutasteride resulted in decreased DHT, E2, and VTG levels, showing a positive correlation. Dutasteride also downregulated the expression of reproduction-related genes (srd5a2, cyp19a1, esr1, esr2a, esr2b, and vtg), with interrelated reductions observed across these levels. Docking studies suggested that dutasteride's effects may operate independently of androgen receptor (AR) and estrogen receptor (ER) interactions. Furthermore, co-exposure of dutasteride (0.5 or 2 μM) with 0.5 μM DHT revealed gene expression levels comparable to the control group. These findings underscore DHT's pivotal role in modulating estrogenic function and the interplay between estrogenic and androgenic responses in vertebrates. Our proposed AOP model offers insights into mechanistic gaps, thereby enhancing current understanding and bridging knowledge disparities.

Development and application of consensus in silico models for advancing high-throughput toxicological predictions

Computational toxicology models have been successfully implemented to prioritize and screen chemicals. There are numerous in silico (quantitative) structure–activity relationship ([Q]SAR) models for the prediction of a range of human-relevant toxicological endpoints, but for a given endpoint and chemical, not all predictions are identical due to differences in their training sets, algorithms, and methodology. This poses an issue for high-throughput screening of a large chemical inventory as it necessitates several models to cover diverse chemistries but will then generate data conflicts. To address this challenge, we developed a consensus modeling strategy to combine predictions obtained from different existing in silico (Q)SAR models into a single predictive value while also expanding chemical space coverage. This study developed consensus models for nine toxicological endpoints relating to estrogen receptor (ER) and androgen receptor (AR) interactions (i.e., binding, agonism, and antagonism) and genotoxicity (i.e., bacterial mutation, in vitro chromosomal aberration, and in vivo micronucleus). Consensus models were created by combining different (Q)SAR models using various weighting schemes. As a multi-objective optimization problem, there is no single best consensus model, and therefore, Pareto fronts were determined for each endpoint to identify the consensus models that optimize the multiple-criterion decisions simultaneously. Accordingly, this work presents sets of solutions for each endpoint that contain the optimal combination, regardless of the trade-off, with the results demonstrating that the consensus models improved both the predictive power and chemical space coverage. These solutions were further analyzed to find trends between the best consensus models and their components. Here, we demonstrate the development of a flexible and adaptable approach for in silico consensus modeling and its application across nine toxicological endpoints related to ER activity, AR activity, and genotoxicity. These consensus models are developed to be integrated into a larger multi-tier NAM-based framework to prioritize chemicals for further investigation and support the transition to a non-animal approach to risk assessment in Canada.

Androgen receptor interactions in endocrine resistant breast cancer - location, location, location

Androgen receptor interactions in endocrine resistant breast cancer - location, location, location

RNA-binding protein 14 promotes phase separation to sustain prostate specific antigen expression under androgen deprivation in human prostate cancer

Castration-resistant prostate cancer (CRPC) is a big challenge in managing prostate cancer patients. The androgen receptor (AR) pathway is a major driver even in CRPC under androgen deprivation. The mechanism in maintaining of the AR pathway under androgen deprivation remains elusive. The recent discovery of biomolecular condensate, a membrane-less intracellular construct formed by liquid-liquid phase separation (LLPS), that facilitate molecular assembly, encouraged the re-screening of our previous microarray data list. We selected Rbm14 as a target molecule for further analysis because it works as a coactivator of nuclear receptors as well as it facilitates formation of biomolecular condensates via its intrinsically disordered region. GFP-tagged Rbm14 transfected into HEK293T cells formed droplet-like puncta, which diminished following treatment with 1,6-hexanediol. Droplet-like structures were also observed in immunofluorescence for endogenous RBM14 of PC-3 and DU145 cells. Luciferase assay revealed that Rbm14 enhanced androgen-responsive element (ARE)-mediated reporter activity in all conditions with or without testosterone and AR. Co-immunoprecipitation confirmed the Rbm14–AR interaction. Long non-coding RNAs, including NEAT1, SRA1, and HOTAIR, were also interacted with Rbm14. Small interfering RNAs of NEAT1 reduced ARE-mediated reporter activity, while transfection of SRA1 and HOTAIR enhance the reporter activity. Treatment with 1,6-hexanediol as well as transfection with a dominant-negative splice variant of Rbm14 reduced expression of prostate specific antigen (PSA), a prototype of androgen-regulated gene, in LNCaP, PC-3, and DU145 cells under androgen deprivation. Immunohistochemically, RBM14 expression was substantially upregulated in prostate cancer tissues after androgen deprivation therapy than in untreated tumors. In conclusion, RBM14 is a novel factor involved in maintenance of PSA expression via phase separation under androgen deprivation in prostate cancer.

