Androgen Receptor Protein Levels Research Articles

Paxillin regulates androgen receptor expression associated with granulosa cell focal adhesions.

Paxillin is a ubiquitously expressed adaptor protein integral to focal adhesions, cell motility, and apoptosis. Paxillin has also recently been implicated as a mediator of nongenomic androgen receptor (AR) signaling in prostate cancer and other cells. We sought to investigate the relationship between paxillin and AR in granulosa cells (GCs), where androgen actions, apoptosis, and focal adhesions are of known importance, but where the role of paxillin is understudied. We recently showed that paxillin knockout in mouse GCs increases fertility in older mice. Here we demonstrate that paxillin knockdown in human granulosa-derived KGN cells, as well as knockout in mouse primary GCs, result in reduced AR protein but not reduced mRNA expression. Further, we find that both AR protein and mRNA half-lives are reduced by approximately one-third in the absence of paxillin, but that cells adapt to chronic loss of paxillin by upregulating AR gene expression. Using co-immunofluorescence and proximity ligation assays, we show that paxillin and AR co-localize at the plasma membrane in GCs in a focal adhesion kinase-dependent way, and that disruption of focal adhesions leads to reduced AR protein level. Our findings suggest that paxillin recruits AR to the GC membrane, where it may be sequestered from proteasomal degradation, and poised for nongenomic signaling, as reported in other tissues. To investigate the physiological significance of this in disorders of androgen excess, we tested the effect of GC-specific paxillin knockout in a mouse model of PCOS induced by chronic postnatal dihydrotestosterone (DHT) exposure. While none of the control mice had estrous cycles, 33% of paxillin knockout mice were cycling, indicating that paxillin deletion may offer partial protection from the negative effects of androgen excess by reducing AR expression. Paxillin-knockout GCs from mice with DHT-induced PCOS also produced more estradiol than GCs from littermate controls. Thus, paxillin may be a novel target in the management of androgen-related disorders in women, such as PCOS.

Abstract 5343: Depletion of gut microbiota with broad spectrum antibiotics drives Pten-null prostate cancer growth in mice

Abstract Gut microbes have the potential to be either promoters or inhibitors of cancer. Use of antibiotics during cancer therapy can lead to dysbiosis, which may affect treatment outcomes. Here, we examine the effects of broad-spectrum antibiotics on cancer progression in mice with Pten-null castration resistant prostate cancer (CRPC). After five or eight weeks, orchiectomized conditional Pten-null knockout (KO) mice aged 50 and 90 weeks were given two biweekly cycles of a broad-spectrum antibody cocktail (BSAbx), respectively. The effects of BSAbx on tumor burden were compared, and molecular and immunological responses were analyzed by quantitative IHC (qIHC) and qRt-PCR at week four after antibiotic treatment. BSAbx treatment resulted in tumor burdens of 1.2 and 2.4-fold higher than in control mice in mice aged 50 and 90 weeks, respectively. qIHC analysis of KI67 showed increased proliferation in stromal and cancer cells. Additionally, androgen receptor (AR) protein and gene expression levels of Ar and Ar target genes (Fkbp5, Tmprss2, and Timp4) were elevated in tumors of BSAbx-treated mice. Restoring gut microbiota by fecal microbiota transplantation (FMT) from KO mice CRPC after BSAbx restored AR transcriptional activity to basal levels and lessened tumor growth. FMT from non-castrated KO with castration-naïve prostate cancer or heathy wildtype mice further reduced AR Transcriptional activity. Moreover, altering the gut microbiota of CRPC mice with FMT from heathy mice resulted in a tendency for lower tumor burden, 21% reduction versus control mice. Immunologically, BSAbx treatment resulted in decreased CD11b+/GR1+ cells in tumors and were associated with decreased interferon-gamma (IFN-gamma), dendritic cell functions and T cell-mediated cytotoxicity gene signatures. Mice that received FMT form health mice showed enhanced T cell mediated immunity as demonstrated by higher IFN-gamma signatures and tumor abundances of CD8+ T cells expression IFN-gamma, TNF-alpha and the cytotoxic granule granzyme B. Our study shows that broad spectrum antibiotics caused dysbiosis and promoted prostate cancer growth in mice with Pten-null prostate cancer. The effects were partly mediated through the upregulation of AR signaling and the impairment of neutrophil and T cell-mediated immune responses in tumors. Citation Format: Marco A. De Velasco, Yurie Kura, Kazuko Sakai, Mitsuhisa Nishimoto, Yasunori Mori, Kazuhiro Yoshimura, Kazutoshi Fujita, Kazuto Nishio, Hirotsugu Uemura. Depletion of gut microbiota with broad spectrum antibiotics drives Pten-null prostate cancer growth in mice [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 5343.

