Androgen Receptor Function Research Articles

Comparative Proteomic and Transcriptomic Analysis of the Impact of Androgen Stimulation and Darolutamide Inhibition.

Several inhibitors of androgen receptor (AR) function are approved for prostate cancer treatment, and their impact on gene transcription has been described. However, the ensuing effects at the protein level are far less well understood. We focused on the AR signaling inhibitor darolutamide and confirmed its strong AR binding and antagonistic activity using the high throughput cellular thermal shift assay (CETSA HT). Then, we generated comprehensive, quantitative proteomic data from the androgen-sensitive prostate cancer cell line VCaP and compared them to transcriptomic data. Following treatment with the synthetic androgen R1881 and darolutamide, global mass spectrometry-based proteomics and label-free quantification were performed. We found a generally good agreement between proteomic and transcriptomic data upon androgen stimulation and darolutamide inhibition. Similar effects were found both for the detected expressed genes and their protein products as well as for the corresponding biological programs. However, in a few instances there was a discrepancy in the magnitude of changes induced on gene expression levels compared to the corresponding protein levels, indicating post-transcriptional regulation of protein abundance. Chromatin immunoprecipitation DNA sequencing (ChIP-seq) and Hi-C chromatin immunoprecipitation (HiChIP) revealed the presence of androgen-activated AR-binding regions and long-distance AR-mediated loops at these genes.

Temperature-dependent contractility of rat tunica dartos muscle: Contribution of cold, menthol-sensitive TRPM8

Tunica dartos smooth muscle (TDSM) lies beneath the scrotal skin, and its contraction leads to scrotum wrinkling upon cooling. However, neither the nature of TDSM cold-sensitivity nor the underlying molecular sensors are well understood. Here we have investigated the role of cold/menthol-sensitive TRPM8 channel in TDSM temperature-dependent contractility. The contraction of isolated male rat TDSM strips was studied by tensiometry. TRPM8 expression was assayed by RT-PCR and fluorescence immunochemistry. Isolated TDSM strips responded to cooling from 33 °C to 20 °C by enhancement of basal tension, and increase of the amplitude and duration of electric field stimulated (EFS) contractions. The effects of cold on basal tension, but not on EFS-contractions, could be 80% inhibited by TRPM8 blockers, capsazepine and BCTC [N-(4‑tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide], and could be partially mimicked by menthol. RT-PCR and immunolabeling showed TRPM8 mRNA and protein expression in TDSM cells with protein labelling being predominantly localized to intracellular compartments. Chemical castration of male rats consequent to the treatment with androgen receptor blocker, flutamide, led to the abrogation of cold effects on TDSM basal tension, but not on EFS-contractions, and to the disappearance of TRPM8 protein expression. We conclude that TRPM8 is involved in the maintenance of basal cold-induced TDSM tonus, but not in sympathetic nerve-mediated contractility, by acting as endoplasmic reticulum Ca2+ release channel whose expression in TDSM cells requires the presence of a functional androgen receptor. Thus, TRPM8 plays a crucial role in scrotal thermoregulation which is important for maintaining normal spermatogenesis and male fertility.

Antagonistic Functions of Androgen Receptor and NF-κB in Prostate Cancer-Experimental and Computational Analyses.

Prostate cancer is very frequent and is, in many countries, the third-leading cause of cancer related death in men. While early diagnosis and treatment by surgical removal is often curative, metastasizing prostate cancer has a very bad prognosis. Based on the androgen-dependence of prostate epithelial cells, the standard treatment is blockade of the androgen receptor (AR). However, nearly all patients suffer from a tumor relapse as the metastasizing cells become AR-independent. In our study we show a counter-regulatory link between AR and NF-κB both in human cells and in mouse models of prostate cancer, implying that inhibition of AR signaling results in induction of NF-κB-dependent inflammatory pathways, which may even foster the survival of metastasizing cells. This could be shown by reporter gene assays, DNA-binding measurements, and immune-fluorescence microscopy, and furthermore by a whole set of computational methods using a variety of datasets. Interestingly, loss of PTEN, a frequent genetic alteration in prostate cancer, also causes an upregulation of NF-κB and inflammatory activity. Finally, we present a mathematical model of a dynamic network between AR, NF-κB/IκB, PI3K/PTEN, and the oncogene c-Myc, which indicates that AR blockade may upregulate c-Myc together with NF-κB, and that combined anti-AR/anti-NF-κB and anti-PI3K treatment might be beneficial.

