Androgen Receptor Coregulators Research Articles

Abstract 6048: Targeting AR positive prostate cancer cells by the novel P300/CBP oral available PROTAC degrader

Abstract Background The Androgen Receptor (AR) serves as the core lineage-specific transcription factor for prostatic epithelial cells. Targeting AR is currently the most promising therapeutic strategy. However, the inevitable development of acquired resistance to anti-androgen treatments underscores the urgent need to devise new therapeutic strategies. Recent studies have revealed that in prostate cancer cells, AR is hijacked by oncogenic transcription factors, such as FOXA1 and HOXB13, which promiscuously bind to distinct enhancer sites, driving the progression of prostate cancer. This process leads to the formation of a substantially rewired AR cancer specific enhanceosome, requiring a variety of coregulators to exhibit oncogenic driver characteristics, which can be potential therapeutic targets for prostate cancer. Histone acetyltransferases (HATs) P300 and CBP are paralogs which play dominant roles in orchestrating high-order chromatin structures and transcription factor complexes by acetylating histone proteins at multiple residuals. Although accumulating evidence suggests that P300/CBP function as AR coregulator and targeting P300/CBP can markedly suppress AR-positive prostate cancer cell growth, including growth of castration-resistant prostate cancer (CRPC), most of the P300/CBP inhibitors have performed poorly in clinical trials. Methods We developed a novel P300/CBP dual oral available PROTAC degrader, namely CBPD-409. The on-target effects of CBPD-409 were assessed by quantitative proteomics (TMT-mass spectrometry) in prostate cancer cell lines. The RNA-seq analysis was conducted to investigate CBPD-409 effects on global transcriptome in prostate cancer cells. The cytotoxicity of CBPD-409 in multiple prostate cancer cell lines was examined by cell-titer-glo and incucyte assays. Conclusion In multiple cell lines, CBPD-409 exhibits remarked potency to degrade both P300 and CBP proteins in time and does dependent manner, without evident off-target effects. By employing CBPD-409, we demonstrate that degrading P300/CBP preferentially suppress AR+ prostate cancer cell growth. The global transcriptomic profiling in CBPD-409 treated prostate cancer cells revealed that loss of P300/CBP results in remarkedly downregulation of AR, Myc, FOXA1 and ERG signaling. Assessment of CBPD-409 cytotoxicity in a panel prostate cancer cell lines suggests that AR positive prostate cancer cells display highest sensitivity to CBPD-409. Citation Format: Jie luo, Abhijit Prolia, Yuanyuan Qiao, Jean Tian, Rahul Mannan, Somnath Mahapatra, Zhixiang Chen, Rithvik Seri, Shaomeng Wang, Arul Chinnaiyan. Targeting AR positive prostate cancer cells by the novel P300/CBP oral available PROTAC degrader [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6048.

Abstract 6603: A TBX2-driven signaling switch from androgen receptor to glucocorticoid receptor confers enzalutamide resistance in prostate cancer

Abstract Background: Recent studies suggest that glucocorticoid receptor (GR) activation can cause enzalutamide resistance in advanced prostate cancer (PCa) via functional bypass of androgen receptor (AR) signaling. However, the specific molecular mechanism(s) driving this process remain unknown. In an effort to identify drivers of prostate cancer progression, in a previous study, we determined that TBX2, a developmental T-box transcription factor (TF) master regulator, is over-expressed in CRPC and drives bone metastatic progression. A recent report confirmed that TBX2 and GR are two of the four TFs that drive enzalutamide resistance in advanced PCa. Our current study demonstrates that TBX2 with known repressor and activator functions, is the molecular switch that represses AR levels while activating GR expression thereby resulting in the replacement of AR signaling to control tumor growth. Methods: We genetically modulated TBX2 using multiple approaches: a) dominant negative, DN, to block TBX2 (TBX2DN), and b) overexpression, OE, to increase TBX2 expression (TBX2OE), c) shRNA mediated knockdown (shTBX2). RNA-seq was performed, and qRT-PCR, Western blot and immunohistochemical (IHC) analyses were used for validation. Further, we used chromatin immunoprecipitation (ChIP) and site directed mutagenesis (SDM) to confirm TBX2 binding on the AR promoter. We also used co-immunoprecipitation (Co-IP) to determine protein partners of TBX2. Results: Mechanistically, our studies revealed that TBX2 bound to the promoters of both AR and GATA2, an AR coregulator, thereby resulting in a bimodal repression of AR expression. Conversely, TBX2 upregulated GR via direct GR promoter binding and TBX2-GR protein-protein interaction. Together, concurrent repression of the AR and activation of GR resulted in enzalutamide resistance. Importantly, we found that SP2509, an allosteric inhibitor of the demethylase-independent function of LSD1, a TBX2-interacting protein in the COREST complex, can disrupt both TBX2-LSD1 and TBX2-GR protein-protein interactions thereby uncovering a unique mode of SP2509 action in CRPC. Together, our study supports a molecular model of CRPC wherein: 1) TBX2, LSD1 and GR proteins interface with each other, and 2) pharmacological inhibition of LSD1 blocks the TBX2-driven AR-to-GR switch through disruption of TBX2-GR interaction. Conclusions: In summary, our study identifies TBX2 as the molecular switch that drives the AR to GR signaling bypass thereby conferring enzalutamide resistance. Further, our study provides key insights into a potential therapeutic modality for targeting the AR to GR signaling switch via disruption of the TBX2-LSD1 and TBX2-GR protein-protein interactions Citation Format: Sayanika Dutta, Girijesh Kumar Patel, Hamed Khedmatgozar, Daniel Latour, Jonathan Welsh, Manisha Tripathi, Srinivas Nandana. A TBX2-driven signaling switch from androgen receptor to glucocorticoid receptor confers enzalutamide resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6603.