Abstract 3013: Alzheimer’s-linked gene BIN1: Unraveling its unexpected role as a tumor suppressor in prostate cancer and influence on androgen receptor signaling

Abstract Androgen-receptor signaling inhibitors (ARSI) have been shown to significantly alter the natural history of castration-resistant PCa (CRPC). Unfortunately, resistance to ARSIs is inevitable. Unraveling the underlying mechanisms to identify potential therapeutic interventions to treat these lethal tumors has become a critical challenge in the post-ARSI era. Recent genomic sequencing has revealed a subclass of PCa harboring deletion of bridging integrator-1 (BIN1) and is associated with increased AR activity. Notably, BIN1 deletion frequently co-occurs with SPOP mutation and deletion of SPOPL, collectively suggesting BIN1 may function as tumor suppressor. To elucidate the role of BIN1 in prostate cells, we generated a prostate-specific BIN1 knockout murine model (PB-CreBIN1FL/FL). This model revealed increased prostate mass at 6 months of age, accompanied by increased cell proliferation. We next crossed this strain to SPOP mutant (SPOPF133V) model to generate a prostate specific BIN1 knockout, SPOP mutant model (PB-CreSPOPF133VBIN1FL/FL) and found increased proliferation and greater prostate mass compared to animals harboring SPOP mutation or BIN1 deletion alone. Next, we generated a doxycycline inducible BIN1 overexpressing PCa model (22Rv1BINOE) to evaluate AR signaling in vitro. We found that overexpression of BIN1 modestly reduced prostate cancer cell proliferation but profoundly suppressed androgen receptor protein level. To evaluate the transcriptional activity of AR and ARv7, we also utilized a luciferase reporter system harboring ARE consensus sequences found in the KLK3 gene (AR-FL target) promoter or in the EDN2 gene (ARv7 target) promoter, respectively. These reporter assays illustrated suppression of both AR-FL and AR-v7 driven gene transcriptional program upon BIN1 overexpression. Co-expression of both BIN1 and SPOPwt showed a synergistic knockdown of AR activity, while SPOPmut did not show this effect. These observations were further confirmed using global RNAseq. The RNAseq analysis also showed downregulation of several AR pathways as well as gene sets associated with cancer progression and metastasis. Interestingly, the RNAseq data showed downregulation of genes that are normally upregulated after SPOP mutation, further suggesting a synergistic role between SPOP and BIN1. Citation Format: Collin McColl, Darlene Skapura, Elisa Ruiz Echartea, Jenny Deng, Aleksandra Rusin, Jinna Shin, Alexey Tyryshkin, Christel Davis, Erik Ehil, Salma Kaochar. Alzheimer’s-linked gene BIN1: Unraveling its unexpected role as a tumor suppressor in prostate cancer and influence on androgen receptor signaling [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 3013.

Abstract 7592: Combined inhibition of CDK4/6 and AKT is highly active against the luminal androgen receptor (LAR) subtype of triple negative breast cancer (TNBC)