Characterization of a novel androgen receptor gene variant identified in an Iranian family with complete androgen insensitivity syndrome (CAIS): a molecular dynamics simulation study

Androgen insensitivity syndrome (AIS) is a common form of 46, XY disorder in sex development disease (DSD). It is due to the androgen receptor (AR) gene mutations and includes clinical subgroups of complete AIS (CAIS) and partial AIS (PAIS), along with a vast area of clinical heterogeneity of completely normal female external genitalia to male infertility. In this study, the Whole Exome Sequencing (WES) was utilized to detect the cause of DSD in a consanguineous Iranian family with two female patients with normal external genitalia and 46, XY karyotype. Sanger sequencing was applied to validate the candidate variant. Next, we predicted the structural alteration induced by the variant on AR protein using bioinformatics analysis such as molecular dynamic (MD) and molecular docking simulations. WES results identified a novel hemizygous p.L763V variant in the AR gene in the proband that was compatible with the X-linked recessive pattern of inheritance. Bioinformatics studies confirmed the loss of AR function. Based on the American College of Medical Genetics and Genomics (ACMG) guidelines, it was categorized as pathogenic. This study broadens the AR mutation spectrum and introduces the novel p.L763V missense pathogenic variant leading to AR failure to bind to its ligand, and the resulting CAIS clinical subgroup. This study presents a prosperous application of WES and bioinformatics analysis to recognize the underlying cause of DSD in Iran, necessary for its clinical/psychological management. Communicated by Ramaswamy H. Sarma

Gene dosage changes in KCTD13 result in penile and testicular anomalies via diminished androgen receptor function.

Despite the high prevalence of hypospadias and cryptorchidism, the genetic basis for these conditions is only beginning to be understood. Using array-comparative-genomic-hybridization (aCGH), potassium-channel-tetramerization-domain-containing-13 (KCTD13) encoded at 16p11.2 was identified as a candidate gene involved in hypospadias, cryptorchidism and other genitourinary (GU) tract anomalies. Copy number variants (CNVs) at 16p11.2 are among the most common syndromic genomic variants identified to date. Many patients with CNVs at this locus exhibit GU and/or neurodevelopmental phenotypes. KCTD13 encodes a substrate-specific adapter of a BCR (BTB-CUL3-RBX1) E3-ubiquitin-protein-ligase complex (BCR (BTB-CUL3-RBX1) E3-ubiquitin-protein-ligase complex (B-cell receptor (BCR) [BTB (the BTB domain is a conserved motif involved in protein-protein interactions) Cullin3 complex RING protein Rbx1] E3-ubiqutin-protein-ligase complex), which has essential roles in the regulation of cellular cytoskeleton, migration, proliferation, and neurodevelopment; yet its role in GU development is unknown. The prevalence of KCTD13 CNVs in patients with GU anomalies (2.58%) is significantly elevated when compared with patients without GU anomalies or in the general population (0.10%). KCTD13 is robustly expressed in the developing GU tract. Loss of KCTD13 in cell lines results in significantly decreased levels of nuclear androgen receptor (AR), suggesting that loss of KCTD13 affects AR sub-cellular localization. Kctd13 haploinsufficiency and homozygous deletion in mice cause a significant increase in the incidence of cryptorchidism and micropenis. KCTD13-deficient mice exhibit testicular and penile abnormalities together with significantly reduced levels of nuclear AR and SOX9. In conclusion, gene-dosage changes of murine Kctd13 diminish nuclear AR sub-cellular localization, as well as decrease SOX9 expression levels which likely contribute in part to the abnormal GU tract development in Kctd13 mouse models and in patients with CNVs in KCTD13.

Exploring anti-androgen therapies in hormone dependent prostate cancer and new therapeutic routes for castration resistant prostate cancer.