Abstract B011: PRAC1 epigenetic silencing in castration resistant prostate cancer and its novel role in androgen receptor biology

Abstract Background Resistance to androgen receptor (AR) directed therapies is one of the key drivers of prostate cancer mortality. Although several molecular alterations involved in castration resistant prostate cancer (CRPC) have been catalogued, a deep mechanistic understanding of the diverse resistance pathways operative in CRPC is still lacking. Here, we describe the role of a previously uncharacterized gene, PRAC1 in treatment resistance and the mechanism that involves epigenetic silencing of the AR co-regulator PRAC1. Methods We applied a reductionist approach by performing whole genome methylation analyses using MBD-seq on paired androgen dependent and castration resistant cell lines to identify candidate gene loci with differential methylation in CRPC. Results from this screen were validated in CRPC cohorts. In vitro and in vivo loss and gain of function experiments were used to delineate molecular interactions, phenotypic and transcriptomic alterations. Results We demonstrate that the PRAC1 gene locus, which encodes for a 6 kDa peptide, frequently undergoes CpG methylation mediated epigenetic silencing. Loss of PRAC1 expression was tightly associated with resistance to AR directed therapies and was present in ~30% of advanced CRPC but not in hormone naïve tumors. Furthermore, tumors with PRAC1 loss showed a significantly shorter time to progression on AR signaling inhibitors (ARSI). Mechanistically, PRAC1 loss allowed for prostate cancer cell growth in the absence of androgens or presence of ARSIs, suggesting that PRAC1 is a key mediator of resistance. Importantly, PRAC1 binds to the AR and was necessary for efficient AR transactivation and using biochemical methods, we showed that these two molecules interact at the molecular level. Additionally, PRAC1 loss dampened canonical AR signaling but induced an AR dependent pro-proliferative expression program. Restoring PRAC1 expression in ARSI resistant models re-established sensitivity to AR directed therapies. Conclusion Our data suggests that PRAC1 is a novel AR co-regulator that undergoes epigenetic silencing in CRPC. Loss of PRAC1confers resistance to AR directed therapies. Restoring PRAC1 function in CRPC could represent a novel therapeutic approach in advanced CRPC. Citation Format: Jin-Yih Low, David M. Esopi, Yiting Lim, Ajay M. Vaghasia, Harrison Tsai, Qizhi Zheng, Jessica Hicks, Nicolas Wyhs, Tamara L. Lotan, William B. Isaacs, Angelo M. DeMarzo, William G. Nelson, Andrew C. Hsieh, Srinivasan Yegnasubramanian, Michael C. Haffner. PRAC1 epigenetic silencing in castration resistant prostate cancer and its novel role in androgen receptor biology [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B011.

The impact of circulating tumor cell HOXB13 RNA detection in men with metastatic castrate-resistant prostate cancer (mCRPC) treated with abiraterone or enzalutamide.