Abstract The LAR subtype of TNBC is enriched for targetable biomarkers such as androgen receptor (AR) expression, PIK3CA mutations, and intact Rb. The purpose of this study was to investigate the most effective combination of CDK4/6, AR, and PI3K-AKT inhibitors in preclinical models of LAR TNBC. The MDAMB453 and MFM223 (AR positive/Rb intact/PIK3CAmutant) LAR TNBC cells were treated with each inhibitor alone or in combination. Drug sensitivity was assessed using colony formation, cell viability, and live-cell analysis. The Loewe additivity equation was used to measure the additive drug effects. Immunoblot analysis was used to evaluate cell cycle and PI3K-AKT signaling and ARE luciferase assay was used to assess AR transcriptional activity. MDAMB453 and MFM223 cells were sensitive to the single agents palbociclib, alpelisib, and capivasertib (IC50, ~500 nM), which inhibited CDK4/6, PI3Kα, and AKT, respectively. Enzalutamide, an AR antagonist, exerted no growth inhibitory activity (IC50, 15-25 μM). Treatment with palbociclib, alpelisib, or capivasertib did not alter the AR protein levels or transcriptional activity. The combination of palbociclib+capivasertib synergistically inhibited the proliferation of MDAMB453 and MFM223 cells (synergistic scores 7.34, p=5.81-11, and 4.78, p=0.012, respectively) better than palbociclib+alpelisib. Enzalutamide did not enhance either combination. Palbociclib+capivasertib inhibited PI3KCA-mutant and Rb-intact LAR TNBC PDX growth more potently than palbociclib+alpelisib (p=0.046). Treatment of LAR TNBC cells with palbociclib suppressed Rb phosphorylation and resulted in adaptive activation of P-AKTS473/T308 and the AKT substrates GSK3β, PRAS40, and FoxO3a after 24 h. Capivasertib inhibited the phosphorylation of AKT and AKT substrates, following CDK4/6 blockade with palbociclib, more potently than alpelisib. PI3Kβ inhibitors (TGX221, GSK2636771) did not block AKT substrate upregulation, suggesting PI3Kβ does not mediate adaptive responses. Rictor knockdown failed to reduce AKT activation following palbociclib treatment, suggesting an mTORC2-independent mechanism. Human Phospho-kinase Array of MDA-MB-453 cells treated with palbociclib showed time dependent upregulation of GSK3βS9, STAT3Y727 and PDFGRβY751. PDGFRβ activation after palbociclib treatment was confirmed in MFM223 cells. PDGFRβ siRNAs in MDAMB453 cells blocked P-AKTS473 upregulation after palbociclib treatment. Treatment with palbociclib and the PDGFRβ small-molecule antagonist CP673451 arrested LAR TNBC growth similar to palbociclib+capivasertib, suggesting an adaptive response involving PDGFRβ signaling. In conclusion, this study supports the clinical testing of the palbociclib and capivasertib combination for LAR TNBC patients and reveals adaptive responses after palbociclib treatment through PDGFRβ signaling. Citation Format: Rosario Chica-Parrado, Gun Min Kim, Yasuaki Uemoto, Dan Ye, Fabiana Napolitano, Chang-Ching Lin, Emmanuel Bikorimana, Kyung-min Lee, Saurabh Mendiratta, Ariella B. Hanker, Carlos L. Arteaga. Combined inhibition of CDK4/6 and AKT is highly active against the luminal androgen receptor (LAR) subtype of triple negative breast cancer (TNBC) [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 7592.

Chronic exposure to low levels of phenanthrene induces histological damage and carcinogenic risk in the uterus of female mice.

Phenanthrene (Phe), a polycyclic aromatic hydrocarbon with low molecular weight, is detected in the environment at high frequency. To study the toxic effects of Phe on the uterine structure and function, female Kunming mice were exposed to Phe (0.05, 0.5, 5ng/mL) for 270days by drinking water. Pathological alterations and their action pathways were analyzed using immunohistochemical and biomolecular technology. Phe significantly increased the percentage of blood vessel area, the number of endometrial neutrophils (indicating the occurrence of inflammation), collagen deposition (indicating fibrosis), and the percentage of Ki-67-positive cells (indicating carcinogenesis) in the uterus. Transcriptome sequencing identified differentially expressed genes that were mainly enriched in some signaling pathways, including inflammation and carcinogenesis, suggesting a carcinogenic risk in the Phe-exposed uterus. Elevated serum estrogen levels and decreased progesterone levels exhibited a disturbance of steroid hormone balance, which might be related to uterine damage. Upregulated protein levels of uterine androgen receptor and estrogen receptor α were linked to the pathological effects. Most of the effects exhibited a nonmonotonic dose response, which might be attributed to the corresponding change in the serum levels of Phe. The results suggest that exposure to low levels of Phe could exert adverse effects on the uterus.

MDM2 regulates the stability of AR, AR-V7, and TM4SF3 proteins in prostate cancer.