Androgen deprivation therapies (ADTs) are important treatments which inhibit androgen-induced prostate cancer (PCa) progression by either preventing androgen biosynthesis (e.g. abiraterone) or by antagonizing androgen receptor (AR) function (e.g. bicalutamide, enzalutamide, darolutamide). A major limitation of current ADTs is they often remain effective for limited durations after which patients commonly progress to a lethal and incurable form of PCa, called castration-resistant prostate cancer (CRPC) where the AR continues to orchestrate pro-oncogenic signalling. Indeed, the increasing numbers of ADT-related treatment-emergent neuroendocrine-like prostate cancers (NePC), which lack AR and are thus insensitive to ADT, represents a major therapeutic challenge. There is therefore an urgent need to better understand the mechanisms of AR action in hormone dependent disease and the progression to CRPC, to enable the development of new approaches to prevent, reverse or delay ADT-resistance. Interestingly the AR regulates distinct transcriptional networks in hormone dependent and CRPC, and this appears to be related to the aberrant function of key AR-epigenetic coregulator enzymes including the lysine demethylase 1 (LSD1/KDM1A). In this review we summarize the current best status of anti-androgen clinical trials, the potential for novel combination therapies and we explore recent advances in the development of novel epigenetic targeted therapies that may be relevant to prevent or reverse disease progression in patients with advanced CRPC.

296 (PB076) - Cellular hormone metabolism is critical for canonical androgen receptor antagonist activity

Background: Second-generation androgen receptor (AR) antagonists and an androgen synthesizing enzyme inhibitor have become the standard of care for advanced prostate cancer (PCa). Clinically-approved AR antagonists bind to the AR ligand binding domain (LBD) and competitively inhibit AR function. Dual action AR inhibitors (DAARIs), which bind to the N-terminal domain (NTD) of the AR inhibit AR signaling and lead AR protein degradation, have been identified by our group as potential therapeutics for PCa.

In silico comparison between the mutated and wild-type androgen receptors and their influence on the selection of optimum androgenic receptor blockers for the treatment of prostate cancer

Background: Prostate cancer is a disease that occurs in men aged more than 50 years. In Iraq, 8.89 men per 100,000 population suffer from prostate cancer, with the incidence being 14,016 cases and mortality being 6,367 cases. Despite advances in treatment against prostate cancer, it can become resistant to drugs. Therefore, the aim of the current study was to search and identify binding sites for the repositioning of drugs by computational methods (docking). Methods: Based on the protein structure of the wild androgen receptor, the analysis parameters (22x22x22 on the X, Y, and Z axes) were established. Results: The interactions of the natural ligands with androgen receptor were 10.0 (testosterone) and 10.8 (dihydrotestosterone) while mutated androgen receptor (T877A) had a low affinity with testosterone and dihydrotestosterone (-5.3 and -6.7, respectively). In the interactions of both receptors with the reported inhibitors (antagonists), a decrease with Bicalutamide (-8.3 and -4.3, respectively) and an increase in affinity with Flutamide and Nilutamide (-7.7 and 8.6, wild AR; -8.7 and -9.3 AR T877A) were observed. As for Enzalutamide and Apalutamide (second-generation antagonists), the change was minimal between wild androgen receptor and T877A (-7.6 and -7.7; -7.3 and -7.3, respectively). The change in the affinity of the ligands with the androgen receptor and androgen receptor T877A shows how a mutation alters the bonds between these molecules. Conclusion: The identification of key sites and potent inhibitors against abnormal androgen receptor functions will enrich prostate cancer treatments.

In silico comparison between the mutated and wild-type androgen receptors and their influence on the selection of optimum androgenic receptor blockers for the treatment of prostate cancer.

