Development and Characterisation of a New Patient-Derived Xenograft Model of AR-Negative Metastatic Castration-Resistant Prostate Cancer.

As the treatment landscape for prostate cancer gradually evolves, the frequency of treatment-induced neuroendocrine prostate cancer (NEPC) and double-negative prostate cancer (DNPC) that is deficient for androgen receptor (AR) and neuroendocrine (NE) markers has increased. These prostate cancer subtypes are typically refractory to AR-directed therapies and exhibit poor clinical outcomes. Only a small range of NEPC/DNPC models exist, limiting our molecular understanding of this disease and hindering our ability to perform preclinical trials exploring novel therapies to treat NEPC/DNPC that are urgently needed in the clinic. Here, we report the development of the CU-PC01 PDX model that represents AR-negative mCRPC with PTEN/RB/PSMA loss and CTNN1B/TP53/BRCA2 genetic variants. The CU-PC01 model lacks classic NE markers, with only focal and/or weak expression of chromogranin A, INSM1 and CD56. Collectively, these findings are most consistent with a DNPC phenotype. Ex vivo and in vivo preclinical studies revealed that CU-PC01 PDX tumours are resistant to mCRPC standard-of-care treatments enzalutamide and docetaxel, mirroring the donor patient's treatment response. Furthermore, short-term CU-PC01 tumour explant cultures indicate this model is initially sensitive to PARP inhibition with olaparib. Thus, the CU-PC01 PDX model provides a valuable opportunity to study AR-negative mCRPC biology and to discover new treatment avenues for this hard-to-treat disease.

Immunoprofiles and Oncologic Outcomes of 15 Patients with Androgen Receptor-Positive Salivary Duct Carcinoma.

BACKGROUND: Salivary duct carcinomas (SDC) are a rare and aggressive subtype of salivary gland neoplasm. They can present with distinct immunoprofiles, such as androgen receptor (AR) and HER-2/Neu-positivity. To date, no consensus exists on how to best manage this entity. METHODS: All patients diagnosed with nonmetastatic AR+ SDC of the parotid from 2013 to 2019 treated with curative intent were included. Immunologic tumor profiling was conducted using 24 distinct markers. Kaplan-Meier analyses were used to estimate locoregional recurrence (LRR), distant control, and overall survival (OS). RESULTS: Fifteen patients were included. Nine (60%) patients presented with T4 disease and eight (53%) had positive ipsilateral cervical lymphadenopathy. Ten (67%) patients underwent trimodality therapy, including surgery followed by adjuvant radiation and concurrent systemic therapy. The median follow-up was 5.5 years (interquartile range, 4.8-6.1). The estimated 5-year rates of LRR, distant progression, and OS were 6%, 13%, and 87%, respectively. CONCLUSION: Despite only including AR+ SDC of the parotid, immunoprofiles, such as expression of HER-2, were highly variable, highlighting the potential to tailor systemic regimens based on individual histologic profiles in the future. Studies with larger patient numbers using tumor-specific molecular profiling and tumor heterogeneity analyses are justified to better understand the biology of these tumors. Molecularly informed treatment approaches, including the potential use of AR- and HER-2/Neu-directed therapies upfront in the definitive setting, may hold future promise to further improve outcomes for these patients.

Anti-COVID-19 Potential of Withaferin-A and Caffeic Acid Phenethyl Ester.