Androgen receptor (AR) and its constitutively active splice variant, AR Variant 7 (AR-V7), regulate genes essential for the development and progression of prostate cancer. Degradation of AR and AR-V7 by the ubiquitination proteasomal pathway is important for the regulation of both their protein stability. Our published results demonstrate that the interaction of TM4SF3 with either AR or AR-V7 leads to mutual stabilization due to a reduction in their ubiquitination and proteasomal degradation. These results led us to search for a common E3 ligase for AR, AR-V7, and TM4SF3. Depletion by siRNA of several E3 ligases identified MDM2 as the common E3 ligase. MDM2 inhibition by siRNA depletion or using a pharmacological inhibitor (MDM2i) of its E3 ligase activity led to elevated levels of endogenous AR, AR-V7, and TM4SF3 in prostate cancer cells. MDM2 knockdown in PC-3 cells, which do not express AR, also increased TM4SF3, demonstrating that MDM2 affects the TM4SF3 protein independent of AR. We further demonstrate that MDM2i treatment reduced the ubiquitination of AR and TM4SF3, suggesting that MDM2 can induce the ubiquitination of these proteins. Increased AR and AR-V7 protein levels induced by MDM2i treatment resulted in the expected increased expression of AR-regulated genes and enhanced proliferation and migration of both LNCaP and Enzalutamide-resistant CWR-22Rv1 prostate cancer cells. Thus, our study expands the known roles of MDM2 in prostate cancer to include its potential involvement in the important mutual stabilization that TM4SF3 exhibits when interacting with either AR or AR-V7.

Does Nanosilver Exposure Modulate Steroid Metabolism in the Testes?-A Possible Role of Redox Balance Disruption.

Silver nanoparticles (AgNPs) are a popular engineered nanomaterial widely used in industry. Despite the benefits they bring to society, AgNPs are not neutral to human health. The aim of this study was to evaluate the effects of a single intravenous dose (5 mg/kg body weight) of 20 nm AgNPs on steroid metabolism and redox balance in the testes of adult rats. The effects were evaluated 1 day or 28 days after intervention and compared with saline-treated animals. Decreased aromatase and estrogen receptor α levels (by 21% and 27%, respectively) were observed 1 day after AgNPs administration, while increased testosterone, increased dihydrotestosterone levels, higher androgen receptors and higher aromatase expression in Leydig cells (by 43%, 50%, 20% and 32%, respectively) as well as lower (by 35%) androgen receptor protein levels were observed 28 days after exposure to AgNPs compared to control groups. The AgNPs treatment resulted in decreased superoxide dismutase activity, decreased GSH/GSSG ratio, and increased glutathione reductase activity (by 23%, 63% and 28%, respectively) compared to control animals, irrespective of the time of measurement. Increased (by 28%) intratesticular lipid hydroperoxides level was observed 1 day after AgNPs exposure, while decreased (by 70%) GSH and increased (by 43%) 7-ketocholesterol levels were observed 28 days after treatment compared to control animals. Conclusions: AgNPs exposure caused redox imbalance in the gonads shortly after AgNPs administration, while a longer perspective AgNPs exposure was associated with impaired androgen metabolism, probably due to increased oxidative stress.

RNA N6-methyladenosine-modified-binding protein YTHDF1 promotes prostate cancer progression by regulating androgen function-related gene TRIM68

PurposeThere is no report about the direct relationship between m6A modification and androgen receptor (AR)-related genes in prostate cancer (PC). We aimed to study the mechanisms of m6A methylation in regulating the pathogenesis of PC from the perspective of AR-related genes.MethodsqRT-PCR was applied to detect the expression of m6A-related genes in PC cell with or without AR inhibitor. The effects of YTHDF1 knockdown on PC cell viability, apoptosis, migration and invasion were investigated using flow cytometry, wound healing and transwell assays, respectively. The mechanism of YTHDF1 action was investigated using m6A RNA immunoprecipitation (MeRIP) sequencing. The biological functions of YTHDF1 were also explored through in vivo experiments.ResultsYTHDF1 was significantly down-regulated in AR inhibitor group. YTHDF1 knockdown significantly decreased AR level, viability and m6A methylation level of PC cells. TRIM68 was identified as a direct target of YTHDF1. Both YTHDF1 and TRIM68 knockdown increased apoptosis, and decreased cell viability, migration, and invasion of PC cells, while TRIM68 overexpression reversed the effects of YTHDF1 knockdown on PC cells. In addition, knockdown of YTHDF1 or TRIM68 significantly decreased the m6A methylation level, and mRNA and protein levels of YTHDF1, TRIM68 and AR in PC cells, while TRIM68 overexpression increased the expression levels above. Furthermore, subcutaneous xenografts of nude mice also revealed that TRIM68 could reverse the effects of YTHDF1 knockdown in PC in vivo.ConclusionThis study suggested the key role of YTHDF1-mediated m6A modification in PC progression by regulating androgen function-related gene TRIM68 in PC.