Background: Prostate cancer is a disease that occurs in men aged more than 50 years. In Iraq, 8.89 men per 100,000 population suffer from prostate cancer, with the incidence being 14,016 cases and mortality being 6,367 cases. Despite advances in treatment against prostate cancer, it can become resistant to drugs. Therefore, the aim of current study was to search and identify binding sites for the repositioning of drugs by computational methods (docking). Methods: Based on the protein structure of the wild androgen receptor, the analysis parameters (22x22x22 on the X, Y, and Z axes) were established. Results: The interactions of the natural ligands with androgen receptor were 10.0 (testosterone) and 10.8 (dihydrotestosterone) while mutated androgen receptor (T877A) had a low affinity with testosterone and dihydrotestosterone (-5.3 and -6.7, respectively). In the interactions of both receptors with the reported inhibitors (antagonists), a decrease with Bicalutamide (-8.3 and -4.3, respectively) and an increase in affinity with Flutamide and Nilutamide (-7.7 and 8.6, wild AR; -8.7 and -9.3 AR T877A) were observed. As for Enzalutamide and Apalutamide (second-generation antagonists), the change was minimal between wild androgen receptor and T877A (-7.6 and -7.7; -7.3 and -7.3, respectively). The change in the affinity of the ligands with androgen receptor and androgen receptor T877A shows how a mutation alters the bonds between these molecules. Conclusion: The identification of key sites and potent inhibitors against abnormal androgen receptor functions will enrich prostate cancer treatments.

Cellular specificity of androgen receptor, coregulators, and pioneer factors in prostate cancer.

Androgen signalling, through the transcription factor androgen receptor (AR), is vital to all stages of prostate development and most prostate cancer progression. AR signalling controls differentiation, morphogenesis, and function of the prostate. It also drives proliferation and survival in prostate cancer cells as the tumour progresses; given this importance, it is the main therapeutic target for disseminated disease. AR is also essential in the surrounding stroma, for the embryonic development of the prostate and controlling epithelial glandular development. Stromal AR is also important in cancer initiation, regulating paracrine factors that excite cancer cell proliferation, but lower stromal AR expression correlates with shorter time to progression/worse outcomes. The profile of AR target genes is different between benign and cancerous epithelial cells, between castrate-resistant prostate cancer cells and treatment-naïve cancer cells, between metastatic and primary cancer cells, and between epithelial cells and fibroblasts. This is also true of AR DNA-binding profiles. Potentially regulating the cellular specificity of AR binding and action are pioneer factors and coregulators, which control and influence the ability of AR to bind to chromatin and regulate gene expression. The expression of these factors differs between benign and cancerous cells, as well as throughout disease progression. The expression profile is also different between fibroblast and mesenchymal cell types. The functional importance of coregulators and pioneer factors in androgen signalling makes them attractive therapeutic targets, but given the contextual expression of these factors, it is essential to understand their roles in different cancerous and cell-lineage states.

Androgen receptor in breast cancer: The "5W" questions.

Androgen receptor (AR) is expressed in 60-70% of breast cancers (BCs) and the availability of anti-AR compounds, currently used for treating prostate cancer, paves the way to tackle specifically AR-positive BC patients. The prognostic and predictive role of AR in BC is a matter of debate, since the results from clinical trials are not striking, probably due to both technical and biological reasons. In this review, we aimed to highlight WHAT is AR, describing its structure and functions, WHAT to test and HOW to detect AR, WHERE AR should be tested (on primary tumor or metastasis) and WHY studying this fascinating hormone receptor, exploring and debating on its prognostic and predictive role. We considered AR and its ratio with other hormone receptors, analyzing also studies including patients with ductal carcinoma in situ and with early and advanced BC, as well. We also emphasized the effects that both other hormone receptors and the newly emerging androgen-inducible non coding RNAs may have on AR function in BC pathology and the putative implementation in the clinical setting. Moreover, we pointed out the latest results by clinical trials and we speculated about the use of anti-AR therapies in BC clinical practice.

HC-1119, a deuterated Enzalutamide, inhibits Migration, Invasion and Metastasis of the AR-positive triple-negative breast Cancer cells

Triple-negative breast cancers (TNBCs) are aggressive, and they develop metastasis at earlier stages, relapse more frequently, and exhibits poorer prognosis than other subtypes of breast cancer. Due to the lack of estrogen receptor for endocrine therapy and HER2 for targeted therapy, new targeted therapies for TNBCs are urgently needed. Enzalutamide is a second-generation androgen receptor (AR) inhibitor, and HC-1119 is a new synthetic deuterated enzalutamide. Owing to the isotope effect, HC-1119 has many advantages over enzalutamide, including slow metabolism, high plasma concentration and low brain exposure. However, the efficacy of HC-1119 in inhibition of AR function in triple-negative breast cancer (TNBC) has not been studied. In this study, we found high-level AR expression in both Hs578T and SUM159PT TNBC cell lines. Activation of AR by dihydrotestosterone (DHT) in both cell lines increased AR protein, induced AR-nuclear localization, enhanced cell migration and invasion in culture, and promoted liver metastasis in mice. Importantly, cotreatment with HC-1119 of these cells efficiently abolished all of these effects of DHT on both Hs578T and SUM159PT cells. These results indicate that HC-1119 is a very effective new second-generation AR antagonist that can inhibit the migration, invasion and metastasis of the AR-positive TNBC cells.