BACKGROUND: The recent COVID-19 (coronavirus disease 2019) pandemic triggered research on the development of new vaccines/drugs, repurposing of clinically approved drugs, and assessment of natural anti-COVID-19 compounds. Based on the gender difference in the severity of the disease, such as a higher number of men hospitalized and in intense care units, variations in sex hormones have been predicted to play a role in disease susceptibility. Cell surface receptors (Angiotensin-Converting Enzyme 2; ACE2 and a connected transmembrane protease serine 2- TMPSS2) are upregulated by androgens. Conversely, androgen antagonists have also been shown to lower ACE2 levels, implying their usefulness in COVID-19 management. OBJECTIVE: In this study, we performed computational and cell-based assays to investigate the anti-- COVID-19 potential of Withaferin-A and Caffeic acid phenethyl ester, natural compounds from Withania somnifera and honeybee propolis, respectively. METHODS: Structure-based computational approach was adopted to predict binding stability, interactions, and dynamics of the two test compounds to three target proteins (androgen receptor, ACE2, and TMPRSS2). Further, in vitro, cell-based experimental approaches were used to investigate the effect of compounds on target protein expression and SARS-CoV-2 replication. RESULTS: Computation and experimental analyses revealed that (i) CAPE, but not Wi-A, can act as androgen antagonist and hence inhibit the transcriptional activation function of androgen receptor, (ii) while both Wi-A and CAPE could interact with ACE2 and TMPRSS2, Wi-A showed higher binding affinity, and (iii) combination of Wi-A and CAPE (Wi-ACAPE) caused strong downregulation of ACE2 and TMPRSS2 expression and inhibition of virus infection. CONCLUSION: Wi-A and CAPE possess multimodal anti-COVID-19 potential, and their combination (Wi-ACAPE) is expected to provide better activity and hence warrant further attention in the laboratory and clinic.

Androgen receptor in breast cancer and its clinical implication.

Breast cancer is a heterogeneous group of diseases characterized by diverse subtypes. Currently, the classification of breast cancer is based on the status of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). In addition to these receptors, the presence of the androgen receptor (AR) in breast cancer cells adds a layer of complexity to our understanding of the disease. The role of AR in breast cancer is intricate, as it can alter diverse signaling pathways in the presence of different hormone receptors (HRs). This complex interplay between signaling pathways affects patient outcomes and prognosis, and the presence of AR has a significant effect. While AR positivity is common in breast cancer, the efficacy of utilizing AR blockade as a monotherapy has been limited, demonstrating only modest results. To address this challenge, substantial efforts have been directed toward comprehending the intricacies of AR's role and pathways in breast cancer development in the hope of understanding its utility as a biomarker or drug target. Multiple ongoing clinical trials are currently investigating combination treatments involving AR inhibitors and other agents to disrupt oncogenic signaling pathways and their crosstalk. Particularly in the context of triple-negative breast cancer (TNBC), where targeted therapeutic options are lacking, extensive research efforts have been dedicated to exploring the potential of AR-related interventions. This review aims to provide an overview of the various breast cancer subtypes with AR signaling mechanisms, and ongoing clinical trials that hold the potential to reshape future clinical approaches.

Semaphorin 3C promotes de novo steroidogenesis in prostate cancer cells.

Intratumoral androgen biosynthesis contributes to castration-resistant prostate cancer progression in patients treated with androgen deprivation therapy. The molecular mechanisms by which castration-resistant prostate cancer acquires the capacity for androgen biosynthesis to bypass androgen deprivation therapy are not entirely known. Here, we show that semaphorin 3C, a secreted signaling protein that is highly expressed in castration-resistant prostate cancer, can promote steroidogenesis by altering the expression profile of key steroidogenic enzymes. Semaphorin 3C not only upregulates enzymes required for androgen synthesis from dehydroepiandrosterone or de novo from cholesterol but also simultaneously downregulates enzymes involved in the androgen inactivation pathway. These changes in gene expression correlate with increased production of androgens induced by semaphorin 3C in prostate cancer model cells. Moreover, semaphorin 3C upregulates androgen synthesis in LNCaP cell-derived xenograft tumors, likely contributing to the enhanced in vivo tumor growth rate post castration. Furthermore, semaphorin 3C activates sterol regulatory element-binding protein, a transcription factor that upregulates enzymes involved in the synthesis of cholesterol, a sole precursor for de novo steroidogenesis. The ability of semaphorin 3C to promote intratumoral androgen synthesis may be a key mechanism contributing to the reactivation of the androgen receptor pathway in castration-resistant prostate cancer, conferring continued growth under androgen deprivation therapy. These findings identify semaphorin 3C as a potential therapeutic target for suppressing intratumoral steroidogenesis.