Peptides disrupting TM4SF3 interaction with AR or AR-V7 block prostate cancer cell proliferation.

Androgen receptor (AR) plays a vital role in the development and progression of prostate cancer from the primary stage to the usually lethal stage known as castration-resistant prostate cancer (CRPC). Constitutively active AR splice variants (AR-Vs) lacking the ligand-binding domain are partially responsible for the abnormal activation of AR and may be involved in resistance to AR-targeting drugs occurring in CRPC. There is increasing consensus on the potential of drugs targeting protein-protein interactions. Our lab has recently identified transmembrane 4 superfamily 3 (TM4SF3) as a critical interacting partner for AR and AR-V7 and mapped the minimal interaction regions. Thus, we hypothesized that these interaction domains can be used to design peptides that can disrupt the AR/TM4SF3 interaction and kill prostate cancer cells. Peptides TA1 and AT1 were designed based on the TM3SF3 or AR interaction domain, respectively. TA1 or AT1 was able to decrease AR/TM4SF3 protein interaction and protein stability. Peptide TA1 reduced the recruitment of AR and TM4SF3 to promoters of androgen-regulated genes and subsequent activation of these AR target genes. Peptides TA1 and AT1 were strongly cytotoxic to prostate cancer cells that express AR and/or AR-V7. Peptide TA1 inhibited the growth and induced apoptosis of both enzalutamide-sensitive and importantly enzalutamide-resistant prostate cancer cells. TA1 also blocked the migration and malignant transformation of prostate cancer cells. Our data clearly demonstrate that using peptides to target the important interaction AR has with TM4SF3 provides a novel method to kill enzalutamide-resistant prostate cancer cells that can potentially lead to new more effective therapy for CRPC.

A Multivalent Peptoid Conjugate Modulates Androgen Receptor Transcriptional Activity to Inhibit Therapy-resistant Prostate Cancer.

Prostate cancers adapt to androgen receptor (AR) pathway inhibitors and progress to castration resistance due to ongoing AR expression and function. To counter this, we developed a new approach to modulate the AR and inhibit castration-resistant prostate cancer (CRPC) using multivalent peptoid conjugates (MPC) that contain multiple copies of the AR-targeting ligand ethisterone attached to a peptidomimetic scaffold. Here, we investigated the antitumor effects of compound MPC309, a trivalent display of ethisterone conjugated to a peptoid oligomer backbone that binds to the AR with nanomolar affinity. MPC309 exhibited potent antiproliferative effects on various enzalutamide-resistant prostate cancer models, including those with AR splice variants, ligand-binding mutations, and noncanonical AR gene expression programs, as well as mouse prostate organoids harboring defined genetic alterations that mimic lethal human prostate cancer subtypes. MPC309 is taken up by cells through macropinocytosis, an endocytic process more prevalent in cancer cells than in normal ones, thus providing an opportunity to target tumors selectively. MPC309 triggers a distinct AR transcriptome compared with DHT and enzalutamide, a clinically used antiandrogen. Specifically, MPC309 enhances the expression of differentiation genes while reducing the expression of genes needed for cell division and metabolism. Mechanistically, MPC309 increases AR chromatin occupancy and alters AR interactions with coregulatory proteins in a pattern distinct from DHT. In xenograft studies, MPC309 produced significantly greater tumor suppression than enzalutamide. Altogether, MPC309 represents a promising new AR modulator that can combat resistant disease by promoting an AR antiproliferative gene expression program.