Androgen receptor α peak expression in retina rather than gonad of Hainan medaka, Oryzias curvinotus

Androgen is a member of the sex steroid hormones family which plays an important role in the development and maturation of the vertebrate reproductive system. The physiological function of androgens is mainly mediated by the androgen receptor (AR). Oryzias curvinotus is a small oviparous fish and a potential model like its sister specie, Oryzias latipes. In this study, two subtypes (α and β) of androgen receptors (ar) genes of O. curvinotus were successfully cloned. Phylogenetic analysis with amino acids showed that arα originates later and differentiates more than arβ. The result of RT-qPCR showed that there were significant differences in the different development stages and different tissues in the expression of two subtypes of ar. Arα remained highly expressed from the eyed stage to the hatching stage, while arβ was lowly expressed. Also, arα was highly expressed in the eyes, liver, and gonads of adults, while arβ remained highly expressed only in adult ovaries. And the result of in situ hybridization confirmed the high expression of arα on the inner nuclear layer of the retina of adult O. curvinotus. Conclusively, the expression and the localization of arα indicated that it plays an important role in both early embryonic development and the eyes of adult O. curvinotus. Further research to enhance the understanding of the new function and mechanism of androgen receptors in medaka is needed.

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.

Androgen receptor functions in pericentral hepatocytes to decrease gluconeogenesis and avoid hyperglycemia and obesity in male mice

BackgroundAlthough the impact of hepatic androgen receptor (AR) pathway on liver pathogenesis was documented, its physiological function in normal liver is remained unclear. This study aims to investigate if hepatic AR acts on metabolism, the major liver function, using a hepatic-specific AR-transgenic (H-ARTG) mouse model. MethodsWe established the albumin promoter driven H-ARTG mice and included wild type (WT) and H-ARKO mice for study. The body weight, specific metabolic parameters and results from various tolerance tests were compared in different groups of mice fed a chow diet, from 2 to 18 months of age. Glucose feeding and insulin treatment were used to study the expression and zonal distribution pattern of AR and related genes in liver at different prandial stages. ResultsThe body weight of H-ARTG mice fed a chow diet was 15 % lower than that of wild-type mice, preceded by lower blood glucose and liver triglyceride levels caused by AR reduced hepatic gluconeogenesis. The opposite phenotypes identified in H-ARKO and castrated H-ARTG mice support the critical role of activated AR in decreasing gluconeogenesis and triglyceride levels in liver. Hepatic AR acting by enhancing the expression of cytosolic glycerol-3-phosphate dehydrogenase (cGPDH), a key of glycerophosphate shuttle, was identified as one mechanism to decrease gluconeogenesis from glycerol. We further found AR normally expressed in zone 3 of hepatic lobules. Its level fluctuates dependent on the demand of glucose, decreased by fasting but increased by glucose uptake or insulin stimulation. ConclusionAR is a newly identified zone 3 hepatic gene with function in reducing blood glucose and body weight in mice. It suggests that stabilization of hepatic AR is a new direction to prevent hyperglycemia, obesity and nonalcoholic fatty liver disease (NAFLD) in males.