5060 Background: It has been shown that HOXB13 is an important androgen receptor (AR) coregulator. The primary objective of this analysis was to correlate HOXB13 expression with clinical outcomes in men with mCRPC. Methods: We conducted a retrospective analysis of the multicenter prospective PROPHECY trial of mCRPC men (n=118) treated abiraterone (abi) or enzalutamide (enza). CTC detection and specific HOXB13 cDNA detection and expression was measured using a modified Adnatest using appropriate controls. CTC HOXB13 status was categorized into 3 groups: undetectable CTCs (CTC=0 based on CTC PSMA & PSA=0); CTC+ but HOXB13 CTC negative (<4 copies) or positive (≥4 copies) where the HOXB13 cutoff point was determined by the maximally selected rank statistic for prognostic associations. The association between HOXB13 and overall survival (OS) and radiographic progression-free survival (rPFS) were explored using the proportional hazards model. We also explored the association between the two endpoints and multigroup of HOXB13 at cutoff 0 and CellSearch CTC at cutoff 1 and 5. Results: 102 men who had sufficient CTC cDNA sample for HOXB13 detection. Of those men with detectable CTCs, 19% (n=19) had no detectable HOXB13, while the majority of men at 67% (n=58) had both CTC and HOXB13 detection. The proportion of patients with CTC=0, CTC+ HOXB13-, and CTC+ HOXB13+ using the optimal HOXB13 threshold were 25%, 31% and 44%, respectively. Men with HOXB13- CTCs were more likely to have low PSA (median 3 vs 79 copies), low CTC PSMA (0 vs 99 copies), low AR-FL (47 vs. 89 copies) and CTC AR-V7 (5.9 vs. 42 copies), and more likely to have liver/lung metastases (41% vs 25%). Median OS were 25.7, 25 and 12.1 months while the median rPFS of the three groups were 9.0, 7.2 and 4.7 months, respectively. Compared to CTC=0, men with HOXB13- CTCs did not have a worse hazard rate for OS (HR: 0.9, 95% CI: 0.4-1.9), while HOXB13+ CTC patients had a worse hazard for OS (HR: 2.0, 95% CI: 1.0-3.9), adjusting for prior abi/enza treatment and Halabi clinical risk score. The positive HOXB13 had also statistically significant increased hazard for progression/death (HR: 2.31, 95% CI: 1.24-4.30). Compared to the group with both HOXB13=0 and CellSearch CTC=0, men with HOXB13>0 and CellSearch CTC≥5 had increased hazard on both OS and rPFS (OS: HR:2.39, 95% CI: 1.06-5.4; rPFS: HR: 2.78, 95% CI: 1.38-5.59). Conclusions: Higher CTC HOXB13 expression is associated with AR dependent biomarkers in CTCs and is adversely prognostic in the context of potent AR inhibition in men with mCRPC, and this assay could be useful to select for patients appropriate for AR or future HOXB13 targeted therapies. Clinical trial information: NCT02269982 .

Abstract 3083: Targeting androgen receptor coregulators to enhance enzalutamide sensitivity in prostate cancer