Abstract 1498: Melanoma antigen gene A6 and androgen receptor interaction in bladder cancer

Abstract The Melanoma Antigen Gene (MAGE) protein family is a large and highly conserved group of proteins that share a common MAGE homology domain. Interestingly, several MAGE proteins are limited in expression to reproductive tissues but are aberrantly expressed in various types of human malignancies. We have recently demonstrated overexpression of MAGE family member A6 in advance-stage bladder cancer (Cancers 13:23, 5931, 2021). However, the mechanisms involved in MAGE gene overexpression and its coregulators are currently unknown. One hypothesis is that MAGEA6 and androgen receptor (AR) signaling is involved in the etiology of bladder cancer progression. In the present study, we determined the functional relationship between MAGEA6 and AR in bladder cancer. Treatment with DHT, an AR agonist, increased AR and MAGEA6 expression in UMUC3 and J82 bladder cancer cells at the protein and transcript level. On the contrary, exposure of cells to enzalutamide, an antiandrogen, resulted in simultaneous reduction of AR and MAGEA6 more prominently in UMUC3 cells, compared to J82 harboring low MAGEA6 transcript. Dual-sgRNA CRISPR/Cas9 knockout of MAGEA6 in UMUC3 and J82 cells resulted in inhibition of cell proliferation, reduced survival, and migration potential in both cell lines. An induction of S-phase and G0/G1 phase cell cycle arrest was noted in UMUC3 and J82 cells. In silico analysis showed Arg20 and Cys557 amino acid residue of AR interacts with Asp236 and Gln106 amino acid residue of MAGEA6 through hydrogen bonds. Further confirmation of physical interaction between AR and MAGEA6, performed by immunoprecipitation of AR and probed for MAGEA6 and vice versa established interaction of AR-MAGEA6 with each other which was further confirmed by mass spectrometry analysis. In conclusion, these findings confirm the oncogenic role of MAGEA6 and its physical interaction with AR may be involved in bladder cancer progression. Citation Format: Prem Prakash Kushwaha, Shiv Verma, Sanjay Gupta. Melanoma antigen gene A6 and androgen receptor interaction in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1498.

Abstract 3441: A multivalent peptoid conjugate that inhibits therapy-resistant prostate cancer cell proliferation by modulating androgen receptor transcriptional activity

Abstract Prostate cancers adapt to androgen receptor (AR) pathway inhibitors and progress to castration resistance despite the continued expression and function of the AR. We developed a new approach to antagonize the proliferative activity of AR and inhibit castration-resistant prostate cancer (CRPC) growth using multivalent peptoid conjugates (MPCs). Our strategy is to display multiple copies of the AR antagonist, ethisterone, on a peptoid scaffold. Such a multivalent display increases the ligand’s effective local concentration, thus targeting AR with high affinity and affecting AR interactions with coregulators. Here, we report a new potent MPC called MPC309, which displays three ethisterone groups and binds to AR with nanomolar affinity. MPC309 reduces the proliferation of enzalutamide-resistant prostate cancer cells, including those harboring AR splice variants, AR ligand binding mutations, and non-canonical AR gene expression programs. We provide evidence that MPC309 enters cells via macropinocytosis, which facilitates the fluid-phase uptake of extracellular macromolecules. Macropinocytotic uptake of MPC309 enhances cancer cell-specific delivery, thus limiting systemic toxicities. MPC309 displays favorable pharmacological properties and produced significantly greater tumor suppression in xenograft studies than enzalutamide, the standard of care anti-androgen in clinical use. Mechanistically, MPC309 inhibits prostate cancer growth by eliciting a unique gene expression program through alterations in AR chromatin occupancy compared to dihydrotestosterone (DHT) or enzalutamide. Thus, MPC309 represents a novel AR antagonist that activates an AR anti-proliferative program to repress the growth of CRPC and supports further investigation of the MPC compound class for treating CRPC. Citation Format: Justine Habault, Jeffrey A. Schneider, Susan Ha, Rachel Ruoff, Joseph Puccini, Dafna Bar-Sagi, Kwok-Kin Wong, Amina Zoubeidi, Frank Claessens, David R. Wise, Susan K. Logan, Kent Kirshenbaum, Michael J. Garabedian. A multivalent peptoid conjugate that inhibits therapy-resistant prostate cancer cell proliferation by modulating androgen receptor transcriptional activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3441.