Abstract SY33-02: The circadian cryptochrome, CRY1, is a pro-tumorigenic factor that rhythmically modulates DNA repair

Abstract The second leading cause of cancer-related death in US men is prostate cancer (PCa), which is an androgen-dependent disease in which the androgen receptor (AR) plays an important role. Recent studies have underscored the importance of AR-mediated DNA repair factor regulation as strongly associated with poor outcome in PCa, yet the mechanistic underpinnings of the molecular and cellular consequence of AR-regulated DNA repair and tumor progression have yet to be fully defined. Mining of comprehensive studies designed to identify AR functions critical for PCa revealed that AR binds to multiple regulatory elements within the CRY1 (cryptochrome 1) locus, as observed in several hormone therapy-sensitive PCa models.Here, the circadian factor CRY1, an evolutionally conserved transcriptional coregulator, is identified as a tumor specific regulator of DNA repair. Intriguingly, the N-terminus of CRY1 is a photolyase homology domain suggesting a potential role for CRY1 in DNA damage response. Circadian factors have been implicated in diverse non-canonical physiological functions such as DNA repair, DNA damage checkpoints, and apoptosis. Given the potential implications of CRY1 and DNA repair interplay, this study sought to delineate the molecular and biological consequences of androgen-responsive CRY1 in cancer through cistromic, transcriptomic, and molecular modeling of CRY1 dysregulation in PCa. Key findings demonstrate that CRY1 expression is androgen-responsive and associates with poor outcome in prostate cancer. Functional studies and first-in-field mapping of the CRY1 cistrome and transcriptome reveal that CRY1 regulates DNA repair and the G2/M transition. DNA damage stabilizes CRY1 in cancer (in vitro, in vivo, and human tumors ex vivo), which proves critical for efficient DNA repair. Further mechanistic investigation shows that stabilized CRY1 temporally regulates expression of genes required for homologous recombination. Collectively, these findings reveal that CRY1 is hormone-induced in tumors, is further stabilized by genomic insult, and promotes DNA repair and cell survival through temporal transcriptional regulation. These studies identify the circadian factor CRY1 as pro-tumorigenic and nominate CRY1 as a new therapeutic target. This work was recently published in Nature Communications (Shafi et al, 2021).Ongoing and future work with elucidate the role of CRY1 in PCa by identifying CRY1-driven pathways impacting DNA damage repair (DDR) regulation and CRY1 function on tumor progression and therapeutic response to discern novel pathways to improve existing therapies. Our ongoing work has the potential for broad implications for understanding and treating lethal PCa. Based on our preliminary data, we hypothesize that CRY1 is key modulator of DDR impacting PCa growth and survival, and that delineating the underlying molecular mechanisms of CRY1 and DDR interplay will allow for improved management of advanced disease. Our data indicate that CRY1 expression is altered in PCa, associated with poor outcome, and elicits pro-tumorigenic phenotypes through direct DDR regulation. Our continued work will define the biology of lethal PCa by delineating the molecular and cellular consequence of tumor-associated CRY1 deregulation, providing insight into the function of CRY1 on DNA repair competency. Also, this study will uncover mechanisms of action and identify novel partners for CRY1 function to transcriptionally regulate DNA repair and cancer progression. Lastly, the translational studies from this research study will elucidate the impact of CRY1 function on tumor progression and therapeutic response. Thus, these findings will reveal potential molecular and clinical biomarkers for patients and will ultimately aid in improving existing therapies and developing effective treatments to reduce death from PCa. The long-term impact of CRY1 as a pro-tumorigenic factor driving PCa progression and lethality, emphasizes the importance of developing treatments targeting this factor to mitigate aggressive disease. This study will be crucial in providing novel insight into CRY1 function in human PCa tumors to identify new avenues for therapeutic intervention. This research represents critical steps to understand how circadian factors, specifically CRY1, mediate DNA repair and promote tumorigenesis. Through our collaboration with circadian and DDR experts, this impactful work will not only provide deep mechanistic insight into CRY1 function in PCa, but also lay the foundation for preclinical studies aimed at developing novel strategies for treating CRY1-deregulated cancers. Thus, this ongoing research has immense translational impact serving as the basis for new investigator-initiated clinical trials and altering regimens to emphasize the importance of chronotherapy enhancing standard-of-care to ultimately improve outcome and reduce lethality of PCa. Citation Format: Ayesha Shafi. The circadian cryptochrome, CRY1, is a pro-tumorigenic factor that rhythmically modulates DNA repair [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 SY33-02.