Inhibition of N-myristoyltransferase activity promotes androgen receptor degradation in prostate cancer.

Even though prostate cancer (PCa) patients initially respond to androgen deprivation therapy, some will eventually develop castration resistant prostate cancer (CRPC). Androgen receptor (AR) mediated cell signaling is a major driver in the progression of CRPC while only a fraction of PCa becomes AR negative. This study aimed to understand the regulation of AR levels by N-myristoyltransferase in PCa cells. Two enantiomers, (1S,2S)- d-NMAPPD and (1R,2R)- d-NMAPPD (LCL4), were characterized by various methods (1 H and 13 C NMR, UHPLC, high-resolution mass spectra, circular dichroism) and evaluated for the ability to bind to N-myristoyltransferase 1 (NMT1) using computational docking analysis. structure-activity relationship analysis of these compounds led to the synthesis of (1R,2R)-LCL204 and evaluation as a potential NMT1 inhibitor utilizing the purified full length NMT1 enzyme. The NMT inhibitory activity wase determined by Click chemistry and immunoblotting. Regulation of NMT1 on tumor growth was evaluated in a xenograft tumor model. (1R,2R)- d-NMAPPD, but not its enantiomer (1S,2S)- d-NMAPPD, inhibited NMT1 activity and reduced AR protein levels. (1R,2R)-LCL204, a derivative of (1R,2R)- d-NMAPPD, inhibited global protein myristoylation. It also suppressed protein levels, nuclear translocation, and transcriptional activity of AR full-length or variants in PCa cells. This was due to enhanced ubiquitin and proteasome-mediated degradation of AR. Knockdown of NMT1 levels inhibited tumor growth and proliferation of cancer cells. Inhibitory efficacy on N-myristoyltransferase activity by d-NMAPPD is stereospecific. (1R,2R)-LCL204 reduced global N-myristoylation and androgen receptor protein levels at low micromolar concentrations in prostate cancer cells. pharmacological inhibition of NMT1 enhances ubiquitin-mediated proteasome degradation of AR. This study illustrates a novel function of N-myristoyltransferase and provides a potential strategy for treatment of CRPC.

Antagonism of androgen receptor signaling by aloe-emodin

Over the past decades, androgen receptor (AR) signaling has been a key driver of both primary and recurrent prostate cancer. In this work, aloe-emodin was identified as a novel AR antagonist, effectively inhibiting AR signaling. Firstly, aloe-emodin can inhibit LNCaP cell growth by promoting apoptosis. Then, the results of Western blot and quantitative real-time PCR further confirmed that aloe-emodin modulated AR protein levels by promoting AR proteasomal degradation, and also inhibited the transcription of the AR downstream target genes, including PSA, KLK2, and TMPRSS2. Furthermore, the result of immunofluorescence showed that aloe-emodin prevented the nuclear translocation of AR. Molecular docking and molecular dynamics simulation suggested that aloe-emodin combined with AR to form stable complexes, which might explain that aloe-emodin prevented the translocation of AR from the cytoplasm to the nucleus by affecting the ligand binding of AR. Therefore, aloe-emodin as a novel AR antagonist may play a crucial role in promoting cancer prevention or complementing pharmacological therapies in the treatment of prostate cancer.