Abstract 4802: Identification of critical hypoxia induced factors in castrate resistant prostate cancer

Abstract Background: Prostate cancer (PCa) is the second most diagnosed cancer in men and the second cause of cancer-related death amongst men worldwide. PCa is a heterogeneous disease, and the outcome is worse for patients when the disease progresses from localized PCa to a castrate-resistant disease. Androgen receptor (AR) signaling is crucial for PCa development and has been a major therapeutic target for decades. Despite the success made with hormone therapy to block AR signaling, castration resistant disease can arise through mutations and amplifications of the androgen receptor (AR) and AR splice variants (AR-Vs). Amongst them, AR-V7 and AR-V12 can promote AR signaling independent of androgen. Furthermore, prostate tumors are highly hypoxic due to abnormal tumor vasculature. Hypoxia influences the efficacy of local and systemic treatment strategies, such as radiotherapy, hormone therapy, and chemotherapy, resulting in poorer clinical outcomes for PCa patients. Therefore, a complete understanding of AR and AR variant function, as well as how they are modulated by hypoxia and therapy is warranted for developing new strategies to treat PCa. Methods: To identify the transcriptional apparatus associated with the AR and its variants under normal and hypoxic conditions, we carried out Bio-ID proximity labeling mass spectrometry (BioID-MS). We generated stable androgen-independent human prostate cancer cell lines, PC-3 and DU145, expressing AR and the AR-V7 and AR-V12 variants fused to a MiniTurboID (MTID) enzyme. To delineate the AR proximal interaction network, we treated cells with androgen or vehicle for 3 hours and affinity-purified biotinylated proteins after the addition of biotin for the final 2 hours. Cells were grown in normoxia (20% O2) or treated with hypoxia (95% N2,5% CO2 and 0.5% O2) for 24 h. Nonspecific interactions were filtered using controls and statistical methods to identify high-confidence interactomes. Results: BioID proximity labeling in PC3 cells identified several AR-associated proteins, including many knowninteractors, validating the approach. Hypoxic conditions led to a striking alteration in proximal interactions and many metabolic proteins were highly enriched with AR upon exposure of cells to hypoxia for 24h. Among these, phosphoglycerate kinase 1 (PGK1) was a top hit. PGK1 has been reported to interact with AR to mediate the expression of genes that regulates cell proliferation and apoptosis. Additionally, PGK1 has an essential role in metabolic reprogramming induced by c-MYC and HIF-1α, leading to enhanced tumorigenesis. Conclusion: Bio-ID mass spectrometry was used to map the proximal interactome of AR and its variants, identifying novel interactions partners for further analysis. We found that hypoxia strongly alters interactors with the AR, and we identified PGK1 as a hypoxia induced AR interaction in PCA. Current work to functionally validate this interaction will be presented. Citation Format: Oloruntoba I. Osagie, Jordann Smakk, Deborah E. Citrin, Travis H. Stracker. Identification of critical hypoxia induced factors in castrate resistant prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4802.

Second generation androgen receptor antagonist, TQB3720 abrogates prostate cancer growth via AR/GPX4 axis activated ferroptosis.