Improved Prostate-Specific Membrane Antigen (PSMA) Stimulation Using a Super Additive Effect of Dutasteride and Lovastatin In Vitro.

Prostate-specific membrane antigen (PSMA)-based imaging improved the detection of primary, recurrent and metastatic prostate cancer. However, in certain patients, a low PSMA surface expression can be a limitation for this promising diagnostic tool. Pharmacological induction of PSMA might be useful to further improve the detection rate of PSMA-based imaging. To achieve this, we tested dutasteride (Duta)-generally used for treatment of benign prostatic enlargement-and lovastatin (Lova)-a compound used to reduce blood lipid concentrations. We aimed to compare the individual effects of Duta and Lova on cell proliferation as well as PSMA expression. In addition, we tested if a combination treatment using lower concentrations of Duta and Lova can further induce PSMA expression. Our results show that a treatment with ≤1 µM Duta and ≥1 µM Lova lead to a significant upregulation of whole and cell surface PSMA expression in LNCaP, C4-2 and VCaP cells. Lower concentrations of Duta and Lova in combination (0.5 µM Duta + 0.5 µM Lova or 0.5 µM Duta + 1 µM Lova) were further capable of enhancing PSMA protein expression compared to a single compound treatment using higher concentrations in all tested cell lines (LNCaP, C4-2 and VCaP).

AndroPred: an artificial intelligence-based model for predicting androgen receptor inhibitors.

Androgen receptor (AR), a steroid receptor, plays a pivotal role in the pathogenesis of prostate cancer (PCa). AR controls the transcription of genes that help cells avoid apoptosis and proliferate, thereby contributing to the development of PCa. Understanding AR molecular mechanisms has led to the development of newer drugs that inhibit androgen production enzymes or block ARs. The FDA has approved a small number of AR-inhibiting drugs for use in PCa thus far, as the identification of novel AR inhibitors is difficult, expensive, time-consuming, and labor-intensive. To accelerate the process, artificial intelligence (AI) algorithms were employed to predict AR inhibitors using a dataset of 2242 compounds. Four machine learning (ML) and deep learning (DL) algorithms were used to train different prediction models based on molecular descriptors (1D, 2D, and molecular fingerprints). The DL-based prediction model outperformed the other trained models with accuracies of 92.18% and 93.05% on the training and test datasets, respectively. Our findings highlight the potential of DL, particularly the DNN model, as an effective approach for predicting AR inhibitors, which could significantly streamline the process of identifying novel AR inhibitors in PCa drug discovery. Further validation of these models using experimental assays and prospective testing of newly designed compounds would be valuable to confirm their predictive power and applicability in practical drug discovery settings.Communicated by Ramaswamy H. Sarma.

Crystal structure determination, molecular docking, and molecular dynamics of arylal dimedones as potential inhibitors for castrate-resistant prostate cancer.

Increased androgen receptor (AR) signaling brought on by higher intratumoral androgen production and AR amplification is associated with castrate-resistant prostate cancer (CRPC). Cell proliferation in this case continues even during low expression of testosterone in the body. Aldo-keto reductase family 1 member C3 (AKR1C3) is one of the most elevated genes in CRPC and catalyzes the formation of powerful AR ligands from inactive forms. The current work aimed to use the x-ray method to investigate the ligand's crystal structure while also conducting molecular docking and molecular dynamics tests on the synthesized molecules against AKR1C3. As per the results obtained, the MM-PBSA binding energies of inhibitors 2,2'-((4-methoxyphenyl)methylene)bis(3,4-hydroxy-5,5-dimethylcyclohex-2-en-1-one is -132.456 kJ mol-1 and 2,2'-(phenylmethylene)bis(3-hydroxy-5,5-dimethylcyclohex-2-en-1-one is -81.017 kJ mol-1 . These results create a promising approach to drug design based on its fit to the structures of the receptor site rather than basing it on analogies to other active structures.

Single-cell multi-omics analysis reveals dysfunctional Wnt signaling of spermatogonia in non-obstructive azoospermia.