OR35-02 Novel Androgen Receptor PROTAC Degrader ARV-110 Ameliorates Androgen Mediated Renal Injury in a Mouse Model of Polycystic Ovary Syndrome

Abstract Disclosure: J. Basnet: None. S. Rezq: None. A.M. Huffman: None. T.E. Asala: None. L.L. Yanes Cardozo: None. D.G. Romero: None. Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women. PCOS women often have increased central adiposity, insulin resistance, and renal injury. Hyperandrogenism is prevalent in PCOS women and is associated with renal injury and dysfunction. Androgens bind to the androgen receptor (AR) to impart downstream transcriptional activity. Targeting the AR may alleviate PCOS complications; however, the use of AR blockers is limited by their severe adverse effects in females; thus, safer alternative approaches are highly needed. The novel AR Proteolysis Targeting Chimera (PROTAC) degrader ARV-110 has shown high efficacy to prevent AR signaling in prostate cancer; however, its efficacy in PCOS is unknown. Using a well-established hyperandrogenemic mouse model of PCOS, we aim to test the hypothesis that decreasing AR protein levels with ARV-110 ameliorates androgen-mediated derangements in PCOS. Methods: Three-week old female mice (C57BL/6N) were implanted with Silastic tubes filled with the androgen dihydrotestosterone (DHT, 8mg, s.c., 90 days) or vehicle (n=6/grp). Four weeks post-DHT administration, mice were treated with ARV-110 (1 mg/kg/day, s.c.) for an additional 8 weeks. Body composition (EchoMRI) and oral glucose tolerance test (OGTT) were assessed. Kidney weight was measured by gravimetry. AR, extracellular matrix (ECM) remodeling marker MMP9, pro-fibrotic marker TGF-β, renal injury markers (urinary albumin to creatinine ration (UACR), KIM1, and NGAL) were assessed by Western-blot or ELISA. Results: DHT mice showed significant (p<0.05) increases in body weight (27.3±0.6 vs 23.9±0.26 g), fat mass (4.2±0.6 vs 2.1±0.1 g), lean mass (21.6±0.2 vs 19.8±0.2 g), as well as kidney weight (491.3±7.69 vs 303.1±5.5 mg) compared to controls. DHT increased fasting glucose (183.2±25.2 vs 141.5±9.9 mg/dL, p<0.05) and impaired OGTT (AUC:34557±6255 vs 23459±1896 mg.min/dL, p<0.05). ARV-110 treatment ameliorated all aforementioned DHT-mediated changes. At the molecular level, DHT-treated mice had higher renal AR (2.5-fold) and MMP9 (1.5-fold) protein expression. DHT upregulated renal TGF-β levels (2-fold, p<0.05), UACR (966.8±160.9 vs 381.5±41.2 ng albumin/mg creatinine), and urinary KIM1 (3.5-fold) and NGAL (6-fold). ARV-110 successfully decreased renal AR protein levels by 70% in DHT mice compared to vehicle-treated DHT. Excitingly, while ARV-110 did not modulate renal TGF-β levels, it completely abolished DHT-mediated increase in renal MMP9 while significantly lowering the levels of urinary renal injury markers UACR, KIM1 and NGAL. Conclusion and significance: Our findings suggest that DHT-mediated kidney hypertrophy, associated ECM remodeling and injury are ameliorated by ARV-110 in DHT-treated mice. AR PROTACs could be a novel therapeutic approach to treat renal dysfunction in PCOS. Supported by NIH P20GM121334. Presentation Date: Sunday, June 18, 2023