Purpose: Prostate cancer (PCa) poses a great threat to humans. The study aimed to evaluate the potential of TQB3720 in promoting ferroptosis to suppress prostate cancer, providing a theoretical basis for PCa therapy. Methods: PCa cells and nude mice models were divided into TQB3720, enzalutamide (ENZ), and control groups. Sulforhodamine B assay, colony formation assessment, organoids culture system, and the CCK8 assay were used for detecting proliferation. Western blot assay was processed to detect the expression of androgen receptor (AR), ferroptosis, and apoptosis-related genes. Flow cytometry was applied to measure the intracellular ROS levels. ELISA was performed to determine the cellular oxidized glutathione (GSSG) and malondialdehyde (MDA) levels. RT-qPCR was conducted to detect the mRNA expression of genes in AR signaling. BODIPYTM™ 581/591 was processed for detection of intracellular lipid peroxidation levels. The interaction of AR with other translational factor complex proteins was explored using Co-immunoprecipitation (Co-IP), and the chromatin immunoprecipitation (ChIP) assay was performed to detect the binding of AR-involved translational complex to downstream genes promoter. Luciferase reporter assay was conducted to examine the translation activity of GPX4 promoter, and immunohistochemistry (IHC) was conducted to analyze the levels of c-MYC, Ki-67 and AR in TQB3720-treated cancer tissues. Results: Here, we found TQB3720 inhibits the growth of prostate cancer in vitro and in vivo. TQB3720 treatment induced intracellular levels of GSSG and MDA significantly, by which hints AR antagonist caused ferroptosis-related cell death. Moreover, molecular evidence shown TQB3720 regulates downstream of AR signaling by binding AR resulting in inhibition of AR entry into the nucleus. Additional, we also proved that TQB3720 abrogates the interaction between AR and SP1 and leads to decrease GPX4 transcription. Conclusion: TQB3720 promotes ferroptosis in prostate cancer cells by reducing the AR/SP1 transcriptional complex binding to GPX4 promoter. As a result, it is suggested to be a potential drug for clinic prostate cancer treatment.

Testis-specific protein Y-encoded 1 regulates androgen receptor expression through the MAPK/ERK pathway in male hepatocellular carcinoma.

To analyze the mechanism of testis-specific protein Y-encoded 1 (TSPY1) in male hepatocellular carcinoma (HCC). This experimental study was carried out at Guangxi Medical University's First Affiliated Hospital, Guangxi, China, between January 2016 and December 2019. The expression of TSPY1, androgen receptor (AR), messenger ribonucleic acids (mRNAs), and proteins were detected by qRT-PCR and Western blotting. The co-localization and interaction of TSPY1 and AR were observed by immunofluorescence assay and co-immunoprecipitation. Hepatocellular carcinoma cells overexpressing and silencing TSPY1 were constructed, and the expression and phosphorylation levels of TSPY1, AR, and mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) signaling pathway-related key molecules ERK1/2, p38, and JNK were also detected. The expression levels of TSPY1, AR mRNAs, and proteins were highly positively correlated in HCC cells in different metastatic potentials with a high correlation coefficient of R=0.929 and R=0.884. Testis-specific protein Y-encoded 1 and AR were then co-localized in the nucleus of HCC cells, and TSPY1 and AR can interact with each other. In addition, the expression of AR and phosphorylation of ERK1/2 were enhanced in TSPY1 overexpressed Huh7 cells. They were reduced in HCCLM3 cells with TSPY1 knockdown expression. In addition, in response to blocking MAPK/ERK signaling activity, AR was reduced in expression. These findings suggested that there was a positive correlation between TSPY1 expression and AR in male HCC cells, and high TSPY1 expression stimulates AR expression, MAPK/ERK signaling pathway may be involved in its mechanism.

Identification of ELK1 interacting peptide segments in the androgen receptor.

Prostate cancer (PCa) growth requires tethering of the androgen receptor (AR) to chromatin by the ETS domain transcription factor ELK1 to coactivate critical cell proliferation genes. Disruption of the ELK1-AR complex is a validated potential means of therapeutic intervention in PCa. AR associates with ELK1 by coopting its two ERK docking sites, through the amino-terminal domain (A/B domain) of AR. Using a mammalian two-hybrid assay, we have now functionally mapped amino acids within the peptide segments 358-457 and 514-557 in the A/B domain as required for association with ELK1. The mapping data were validated by GST (glutathione S-transferase)-pulldown and BRET (bioluminescence resonance energy transfer) assays. Comparison of the relative contributions of the interacting motifs/segments in ELK1 and AR to coactivation of ELK1 by AR suggested a parallel mode of binding of AR and ELK1 polypeptides. Growth of PCa cells was partially inhibited by deletion of the upstream segment in AR and nearly fully inhibited by deletion of the downstream segment. Our studies have identified two peptide segments in AR that mediate the functional association of AR with its two docking sites in ELK1. Identification of the ELK1 recognition sites in AR should enable further structural studies of the ELK1-AR interaction and rational design of small molecule drugs to disrupt this interaction.