Abstract Steroid hormone signaling axes drive proliferation and survival in prostate cancer (PCa), breast and other cancers. In late stage PCa, the androgen receptor (AR) is therapeutically antagonized with success, for example with Enzalutamide (Enza). Although initially successful, resistance emerges associated with tissue plasticity and patient mortality. Enza-resistance mechanisms include altered AR coregulator interactions. Identifying which coregulators change Enza sensitivity is attractive as they may represent new drug targets but challenging given the multitude of coregulators expressed in PCa and the diverse roles they play in transcription and translation.To meet this challenge, we designed a bioinformatic workflow to identify under-investigated yet clinically-impactful coregulators. We tested coactivators (CoA, n~750), corepressors (CoR, n ~600), and mixed function coregulators (Mixed, n~600) associations with patient outcome in TCGA PCa cohorts; applied partial correlation analyses to reveal coregulators that impacted AR target genes; utilized DepMap essentiality scores to rank importance; and examined interactions in publicly available AR RIME data. The impact of the top ranked coregulators (n=18) on Enza sensitivity was examined using shRNA or siRNA approaches, and with inhibitor drugs in Enza-resistant PCa cell lines (LNCaP C42B, 22Rv1). Finally, coregulator expression was examined in single cell RNA-Seq from PCa 3D organoids derived from the PBCre4:Ptenf/f:Rb1f/f (DKO) and the PBCre4:Ptenf/f:Rb1f/f:Trp53f/f (TKO) PCa mouse model, which recapitulate the loss of Enza sensitivity and lineage plasticity. ShRNA targeting coregulators was undertaken in organoids. The bioinformatics workflow identified 18 coregulators, including CoA (e.g., WDR5, KAT7, BLM, SMARCC1), CoR (e.g., SETDB1, PSPC1), and Mixed (e.g., KDM5A, BPTF, TCEGR1) to screen for impact on Enza. KAT7 shRNA modulated Enza sensitivity, regulated expression of AR target genes, and synergized with a KAT7 inhibitor (WM3835) to enhance Enza sensitivity. Similarly, a BLM inhibitor (ML216) synergized with Enza. Interestingly, although WDR5 siRNA alone did not alter Enza responses, the combination of KAT7 shRNA and WDR5 siRNA significantly potentiated Enza responses. Similarly, SMARCC1 shRNA cooperated with siRNA to several targets (TBX3, BPTF, or SETDB1) to enhance Enza responses. Smarcc1 and Tbx3 were upregulated in Enza-resistant neuroendocrine PCa, and the recently identified Pou2f3 Enza-resistant phenotypes, and Bptf was upregulated in the Tff3 Enza-resistant phenotype. We have shRNA targeted Blm, Smarcc1, Tbx3, and Bptf in the DKO and TKO organoids and are measuring the impact of Enza responses. Overall, we have identified potentially novel AR coregulators that associate with the development of therapy resistance and shown that targeting these coregulators may augment Enza sensitivity in PCa. Citation Format: Rayzel C. Fernandes, Debasis Nayak, Damien A. Leach, Meenalakshmi Chinnam, Wilbert Zwart, David W. Goodrich, Moray J. Campbell, Charlotte L. Bevan. Targeting androgen receptor coregulators to enhance enzalutamide sensitivity in 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 3083.

LSD1/PRMT6-targeting gene therapy to attenuate androgen receptor toxic gain-of-function ameliorates spinobulbar muscular atrophy phenotypes in flies and mice

Spinobulbar muscular atrophy (SBMA) is caused by CAG expansions in the androgen receptor gene. Androgen binding to polyQ-expanded androgen receptor triggers SBMA through a combination of toxic gain-of-function and loss-of-function mechanisms. Leveraging cell lines, mice, and patient-derived specimens, we show that androgen receptor co-regulators lysine-specific demethylase 1 (LSD1) and protein arginine methyltransferase 6 (PRMT6) are overexpressed in an androgen-dependent manner specifically in the skeletal muscle of SBMA patients and mice. LSD1 and PRMT6 cooperatively and synergistically transactivate androgen receptor, and their effect is enhanced by expanded polyQ. Pharmacological and genetic silencing of LSD1 and PRMT6 attenuates polyQ-expanded androgen receptor transactivation in SBMA cells and suppresses toxicity in SBMA flies, and a preclinical approach based on miRNA-mediated silencing of LSD1 and PRMT6 attenuates disease manifestations in SBMA mice. These observations suggest that targeting overexpressed co-regulators can attenuate androgen receptor toxic gain-of-function without exacerbating loss-of-function, highlighting a potential therapeutic strategy for patients with SBMA.

N/C Interactions Are Dispensable for Normal In Vivo Functioning of the Androgen Receptor in Male Mice.

The androgen receptor (AR) plays a central role in the development and maintenance of the male phenotype. The binding of androgens to the receptor induces interactions between the carboxyterminal ligand-binding domain and the highly conserved 23FQNLF27 motif in the aminoterminal domain. The role of these so-called N/C interactions in AR functioning is debated. In vitro assays show that mutating the AR in the 23FQNLF27 motif (called ARNoC) attenuates the AR transactivation of reporter genes, has no effect on ligand binding, but does affect protein-protein interactions with several AR coregulators. To test the in vivo relevance of the N/C interaction, we analyzed the consequences of the genomic introduction of the ARNoC mutation in mice. Surprisingly, the ARNoC/Y mice show a normal male development, with unaffected male anogenital distance and normal accessory sex glands, male circulating androgen levels, body composition, and fertility. The responsiveness of androgen target genes in kidney, prostate, and testes was also unaffected. We thus conclude that the N/C interactions in the AR are not essential for the development of a male phenotype under normal physiological conditions.

TRIM33 drives prostate tumor growth by stabilizing androgen receptor from Skp2-mediated degradation.