Background: Non-obstructive azoospermia (NOA) is the most severe type that leads to 1% of male infertility. Wnt signaling governs normal sperm maturation. However, the role of Wnt signaling in spermatogonia in NOA has incompletely been uncovered, and upstream molecules regulating Wnt signaling remain unclear. Methods: Bulk RNA sequencing (RNA-seq) of NOA was used to identify the hub gene module in NOA utilizing weighted gene co-expression network analyses (WGCNAs). Single-cell RNA sequencing (scRNA-seq) of NOA was employed to explore dysfunctional signaling pathways in the specific cell type with gene sets of signaling pathways. Single-cell regulatory network inference and clustering (pySCENIC) for Python analysis was applied to speculate putative transcription factors in spermatogonia. Moreover, single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) determined the regulated genes of these transcription factors. Finally, spatial transcriptomic data were used to analyze cell type and Wnt signaling spatial distribution. Results: The Wnt signaling pathway was demonstrated to be enriched in the hub gene module of NOA by bulk RNA-seq. Then, scRNA-seq data revealed the downregulated activity and dysfunction of Wnt signaling of spermatogonia in NOA samples. Conjoint analyses of the pySCENIC algorithm and scATAC-seq data indicated that three transcription factors (CTCF, AR, and ARNTL) were related to the activities of Wnt signaling in NOA. Eventually, spatial expression localization of Wnt signaling was identified to be in accordance with the distribution patterns of spermatogonia, Sertoli cells, and Leydig cells. Conclusion: In conclusion, we identified that downregulated Wnt signaling of spermatogonia in NOA and three transcription factors (CTCF, AR, and ARNTL) may be involved in this dysfunctional Wnt signaling. These findings provide new mechanisms for NOA and new therapeutic targets for NOA patients.

Trichorhinophalangeal syndrome type 1 (TRPS1) expression in male breast carcinoma.

Currently, there is a paucity of highly specific and sensitive markers to identify breast carcinoma in male patients. Immunohistochemical stains commonly used for unmasking primary breast carcinomas include estrogen receptor (ER) and GATA3. However, these markers are commonly expressed in carcinomas originating from other organ systems and can be reduced in breast carcinomas with higher histologic grades. Androgen receptor (AR) may be used to highlight primary male breast cancer, but this marker can also be expressed in other carcinomas. We evaluated TRPS1, a highly sensitive and specific marker for female breast carcinoma, in cases of male breast carcinoma. Through an institutional database search, we identified 72 cases of primary invasive breast carcinoma in male patients. Among ER/progesterone receptor (PR)-positive cancers, 97% showed intermediate or high positivity for both TRPS1 and GATA3. Among HER2-positive cancers, 100% showed intermediate or high positivity for TRPS1 and GATA3. One case of triple-negative breast cancer was collected, showing high positivity for TRPS1 and negativity for GATA3. AR staining was non-specific and heterogeneous: 76% showed high positivity, but the remaining 24% showed low or intermediate positivity. Additionally, among 29 cases of metastatic carcinoma to male breast tissue, 93% were negative for TRPS1, and the remaining 2 cases (7%), which were carcinomas from salivary gland primary tumors, were intermediate positive. TRPS1 is a sensitive and specific marker in the unmasking of male primary invasive breast carcinoma across different subtypes. Additionally, TRPS1 is not expressed in metastatic carcinomas of multiple primaries, with the exception of salivary gland primaries.

Neferine attenuates development of testosterone-induced benign prostatic hyperplasia in mice by regulating androgen and TGF-ß/Smad signaling pathways.