1456-P: Molecular Architecture of AR Action in Insulin Secretion in Male Pancreatic ß-Cells

Male mice lacking the androgen receptor (AR) in islet β cells exhibit blunted glucose-stimulated insulin secretion (GSIS) , leading to hypoinsulinemia and hyperglycemia. We showed that dihydrotestosterone (DHT) activation of an extranuclear AR in β cells potentiates GSIS by amplifying the insulinotropic action of GLP-1 in mouse and human islets. To gain information on DHT-stimulated signaling networks promoting GSIS, we combined functional proteomics with highly selective pharmacological inhibitors in male human islets, and islets from genetic mouse models. We found that DHT-stimulated GSIS in human islets involved a signaling cascade including focal adhesion kinases, the nonreceptor tyrosine kinase SRC, phosphoinositide 3-kinase, and the activation of mTOR complex 2. To define the early network of AR interacting proteins, we performed DHT-stimulated AR immunoprecipitation in INS1 832/3 cells followed by mass spectrometry-based proteomics. We will discuss the AR interactome including: 1) adenylate cyclase-modulating G proteins Gαs and Gαi, cAMP-dependent PKA, and the cAMP-dependent protein LASP1 regulating actin-based cytoskeletal activities; 2) Actin cytoskeleton remodeling proteins promoting actin depolymerization, and 3) Vesicular maturation including the lysosomal vacuolar-type ATPase acidifying insulin secretory granules and the prohormone convertase 2 involved in proinsulin processing. Notably, GLP-1 addition to DHT decreased AR interaction with all these proteins and promoted AR interaction with microtubule motor proteins promoting vesicular transport, proteins involved in exocytic vesicle docking to the plasma membrane, as well as small GTPase activating proteins and guanine nucleotide exchange factors involved in actin remodeling, as well as vesicle trafficking. Together these experiments provide novel insights into the architecture of AR action in enhancing GSIS. Disclosure P.Mota de sa: None. M.Qadir: None. M.Blandino-rosano: None. J.Fan: None. W.Xu: None. E.Bernal-mizrachi: None. F.Mauvais-jarvis: Consultant; Mithra Pharmaceutical. Funding National Institutes of Health (DK107444 and DK074970) U.S. Department of Veterans Affairs Merit Award (BX003725)

The effect of PARP inhibition on androgen receptor localization and activity in castration resistant prostate cancer.

e17037 Background: The poly(ADP-ribose) polymerase inhibitors (PARPi) olaparib and rucaparib have been approved for the treatment of metastatic castration resistant prostate cancer (mCRPC) in the setting of homologous recombination deficiency (HRD). Additionally, PARPi have been shown to modulate androgen receptor (AR) signaling, with a recent report demonstrating mono-ADP-ribosylation of cysteine residues in AR by Parp7. Here, we further evaluated the effect of PARPi on AR activity and localization. Methods: The effect of PARPi on cell growth and survival of CRPC cell lines was evaluated in vitro and in vivo. AR ribosylation was assessed in CRPC cell lines by immunoprecipitation (IP) assays and proximity ligation assays (PLA). The subcellular localization of AR was determined by quantitative immunofluorescence microscopy in CRPC cell lines and xenograft models. Changes in AR activity with PARPi treatment were evaluated by a luciferase reporter assay and AR target gene expression in a PDX model. Finally, PARPi mediated alteration in the AR protein interactome was evaluated by liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics. Results: The PARPi olaparib and talazoparib, and to a lesser extent veliparib, inhibited CRPC cell growth. Evidence of AR ribosylation was seen by IP and PLA. PARPi treatment of multiple in vitro and in vivo prostate cancer models resulted in a shift of AR subcellular localization, from predominantly nuclear to cytoplasmic compartments. In luciferase reporter assays, AR transactivation activity was decreased after PARPi treatment in a dose dependent manner. In vivo, in prostate cancer xenograft models, decreased AR target gene expression was seen upon PARPi treatment. LC-MS/MS proteomic studies revealed that PARP inhibition resulted in significant changes in the composition of AR interaction partners, in particular of proteins related to intracellular trafficking and nuclear transport. This suggests a potential link between altered AR complex assembly and the observed changes in AR subcellular localization in the context of PARPi treatment. Conclusions: We describe a novel sequela of PARPi therapy to alter AR localization and activity in CRPC. Single agent PARP inhibition can alter prostate cancer cell growth in vitro and in vivo. With PARPi treatment, AR localization is shifted to the cytoplasm, the AR interactome is altered, and AR transcriptional activity decreased. These findings implicate a collateral mechanism of PARPi in preventing prostate cancer cell growth/survival that may augment previously described mechanisms related to HDR and synthetic lethality. It is essential to understand the role of PARPi beyond synthetic lethality in the context of HRD in order to better define the spectrum of response to PARPi across patients, and for development of biology-informed combination therapies.