Androgen receptor (AR) is a master transcription factor that drives prostate cancer (PCa) development and progression. Alterations in the expression or activity of AR coregulators significantly impact the outcome of the disease. Using a proteomics approach, we identified the tripartite motif-containing 33 (TRIM33) as a novel transcriptional coactivator of AR. We demonstrate that TRIM33 facilitates AR chromatin binding to directly regulate a transcription program that promotes PCa progression. TRIM33 further stabilizes AR by protecting it from Skp2-mediated ubiquitination and proteasomal degradation. We also show that TRIM33 is essential for PCa tumor growth by avoiding cell-cycle arrest and apoptosis, and TRIM33 knockdown sensitizes PCa cells to AR antagonists. In clinical analyses, we find TRIM33 upregulated in multiple PCa patient cohorts. Finally, we uncover an AR-TRIM33-coactivated gene signature highly expressed in PCa tumors and predict disease recurrence. Overall, our results reveal that TRIM33 is an oncogenic AR coactivator in PCa and a potential therapeutic target for PCa treatment.

Abstract 1483: RNA interference seed-based approach to inhibit androgen signaling and induce prostate cancer cell death

Abstract The goal of this study is to identify RNA interference (RNAi) seed sequences that inhibit androgen signaling and induce toxicity in prostate cancer (PCa) cells. The high prevalence of acquired resistance to androgen deprivation therapy, as well as first and second generation androgen receptor (AR) antagonists, drives the development of lethal castration resistant prostate cancer (CRPC). As a result, PCa remains one of the leading causes of cancer related deaths in men. AR amplification, gain of function mutations and constitutively active splice isoforms of the AR, and the overexpression of AR coregulators, play considerable roles in promoting CRPC by sustaining AR activity in the presence of the low androgen environment and/or AR antagonists. The development of specific small molecule inhibitors of AR splice isoforms has been largely unsuccessful, and inhibitors of individual AR coregulators have not translated well into the clinic, likely due to their large number and functional redundancies. Therefore, novel therapeutic strategies for targeting androgen signaling in CRPC are necessary. Previous studies in our lab have demonstrated RNAi-mediated PCa cell death through seed region (nucleotides 2-8) complementarity of shRNA/siRNA guide strands to the 3’UTR of the AR and multiple AR coregulators and essential genes. In the current study, we used an unbiased novel seed-based shRNA screen to identify seed sequences that are toxic to different PCa cells, including CRPC cells expressing the clinically relevant constitutively active AR variant 7 (AR-V7). Common motifs were identified among toxic seed sequences, and the miRDB target prediction database was used to select those seeds that were predicted to target the AR and AR coregulators. Using RNA-seq analyses we confirmed that selected seed sequences inhibited global androgen signaling and identified potential direct targets known to be relevant to PCa. Conclusions: These studies have identified androgen signaling inhibitory sequences that have the potential to be used in RNAi-seed based therapies for PCa. The ability to inhibit the expression of the AR and multiple essential AR coregulators simultaneously with one agent, and the reduced likelihood of developed resistance, are considerable benefits of this approach. Citation Format: Joshua M. Corbin, Maria J. Ruiz-Echevarria. RNA interference seed-based approach to inhibit androgen signaling and induce prostate cancer cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1483.

MiR-27 and miR-124 target AR coregulators in prostate cancer: Bioinformatics and in vitro analysis.

The inadequate efficacy of the current treatments for metastatic prostate cancer has directed efforts to the discovery of novel therapies. MicroRNAs (miRNAs) have been considered potential therapeutic agents due to their ability to control gene expression and cellular pathways. The accurate identification of genes and pathways which are targeted by a miRNA is the first step in the therapeutic use of these molecules. In this regard, there are multiple experimental and computational methods to predict and confirm the miRNA-mRNA relationships. The targeting the androgen receptor (AR) indirectly as the most important mediator of prostate cancer has been posited to both control the disease and prevent resistance to treatment. This study aimed to identify miRNAs targeting AR coregulators. For this purpose, we examined target genes by combining miRNA-mRNA computational and experimental data from various databases. miR-27a-3p and miR-124 displayed the highest scores and were selected as miRNAs with the potential to target candidate genes. Next, three cell lines of prostate cancer including PC3, LNCAP, and DU145 were transfected with plasmids which were expressed these selected miRNAs. Then, the gene expression and cell cycle analysis were performed. A decrease was observed in cell viability in all three cell lines than the cells transfected with backbone plasmid. Furthermore, the findings indicated that miR-27a-3p and miR-124 led to a significant decrease in the expression of all genes that were studied in PC3 cell line. In addition, miR-124 caused significant the cellular arrest in the G0/G1 stage, while for miR-27a-3p, this arrest occurred was in the G2/M stage. Our results indicated that the function of a unique miRNA could be different in different cell lines with particular cancer phenotype based on the cell line stage. These findings offer the possibility of employing the miR-124 and miR-27a-3p as therapeutic agents for prostate cancer treatment.