Benign prostatic hyperplasia (BPH) is a common urinary disease among the elderly, characterized by abnormal prostatic cell proliferation. Neferine is a dibenzyl isoquinoline alkaloid extracted from Nelumbo nucifera and has antioxidant, anti-inflammatory and anti-prostate cancer effects. The beneficial therapeutic effects and mechanism of action of neferine in BPH remain unclear. A mouse model of BPH was generated by subcutaneous injection of 7.5 mg/kg testosterone propionate (TP) and 2 or 5 mg/kg neferine was given orally for 14 or 28 days. Pathological and morphological characteristics were evaluated. Prostate weight, prostate index (prostate/body weight ratio), expression of type Ⅱ 5α-reductase, androgen receptor (AR) and prostate specific antigen were all decreased in prostate tissue of BPH mice after administration of neferine. Neferine also downregulated the expression of pro-caspase-3, uncleaved PARP, TGF-ß1, TGF-ß receptor Ⅱ (TGFBR2), p-Smad2/3, N-cadherin and vimentin. Expression of E-cadherin, cleaved PARP and cleaved caspase-3 was increased by neferine treatment. 1-100 µM neferine with 1 µM testosterone or 10 nM TGF-ß1 were added to the culture medium of the normal human prostate stroma cell line, WPMY-1, for 24 h or 48 h. Neferine inhibited cell growth and production of reactive oxygen species (ROS) in testosterone-treated WPMY-1 cells and regulated the expression of androgen signaling pathway proteins and those related to epithelial-mesenchymal transition (EMT). Moreover, TGF-ß1, TGFBR2 and p-Smad2/3, N-cadherin and vimentin expression were increased but E-cadherin was decreased after 24 h TGF-ß1 treatment in WPMY-1 cells. Neferine reversed the effects of TGF-ß1 treatment in WPMY-1 cells. Neferine appeared to suppress prostate growth by regulating the EMT, AR and TGF-ß/Smad signaling pathways in the prostate and is suggested as a potential agent for BPH treatment.

Racial Differences in Androgen Receptor (AR) and AR Splice Variants (AR-SVs) Expression in Treatment-Naïve Androgen-Dependent Prostate Cancer.

Androgen receptor splice variants (AR-SVs) contribute to the aggressive growth of castration-resistant prostate cancer (CRPC). AR-SVs, including AR-V7, are expressed in ~30% of CRPC, but minimally in treatment-naïve primary prostate cancer (PCa). Compared to Caucasian American (CA) men, African American (AA) men are more likely to be diagnosed with aggressive/potentially lethal PCa and have shorter disease-free survival. Expression of a truncated AR in an aggressively growing patient-derived xenograft developed with a primary PCa specimen from an AA patient led us to hypothesize that the expression of AR-SVs could be an indicator of aggressive growth both in PCa progression and at the CRPC stage in AA men. Tissue microarrays (TMAs) were created from formalin-fixed paraffin-embedded (FFPE) prostatectomy tumor blocks from 118 AA and 115 CA treatment-naïve PCa patients. TMAs were stained with AR-V7-speicifc antibody and with antibodies binding to the N-terminus domain (NTD) and ligand-binding domain (LBD) of the AR. Since over 20 AR-SVs have been identified, and most AR-SVs do not as yet have a specific antibody, we considered a 2.0-fold or greater difference in the NTD vs. LBD staining as indication of potential AR-SV expression. Two AA, but no CA, patient tumors stained positively for AR-V7. AR staining with NTD and LBD antibodies was robust in most patients, with 21% of patients staining at least 2-fold more for NTD than LBD, indicating that AR-SVs other than AR-V7 are expressed in primary treatment-naïve PCa. About 24% of the patients were AR-negative, and race differences in AR expression were not statistically significant. These results indicate that AR-SVs are not restricted to CRPC, but also are expressed in primary PCa at higher rate than previously reported. Future investigation of the relative expression of NTD vs. LBD AR-SVs could guide the use of newly developed treatments targeting the NTD earlier in the treatment paradigm.

Megakaryocyte-Derived IL-8 Acts as a Paracrine Factor for Prostate Cancer Aggressiveness through CXCR2 Activation and Antagonistic AR Downregulation.