Antitumor mechanism of combination of Angelica gigas and Torilis japonica in LNCaP prostate cancer cells via G1 arrest and inhibition of Wnt/β-catenin and androgen receptor signaling.

The goal of the current study is to assess the antitumor mechanism by the combination (7:3) of Angelica gigas and Torilis japonica (AT) that was found most effective through screening against prostate-specific antigen (PSA) in LNCaP prostate cancer cells. Here, AT reduced the viability and the number of colonies in androgen-dependent LNCaP cells more than in androgen independent PC3 and DU145 cells. Also, AT induced G1 phase arrest, cleaved PARP and caspase 3, activated p27 and decreased the expression of Cyclin D1, Cyclin E, cdk2 in LNCaP cells. Furthermore, AT decreased the expression of PSA and androgen receptor (AR) at mRNA and protein levels in LNCaP cells. Interestingly, AT attenuated the expression of AR, PSA and Wnt-3a and the stability of AR and PSA in LNCaP cells. Furthermore, AT reversed dihydrotestosterone (DHT)-induced upregulation of AR and PSA in LnCaP cells. Notably, AT disrupted the protein-protein interaction, nuclear translocation and fluorescent expression of β-catenin and AR in LNCaP cells. Consistently, β-catenin depletion enhanced the decreased expression of AR in AT treated LNCaP cells. Taken together, our findings highlight evidence that AT suppresses the proliferation of LNCaP cells via G1 arrest and inhibition of β-catenin and AR as a potential anticancer agent.

Structural studies of an androgen receptor complex reveal modes of allosteric regulation

The androgen receptor (AR) is the master transcription factor governing gene expression programs required for prostate epithelium development and male phenotype maintenance. AR misregulation is a hallmark of prostate cancer where AR hyperactivation and transcriptome expansion occur in part through AR amplification and interaction with oncoprotein cofactors. Despite its biological importance, how AR’s domains and cofactors cooperate to bind DNA have remained elusive. We isolated three conformations of AR bound to DNA showing AR forms a non‐obligate dimer, with the buried dimer interface utilized by ancestral steroid receptors repurposed to facilitate cooperative DNA binding. We identify novel allosteric surfaces which are compromised in androgen insensitivity syndrome, and reinforced by AR’s oncoprotein cofactor, ERG, and DNA binding motifs. Finally, we present evidence this plastic dimer interface was adopted for transactivation at the expense of DNA binding. Our work highlights how fine‐tuning AR’s cooperative interactions translate to consequences in development and disease.

Allosteric interactions prime androgen receptor dimerization and activation

The androgen receptor (AR) is a nuclear receptor that governs gene expression programs required for prostate development and male phenotype maintenance. Advanced prostate cancers display AR hyperactivation and transcriptome expansion, in part, through AR amplification and interaction with oncoprotein cofactors. Despite its biological importance, how AR domains and cofactors cooperate to bind DNA has remained elusive. Using single-particle cryo-electron microscopy, we isolated three conformations of AR bound to DNA, showing that AR forms a non-obligate dimer, with the buried dimer interface utilized by ancestral steroid receptors repurposed to facilitate cooperative DNA binding. We identify novel allosteric surfaces which are compromised in androgen insensitivity syndrome and reinforced by AR's oncoprotein cofactor, ERG, and by DNA-binding motifs. Finally, we present evidence that this plastic dimer interface may have been adopted for transactivation at the expense of DNA binding. Our work highlights how fine-tuning AR's cooperative interactions translate to consequences in development and disease.

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.