Epigenetic Coregulation of Androgen Receptor Signaling.

The androgen receptor (AR) is a ligand-activated transcription factor belonging to the nuclear receptor (NR) superfamily. As with other members of the NR family, transcriptional activity of the AR is regulated by interactions with coregulatory proteins, which either enhance (coactivators) or repress (corepressors) its transcriptional activity. AR associated coregulators are functionally diverse, but a large fraction are epigenetic histone and chromatin modifiers. Epigenetic coregulators are recruited to gene regulatory regions as part of multi-protein complexes, often acting in a dynamic and inter-dependent manner to remodel chromatin, thereby allowing or inhibiting the access of AR-associated transcriptional machinery to target genes; functional consequences being regulation of transcriptional output. Epigenetic modifiers, including those that function as AR coregulators, are frequently mutated or aberrantly expressed in prostate cancer and are implicated in disease progression. Some of these modifiers are being investigated as therapeutic targets in several cancer types and could potentially be used to modulate aberrant AR activity in prostate cancer. In this chapter we will summarise the functional role of epigenetic coregulators in AR signalling, their dysregulation during prostate cancer progression and the current status of drugs targeting these enzymes.

PIM1 phosphorylation of the androgen receptor and 14-3-3 \u03b6 regulates gene transcription in prostate cancer

PIM1 is a serine/threonine kinase over-expressed in prostate cancer. We have previously shown that PIM1 phosphorylates the androgen receptor (AR), the primary therapeutic target in prostate cancer, at serine 213 (pS213), which alters expression of select AR target genes. Therefore, we sought to investigate the mechanism whereby PIM1 phosphorylation of AR alters its transcriptional activity. We previously identified the AR co-activator, 14-3-3 ζ, as an endogenous PIM1 substrate in LNCaP cells. Here, we show that PIM1 phosphorylation of AR and 14-3-3 ζ coordinates their interaction, and that they extensively occupy the same sites on chromatin in an AR-dependent manner. Their occupancy at a number of genes involved in cell migration and invasion results in a PIM1-dependent increase in the expression of these genes. We also use rapid immunoprecipitation and mass spectrometry of endogenous proteins on chromatin (RIME), to find that select AR co-regulators, such as hnRNPK and TRIM28, interact with both AR and 14-3-3 ζ in PIM1 over-expressing cells. We conclude that PIM1 phosphorylation of AR and 14-3-3 ζ coordinates their interaction, which in turn recruits additional co-regulatory proteins to alter AR transcriptional activity.

Androgen receptor coregulator long noncoding RNA CTBP1-AS is associated with polycystic ovary syndrome in Kashmiri women.

Polycystic ovary syndrome (PCOS) is one of the most common reproductive, endocrine, and metabolic disorder in premenopausal women. Even though the pathophysiology of PCOS is complex and obscure, the disorder is prominently considered as the syndrome of hyperandrogenism. C-Terminal binding protein 1 antisense (CTBP1-AS) acts as a novel androgen receptor regulating long noncoding RNA (lncRNA). Therefore, the present study was aimed to establish the possible association of androgen receptor regulating long noncoding RNA CTBP1-AS with PCOS. A total of 178 subjects including 105 PCOS cases and 73 age-matched healthy controls were recruited for the study. The anthropometric, hormonal, and biochemical parameters of all subjects were analyzed. Total RNA was isolated from peripheral venous blood and expression analysis was done by quantitative real-time PCR. The correlation analysis was performed to evaluate the association between and various clinical parameters and lncRNA CTBP1-AS expression. The mean expression level of CTBP1-AS was found to be significantly higher in the PCOS women than in the healthy controls (-lnCTBP1-AS, 4.23 ± 1.68 versus 1.24 ± 0.29, P < 0.001). Furthermore, subjects with higher expression level of CTBP1-AS had significantly higher risk of PCOS compared to subjects with low levels of CTBP1-AS expression (actual OR = 11.36, 95% CI = 5.59-23.08, P < 0.001). The area under receiver operator characteristic (ROC) curve was 0.987 (SE 0.006, 95% CI 0.976-0.99). However, lncRNA CTBP1-AS was found to have no association with different clinical characteristics of PCOS. In conclusion, androgen receptor coregulating lncRNA CTBP1-AS is associated with PCOS women and high expression of CTBP1-AS is a risk factor for PCOS in Kashmiri women.