Prostate cancer is the fifth leading cause of cancer-related mortality in men, primarily because of treatment resistance, recurrence, and metastasis. In the present study, we investigated the role of paracrine interleukin-8 (IL-8) in the antagonistic expression of IL-8 and androgen receptor (AR), and the contribution of IL-8 to prostate cancer aggressiveness. In hormone-responsive LNCaP cells that do not express IL-8, recombinant IL-8 treatment significantly increased expressions of IL-8, CXC chemokine receptor 2 (CXCR2), matrix metalloproteinase (MMP)-2/9, Snail, and vimentin. IL-8 treatment significantly decreased AR and E-cadherin expression. IL-8-induced gene expression changes were suppressed by navarixin, a CXCR1/2 inhibitor, and gallein, a Gßγ inhibitor. In PC-3 androgen-refractory prostate cancer cells, IL-8 knockdown reduced expressions of CXCR2, MMP-2/9, Snail, and vimentin, and increased AR and E-cadherin expressions at the mRNA and protein levels. Co-culture with MEG-01 human megakaryocytic cells secreting high levels of IL-8 induced gene expression changes in both LNCaP and PC-3 cells, similar to those induced by IL-8 treatment. The altered gene expressions were accompanied by significant activation of transcription factor Snail in LNCaP and PC-3 cells. Treatment with the CXCR blocker navarixin inhibited the invasion of PC-3 cells but not LNCaP cells. However, invasion induced by MEG-01 was inhibited by navarixin in both LNCaP and PC-3 cells. The collective findings demonstrate that IL-8 enhances CXCR2 expression, which antagonistically regulates AR expression. More importantly, through changes in IL-8/CXCR2-regulated gene expression, IL-8 induces antiandrogen therapy resistance and epithelial-mesenchymal transition in prostate cancer.

Improvement of Cardiovascular Function in Aging Females by the Prolonged Activation of G Protein-Coupled Estrogen Receptor.

Ample evidence suggests that estrogen replacement therapy is associated with beneficial effects with regard to cardiovascular diseases when the therapy is initiated temporally close to menopause but not when it is initiated later. Little is known about the complex interactions between hormone receptors after menopause. Coronary artery function and cardiac function were measured in rats that had either received no treatment or had been pretreated with an androgen receptor (AR) antagonist and/or a GPER agonist G-1. ICI 182,780 was used to block the classical estrogen receptors (ERs) to investigate their complex interactions with GPER. The beneficial effects of GPER were only observed by blocking ARs and classical ERs in aged female rats. The results demonstrate that GPER activation is a potential therapeutic target for the inhibition of age-dependent coronary artery dysfunction and cardiac dysfunction under the condition of blocking ARs and classical ERs after menopause. CLINICAL RELEVANCE: The risk of cardiovascular disease in postmenopausal women significantly increased. The role of sex hormones and their receptors during this process is still complicated. Our present study demonstrated that the imbalance of androgen and estrogen may contribute to the impairment of vascular reactivity and subsequent cardiac function. Treatment with GPER agonist G1 combined with the inhibition of ERα and ERß could improve vascular function and reduce the myocardial ischemia reperfusion injury. These findings may provide the novel and effective strategy for the treatment of cardiovascular diseases in postmenopausal women.

Four novel mutations in the androgen receptor gene from Vietnamese patients with androgen insensitivity syndrome.

BACKGROUND: Androgens and androgen receptor (AR) are critical regulators of the masculinization process in male sexual development. The absence of a functioning AR results in the development of the androgen insensitivity syndrome (AIS), a rare disorder of sexual development (DSD) characterized by the external genitalia feminization, gynecomastia, and impaired spermatogenesis. OBJECTIVE: To determine the AR gene mutations associated with male DSD in four unrelated Vietnamese patients. METHODS: To detect the disease-causing mutations, whole exome sequencing (WES) was performed on four patients diagnosed with AIS. Sanger sequencing was then used for validation of the identified mutations. Finally, 12 web-based tools, three-dimensional protein modeling software, and the guidelines issued by the American College of Medical Genetics and Genomics were used to assess the potential pathogenicity of these mutations. RESULTS: Four distinct novel mutations, namely c.1834T > A (p.Cys612Ser), c.2122 C > G (p.Leu708Val), c.2630T > G (p.Phe877Cys), and c.2641 C > A (p.Leu881Met) in the AR gene, were identified in four AIS patients using WES. The in silico analysis results revealed that the Cys612, Leu708, Phe877, and Leu881 sites are important for an appropriate response to androgens of the AR, and mutation at these sites can have adverse effects on the AR functions, androgen-AR interaction, and AR signaling pathway. CONCLUSIONS: WES and in silico analyses strongly suggested that four novel AR mutations are pathogenic and have led to the development of AIS in the four Vietnamese patients under consideration.