Abstract LB038: Identification of androgen receptor co-chaperone interactors implicated in castration resistant prostate cancer

Abstract Background: Prostate cancer (PCa) is the most common non-cutaneous cancer afflicting men, both in the United States and worldwide. Aberrant molecular signaling mechanisms mediated by the Androgen Receptor (AR) are attributed as the main culprits for the initiation and progression of PCa, and research efforts have focused on developing strategies to directly target the AR. In an alternative approach, molecular chaperones that are critical for the maturation and signaling of the AR have emerged as potential therapeutic targets. Our lab has demonstrated the potential of inhibitors that target AR cochaperones, FKBP51 and FKBP52, in PCa cells. Previously, FKBP51 and FKBP52 have been shown to play a role in cell proliferation and survival, steroid hormone receptor maturation, and other mechanisms highly implicated in prostate carcinogenesis. Therefore, it is imperative to understand the protein-protein interactions that regulate FKBP51 and FKBP52-mediated AR function in PCa. We hypothesize that unknown interactors cooperate with FKBP51 and FKBP52 to promote PCa progression via the AR signaling pathways. Here, we performed a series of experiments aimed to identify and characterize auxiliary proteins influencing FKBP51- and FKBP52-mediated AR activity. Ultimately, our goal is to define novel protein interactions involved in AR-mediated PCa progression for therapeutic intervention. Methods: We implemented tandem affinity purification followed by mass spectrometry analysis in 22RV1 PCa cells using FKBP51 and FKBP52 as bait. The spectral counts of proteins identified were then used to analyze protein-protein interactions through the IDEP workflow software. Gene Ontology (GO) term enrichment analysis was used to generate a STRING interaction network. Protein interactions of interest were then validated via reciprocal immunoprecipitations. Results: This interactome analysis resulted in 353 IDs for FKBP51 and FKBP52 interactors. Heatmaps representing top 100 interactors were generated for both bait proteins. Members of the peroxiredoxin family, implicated in development of PCa and resistance to treatment, are among the top candidates of interest resulting from this interactome. Conclusion: This analysis aims to provide important insights into the androgen signaling milieu, which can be applied in the development of novel treatments targeting AR interacting partners in PCa progression. This study contributes to the growing list of functionally diverse proteins that help modulate AR transcriptional activity, and supports the idea that AR signaling is dynamic with numerous molecules involved in the aberrant activation of AR in prostate-tumor cells Citation Format: Olga B. Soto, Abhijeet R. Patil, Sourav Roy, Marc B. Cox. Identification of androgen receptor co-chaperone interactors implicated in castration resistant prostate cancer [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 LB038.

Anticancer function of microRNA‐30e is mediated by negative regulation of HELLPAR, a noncoding macroRNA, and genes involved in ubiquitination and cell cycle progression in prostate cancer

Prostate cancer (PCa) progression relies on androgen receptor (AR) function, making AR a top candidate for PCa therapy. However, development of drug resistance is common, which eventually leads to development of castration‐resistant PCa. This warrants a better understanding of the pathophysiology of PCa that facilitates the aberrant activation of key signaling pathways including AR. MicroRNAs (miRNAs) function as regulators of cancer progression as they modulate various cellular processes. Here, we demonstrate a multidimensional function of miR‐30e through the regulation of genes involved in various signaling pathways. We noted loss of miR‐30e expression in prostate tumors, which, when restored, led to cell cycle arrest, induction of apoptosis, improved drug sensitivity of PCa cells and reduced tumor progression in xenograft models. We show that experimental upregulation of miR‐30e reduces expression of mRNAs including AR, FBXO45, SRSF7 and MYBL2 and a novel long noncoding RNA (lncRNA) HELLPAR, which are involved in cell cycle, apoptosis and ubiquitination, and the effects could be rescued by inhibition of miR‐30e expression. RNA immunoprecipitation analysis confirmed direct interactions between miR‐30e and its RNA targets. We noted a newly identified reciprocal relationship between miR‐30e and HELLPAR, as inhibition of HELLPAR improved stabilization of miR‐30e. Transcriptome profiling and quantitative real‐time PCR (qRT‐PCR) validation of miR‐30e‐expressing PCa cells showed differential expression of genes involved in cell cycle progression, apoptosis and ubiquitination, which supports our in vitro study. This study demonstrates an integrated function of miR‐30e on dysregulation of miRNA/lncRNA/mRNA axes that may have diagnostic and therapeutic significance in aggressive PCa.