ING Tumour Suppressors and ING Splice Variants as Coregulators of the Androgen Receptor Signalling in Prostate Cancer.

Prevention and overcoming castration resistance of prostate cancer (PC) remains one of the main unsolved problems in modern oncology. Hence, many studies are focused on the investigation of novel androgen receptor (AR) regulators that could serve as potential drug targets in disease therapy. Among such factors, inhibitor of growth (ING) proteins were identified. Some ING proteins act as AR transcriptional coregulators, indicating their relevance for PC research. The ING family consists of five protein-coding genes from ING1 to ING5 and pseudogene INGX. The ING genes were revealed through their sequence homology to the first identified ING1 from an in vivo screen. ING factors are a part of histone modification complexes. With the help of the conserved plant homeodomain (PHD) motif, ING factors bind to Histone 3 Lysine 4 (H3K4) methylation mark with a stronger affinity to the highest methylation grade H3K4me3 and recruit histone acetyltransferases (HAT) and histone deacetylases (HDAC) to chromatin. ING1 and ING2 are core subunits of mSIN3a-HDAC corepressor complexes, whereas ING3–5 interact with different HAT complexes that serve as coactivators. ING members belong to type II tumour suppressors and are frequently downregulated in many types of malignancies, including PC. As the family name indicates, ING proteins are able to inhibit cell growth and tumour development via regulation of cell cycle and cancer-relevant pathways such as apoptosis, cellular senescence, DNA repair, cell migration, invasion, and angiogenesis. Many ING splice variants that enhance the diversity of ING activity were discovered. However, it seems that the existence of multiple ING splice variants is underestimated, since alternative splice variants, such as the AR coregulators ING1 and ING3, counteract full-length ING and thus play an opposite functional role. These results open a novel prospective investigation direction in understanding ING factors biology in PC and other malignancies.

Identification and Functional Characterization of a Novel Androgen Receptor Coregulator, EAP1.

The androgen receptor (AR) plays an essential role in the development of prostate cancer, and androgen-deprivation therapy is used as a first-line treatment for prostate cancer. However, under androgen-deprivation therapy, castration-resistant prostate cancer inevitably arises, suggesting that the interacting transcriptional coregulators of AR are promising targets for developing novel therapeutics. In this study, we used novel proteomic techniques to evaluate the AR interactome, including biochemically labile binding proteins, which might go undetected by conventional purification methods. Using rapid immunoprecipitation mass spectrometry of endogenous proteins, we identified enhanced at puberty 1 (EAP1) as a novel AR coregulator, whereas its interaction with AR could not be detected under standard biochemical conditions. EAP1 enhanced the transcriptional activity of AR via the E3 ubiquitin ligase activity, and its ubiquitination substrate proteins included AR and HDAC1. Furthermore, in prostate cancer specimens, EAP1 expression was significantly correlated with AR expression as well as a poor prognosis of prostate cancer. Together, these results suggest that EAP1 is a novel AR coregulator that promotes AR activity and potentially plays a role in prostate cancer progression.

Does and duration of exposure time dependent regulation of proteins in the urinary bladder of hamsters exposed to sodium arsenite

Does and duration of exposure time dependent regulation of proteins in the urinary bladder of hamsters exposed to sodium arsenite

Regulation of transgelin and GST-pi proteins in the tissues of hamsters exposed to sodium arsenite

Regulation of transgelin and GST-pi proteins in the tissues of hamsters exposed to sodium arsenite

Chromatin-directed proteomics-identified network of endogenous androgen receptor in prostate cancer cells