Metastatic prostate cancer diagnosed by fine-needle aspiration: Contemporary cytopathologic and biomarker assessment with clinical correlates.

INTRODUCTION: The diagnosis of metastatic prostatic cancer (MPC) by fine needle aspiration (FNA) can usually be rendered by typical cytomorphologic and immunohistochemical (IHC) features. However, MPC diagnosis may be complicated by transformation to atypical phenotypes such as small cell carcinoma, typically under pressure from androgen deprivation therapy (ADT). Predictive and prognostic biomarkers can also be assessed by IHC. This study illustrates how careful assessment of cytologic and biomarker features may provide therapeutic and prognostic information in MPC. DESIGN: We reviewed our anatomic pathology archives for MPC diagnosed by FNA from January 2014 to June 2021. Clinical histories, cytology slides, and cell blocks were reviewed. Extensive IHC biomarker workup was performed, including markers of prostate lineage, cell-cycle dysfunction, Ki-67, neuroendocrine markers, PDL1, and androgen receptor splice variant 7. Cases were reclassified into three categories: conventional type, intermediary type, and high-grade neuroendocrine carcinoma (HGNC). RESULTS: Eighteen patients were identified. Twelve had conventional MPC, including six of six ADT-naive patients. Six of twelve (50%) with prior ADT were reclassified as intermediary or HGNC. Four intermediary cases included two with squamous differentiation and two with pro-proliferative features. Two HGNC cases had typical small cell carcinoma cytomorphology. Expression of PDL1 was identified in two cases and ARv7 in three cases. Five of five intermediary and HGNC patients died of disease versus six of eleven with with conventional type. CONCLUSIONS: Aggressive cytomorphologic variants were commonly identified in patients with prior ADT. Identification of nonconventional cytomorphology and increased proliferation can provide important prognostic information. Recognition of these changes is important for an accurate diagnosis, and the identification of high-grade variants can affect therapeutic decision-making. Clinically actionable biomarkers such as PDL1 and ARv7 can be assessed by IHC.

Inhibiting androgen receptor splice variants with cysteine-selective irreversible covalent inhibitors to treat prostate cancer.

Androgen receptor (AR) and its splice variants (AR-SVs) promote prostate cancer (PCa) growth by orchestrating transcriptional reprogramming. Mechanisms by which the low complexity and intrinsically disordered primary transactivation domain (AF-1) of AR and AR-SVs regulate transcriptional programming in PCa remains poorly defined. Using omics, live and fixed fluorescent microscopy of cells, and purified AF-1 and AR-V7 recombinant proteins we show here that AF-1 and the AR-V7 splice variant form molecular condensates by liquid-liquid phase separation (LLPS) that exhibit disorder characteristics such as rapid intracellular mobility, coactivator interaction, and euchromatin induction. The LLPS and other disorder characteristics were reversed by a class of small-molecule-selective AR-irreversible covalent antagonists (SARICA) represented herein by UT-143 that covalently and selectively bind to C406 and C327 in the AF-1 region. Interfering with LLPS formation with UT-143 or mutagenesis resulted in chromatin condensation and dissociation of AR-V7 interactome, all culminating in a transcriptionally incompetent complex. Biochemical studies suggest that C327 and C406 in the AF-1 region are critical for condensate formation, AR-V7 function, and UT-143's irreversible AR inhibition. Therapeutically, UT-143 possesses drug-like pharmacokinetics and metabolism properties and inhibits PCa cell proliferation and tumor growth. Our work provides critical information suggesting that clinically important AR-V7 forms transcriptionally competent molecular condensates and covalently engaging C327 and C406 in AF-1, dissolves the condensates, and inhibits its function. The work also identifies a library of AF-1-binding AR and AR-SV-selective covalent inhibitors for the treatment of PCa.