Exploring the Role of Posttranslational Modifications in Spinal and Bulbar Muscular Atrophy.

Spinal and Bulbar Muscular Atrophy (SBMA) is an X-linked adult-onset progressive neuromuscular disease that affects the spinal and bulbar motor neurons and skeletal muscles. SBMA is caused by expansion of polymorphic CAG trinucleotide repeats in the Androgen Receptor (AR) gene, resulting in expanded glutamine tract in the AR protein. Polyglutamine (polyQ) expansion renders the mutant AR protein toxic, resulting in the formation of mutant protein aggregates and cell death. This classifies SBMA as one of the nine known polyQ diseases. Like other polyQ disorders, the expansion of the polyQ tract in the AR protein is the main genetic cause of the disease; however, multiple other mechanisms besides the polyQ tract expansion also contribute to the SBMA disease pathophysiology. Posttranslational modifications (PTMs), including phosphorylation, acetylation, methylation, ubiquitination, and SUMOylation are a category of mechanisms by which the functionality of AR has been found to be significantly modulated and can alter the neurotoxicity of SBMA. This review summarizes the different PTMs and their effects in regulating the AR function and discusses their pathogenic or protective roles in context of SBMA. This review also includes the therapeutic approaches that target the PTMs of AR in an effort to reduce the mutant AR-mediated toxicity in SBMA.

TALAPRO-3: A phase 3, double-blind, randomized study of enzalutamide (ENZA) plus talazoparib (TALA) versus placebo plus ENZA in patients with DDR gene–mutated, metastatic castration-sensitive prostate cancer (mCSPC).

TPS5096 Background: TALA is a poly(ADP-ribose) polymerase inhibitor (PARPi) approved as monotherapy for germline BRCA1/2-mutated HER2-negative advanced breast cancer. Clinical efficacy in metastatic castration-resistant prostate cancers (mCRPC) with alterations in DNA damage response (DDR) genes involved directly or indirectly in homologous recombination repair (HRR) has been demonstrated with some PARPi. Phase 3 study findings (de Bono et al. N Engl J Med, 2020;382:2091-2102) resulted in the approval of olaparib for mCRPC. ENZA is an androgen receptor (AR) inhibitor and established therapy for mCSPC. Since PARP activity has been shown to support AR function, PARP inhibition may increase sensitivity to AR-directed therapies. In addition, AR blockade downregulates HRR gene regulation, which has been hypothesized to induce a “ BRCAness” phenotype. A Phase 2 study of TALA monotherapy (TALAPRO-1) demonstrated robust antitumor activity in men with heavily pretreated, HRR-mutated mCRPC. The Phase 3, double-blind, randomized trial TALAPRO-3 (NCT04821622) herein presented will compare the combination of TALA plus ENZA vs placebo plus ENZA in men with mCSPC with DDR/HRR alterations. Methods: Approximately 550 patients with mCSPC harboring DDR/HRR alterations will be randomized to TALA (0.5 mg once daily) plus ENZA (160 mg once daily) or placebo (once daily) plus ENZA (160 mg once daily). Patients will be stratified according to de novo mCSPC vs relapsed mCSPC, high-volume disease vs low-volume disease, where high-volume disease is defined as the presence of visceral metastases or ≥4 bone lesions with ≥1 beyond the vertebral bodies and pelvis and BRCA vs non-BRCA mutational status. Key eligibility criteria include age ≥18 years; histological diagnosis of prostate cancer; alterations in at least one of 12 DDR/HRR genes known to sensitize patients to PARPi ( ATM, ATR, BRCA1, BRCA2, CDK12, CHEK2, FANCA, MLH1, MRE11A, NBN, PALB2, RAD51C); and metastatic disease (no brain metastases). Primary endpoint is rPFS (time to radiographic progression in soft tissue per RECIST 1.1 or in bone per PCWG3 criteria by investigator, or death). Secondary endpoints include overall survival, safety, and patient-reported outcomes. Patient recruitment is planned at approximately 285 sites in 27 countries, including the US and Europe, South America, South Africa, and Asia-Pacific. This study was approved by an Institutional Review Board. Clinical trial information: NCT04821622.