Treatment of prostate cancer confronts resistance to androgen receptor (AR)-targeted therapies. AR-associated coregulators and chromatin proteins hold a great potential for novel therapy targets. Here, we employed a powerful chromatin-directed proteomics approach termed ChIP-SICAP to uncover the composition of chromatin protein network, the chromatome, around endogenous AR in castration resistant prostate cancer (CRPC) cells. In addition to several expected AR coregulators, the chromatome contained many nuclear proteins not previously associated with the AR. In the context of androgen signaling in CRPC cells, we further investigated the role of a known AR-associated protein, a chromatin remodeler SMARCA4 and that of SIM2, a transcription factor without a previous association with AR. To understand their role in chromatin accessibility and AR target gene expression, we integrated data from ChIP-seq, RNA-seq, ATAC-seq and functional experiments. Despite the wide co-occurrence of SMARCA4 and AR on chromatin, depletion of SMARCA4 influenced chromatin accessibility and expression of a restricted set of AR target genes, especially those involved in cell morphogenetic changes in epithelial-mesenchymal transition. The depletion also inhibited the CRPC cell growth, validating SMARCA4’s functional role in CRPC cells. Although silencing of SIM2 reduced chromatin accessibility similarly, it affected the expression of a much larger group of androgen-regulated genes, including those involved in cellular responses to external stimuli and steroid hormone stimulus. The silencing also reduced proliferation of CRPC cells and tumor size in chick embryo chorioallantoic membrane assay, further emphasizing the importance of SIM2 in CRPC cells and pointing to the functional relevance of this potential prostate cancer biomarker in CRPC cells. Overall, the chromatome of AR identified in this work is an important resource for the field focusing on this important drug target.

The MYH9 Cytoskeletal Protein Is a Novel Corepressor of Androgen Receptors.

In the progression of castration-resistant prostate cancer (CRPC), the androgen receptor (AR) that serves as a transcription factor becomes the most remarkable molecule. The transcriptional activity of AR is regulated by various coregulators. As a result, altered expression levels, an aberrant location or activities of coregulators promote the development of prostate cancer. We describe herein results showing that compared with androgen-dependent prostate cancer (ADPC) cells, AR nuclear translocation capability is enhanced in androgen-independent prostate cancer (AIPC) cells. To gain insight into whether AR coregulators are responsible for AR translocation capability, we performed coimmunoprecipitation (CO-IP) coupled with LC-MS/MS to screen 27 previously reported AR cofactors and 46 candidate AR cofactors. Furthermore, one candidate, myosin heavy chain 9 (MYH9), was identified and verified as a novel AR cofactor. Interestingly, the distribution of MYH9 was in both the cytoplasmic and nuclear compartments yet was enriched in the nucleus when AR was knocked down by AR shRNA, suggesting that the nuclear translocation of MYH9 was negatively regulated by AR. In addition, we found that blebbistatin, an inhibitor of MYH9, not only promoted AR nuclear translocation but also enhanced the expression of the AR target gene PSA, which indicates that MYH9 represses nuclear AR signaling. Taken together, our findings reveal that MYH9 appears to be a novel corepressor of AR plays a pivotal role in the progression of CRPC.

BRD9 Is a Critical Regulator of Androgen Receptor Signaling and Prostate Cancer Progression.

Switch/sucrose-nonfermentable (SWI/SNF) chromatin-remodeling complexes are critical regulators of chromatin dynamics during transcription, DNA replication, and DNA repair. A recently identified SWI/SNF subcomplex termed GLTSCR1/1L-BAF (GBAF; or "noncanonical BAF", ncBAF) uniquely contains bromodomain-containing protein BRD9 and glioma tumor suppressor candidate region 1 (GLTSCR1) or its paralog GLTSCR1-like (GLTSCR1L). Recent studies have identified a unique dependency on GBAF (ncBAF) complexes in synovial sarcoma and malignant rhabdoid tumors, both of which possess aberrations in canonical BAF (cBAF) and Polybromo-BAF (PBAF) complexes. Dependencies on GBAF in malignancies without SWI/SNF aberrations, however, are less defined. Here, we show that GBAF, particularly its BRD9 subunit, is required for the viability of prostate cancer cell lines in vitro and for optimal xenograft tumor growth in vivo. BRD9 interacts with androgen receptor (AR) and CCCTC-binding factor (CTCF), and modulates AR-dependent gene expression. The GBAF complex exhibits overlapping genome localization and transcriptional targets as bromodomain and extraterminal domain-containing (BET) proteins, which are established AR coregulators. Our results demonstrate that GBAF is critical for coordinating SWI/SNF-BET cooperation and uncover a new druggable target for AR-positive prostate cancers, including those resistant to androgen deprivation or antiandrogen therapies. SIGNIFICANCE: Advanced prostate cancers resistant to androgen receptor antagonists are still susceptible to nontoxic BRD9 inhibitors, making them a promising alternative for halting AR signaling in progressed disease.