Targeting androgen receptor degradation with PROTACs from bench to bedside.

Inhibition of androgen receptor (AR) has been extensively investigated to treat prostate cancer. Resistance mechanisms such as increased levels of androgen production, increased AR gene, enhancer expression and AR point mutations always reduce the clinical efficacy. Design and discovery of small-molecule PROTAC AR degraders have been pursued as a new therapeutic strategy to overcome common resistance mechanisms developed during prostate cancer treatment. In the last two decades, potent and efficacious PROTAC AR degraders have been gotten rapid development and several such compounds have been advanced into preclinical phase and phase I/II trials for the treatment of human prostate cancers. Especially, the first PROTAC to enter the clinic, ARV-110, has shown good clinical effects in patients with mCRPC. This fully demonstrates the high clinical value of PROTAC strategy in treatment of human diseases. Here, we summarized the recent advances in the development of these potential clinical-stage PROTAC AR degraders.

UGT2B28 accelerates prostate cancer progression through stabilization of the endocytic adaptor protein HIP1 regulating AR and EGFR pathways.

The androgen inactivating UGT2B28 pathway emerges as a predictor of progression in prostate cancer (PCa). However, the clinical significance of UGT2B28 tumoral expression and its contribution to PCa progression remain unclear. Using the Canadian Prostate Cancer Biomarker Network biobank (CPCBN; n = 1512), we analyzed UGT2B28 tumor expression in relation to clinical outcomes in men with localized PCa. UGT2B28 was overexpressed in tumors compared to paired normal adjacent prostatic tissue and was associated with inferior outcomes. Functional analyses indicated that UGT2B28 promoted cell proliferation, and its expression was regulated by the androgen receptor (AR)/ARv7. Mechanistically, UGT2B28 was shown to be a protein partner of the endocytic adaptor protein huntingtin-interacting protein 1 (HIP1), increasing its stability and priming AR/epidermal growth factor receptor (EGFR) pathways, leading to ERK1/2 activation triggering cell proliferation and epithelial-to-mesenchymal transition (EMT). HIP1 knockdown in UGT2B28 positive cells, and dual pharmacological targeting of AR and EGFR pathways, abolished cell proliferative advantages conferred by UGT2B28. In conclusion, UGT2B28 is a prognosticator of progression in localized PCa, regulates both AR and EGFR oncogenic signaling pathways via HIP1, and therefore can be therapeutically targeted by using combination of existing AR/EGFR inhibitors.

Correlation of expression of Major Vault Protein with androgen receptor and immune checkpoint protein B7-H3, and with poor prognosis in prostate cancer.

Prostate cancer diagnosis and early stratification is an important aspect to avoid undertreatment of high-risk prostate cancer patients. Major Vault Protein (MVP) has been proposed as a prognostic biomarker in prostate cancer. PTEN and the immune checkpoint protein B7-H3 interact with MVP and are important in prostate cancer progression and therapy response. We evaluated the expression of MVP by immunohistochemistry of tissue microarray samples from a retrospective cohort consisting of 119 prostate cancer patients. We correlated the protein expression of MVP with clinicopathological characteristics, and protein expression of androgen receptor (AR), PTEN, immune checkpoint proteins B7-H3 and PD-L1. We found MVP to be expressed in 53 % of prostate tumors, and correlated positively with biochemical recurrence (ρ = 0.211/p = 0.021). Furthermore, we found positive correlation of MVP expression with expression of AR (ρ = 0.244/p = 0.009) and the immune checkpoint protein B7-H3 (ρ = 0.200/p = 0.029), but not with PD-L1 (ρ = 0.152/p = 0.117) or PTEN expression (ρ = - 0.034/p = 0.721). Our findings support the notion that expression of MVP is associated with poor prognosis in prostate cancer. The correlation between MVP and immune checkpoint protein B7-H3 in prostate cancer suggests a role for MVP in immunoregulation and drug resistance.