THU573 Paxillin Regulates Androgen Receptor In Granulosa Cells

Abstract Disclosure: A. Weidner: None. A. Roy: None. K. Vann: None. O. Astapova: None. Paxillin is a ubiquitously-expressed adaptor protein that is integral in focal adhesions and plays an active role in the regulation of cell motility and apoptosis. Recently, paxillin was shown to mediate pro-tumorigenic androgen signaling in prostate cancer cells, where it is necessary for rapid cytoplasmic kinase signaling triggered by activation of membrane-bound androgen receptor. We sought to investigate the relationship between paxillin and androgen receptor (AR) in granulosa cells, where androgen actions, apoptosis and focal adhesions are of known importance, but the role of paxillin is understudied. When paxillin expression was silenced using siRNA in human granulosa-derived KGN cells, AR protein expression was significantly reduced, while its RNA expression was unaffected. Similarly, in granulosa cell-specific paxillin knockout mice, AR protein expression in granulosa cells was reduced. Further, we found that AR and paxillin co-localize in focal adhesions in mouse primary granulosa cells and in KGN cells, and their interaction requires the activity of focal adhesion kinase. Disruption of focal adhesion with a focal adhesion kinase inhibitor also leads to reduced AR protein levels. To further explore the mechanisms by which paxillin regulates AR expression, we created paxillin-null KGN cell lines using CRISPR-Cas9 gene editing. While paxillin loss resulted in reduced AR protein expression early (days) in the gene-editing process, this effect disappeared after weeks of clonal expansion from selected single cells. Expanded paxillin-null KGN clones showed normal AR protein expression, but increased AR mRNA expression, suggesting an adaptive increase in AR gene transcription to compensate for reduced AR protein levels in cells that survived paxillin deletion. Studies of AR protein degradation using cycloheximide to block protein synthesis showed that the half-life of AR was reduced from 6.7 to 4.5 hours (approximately 33%) by paxillin deletion, indicating accelerated AR protein degradation in cells lacking paxillin. Based on our results, we hypothesize that paxillin brings AR into focal adhesions in granulosa cells, thereby sequestering it from proteasomal degradation in the cytoplasm. Thus, paxillin may be a novel target in the management of androgen-related disorders in women, such as polycystic ovary syndrome. Presentation Date: Thursday, June 15, 2023

Pinostilbene inhibits full-length and splice variant of androgen receptor in prostate cancer

Prostate cancer is the most prevalent cancer in men worldwide and is promoted by the sex hormone androgen. Expression of androgen from the testis can be significantly reduced through castration. However, as most prostate cancer patients acquire castration resistance, additional therapeutic solutions are necessary. Although anti-androgens, such as enzalutamide, have been used to treat castration-resistant prostate cancer (CRPC), enzalutamide-resistant CRPC (Enz-resistant CRPC) has emerged. Therefore, development of novel treatments for Enz-resistant CRPC is urgent. In this study, we found a novel anti-androgen called pinostilbene through screening with a GAL4-transactivation assay. We confirmed that pinostilbene directly binds to androgen receptor (AR) and inhibits its activation and translocalization. Pinostilbene treatment also reduced the protein level and downstream gene expression of AR. Furthermore, pinostilbene reduced the protein level of AR variant 7 in the Enz-resistant prostate cancer cell line 22Rv1 and inhibited cell viability and proliferation. Our results suggest that pinostilbene has the potential to treat Enz-resistant CRPC.

Discovery of ARD-1676 as a Highly Potent and Orally Efficacious AR PROTAC Degrader with a Broad Activity against AR Mutants for the Treatment of AR + Human Prostate Cancer.

We report herein the discovery and extensive characterization of ARD-1676, a highly potent and orally efficacious PROTAC degrader of the androgen receptor (AR). ARD-1676 was designed using a new class of AR ligands and a novel cereblon ligand. It has DC50 values of 0.1 and 1.1 nM in AR+ VCaP and LNCaP cell lines, respectively, and IC50 values of 11.5 and 2.8 nM in VCaP and LNCaP cell lines, respectively. ARD-1676 effectively induces degradation of a broad panel of clinically relevant AR mutants. ARD-1676 has an oral bioavailability of 67, 44, 31, and 99% in mice, rats, dogs, and monkeys, respectively. Oral administration of ARD-1676 effectively reduces the level of AR protein in the VCaP tumor tissue in mice and inhibits tumor growth in the VCaP mouse xenograft tumor model without any sign of toxicity. ARD-1676 is a highly promising development candidate for the treatment of AR+ human prostate cancer.

Abstract A006: Loss of PMEPA1 gene isoform facilitates the development of hormone and radiation therapies resistance in prostate cancer cells

Abstract Introduction: Nearly all the patients receiving hormone treatment eventually progress into castration resistance stage. In addition, approximately 30% to 40% of patients receiving radiation treatment will develop resistance. The aberrant alterations of androgen receptor (AR) and transforming growth factor-β (TGF-β) signaling play critical roles in such processes. Our studies identified five PMEPA1 (Prostate Transmembrane Protein Androgen Induced 1) gene isoforms in prostate cancer cells. PMEPA1-b isoform was androgen responsive and inhibited androgen signaling. In contrast, PMEPA1 isoforms a and d were TGF-β inducible and suppressed TGF-β signaling. Loss or reduced expressions of PMEPA1 was detected in over 60% of prostate cancer samples. In this study, we investigated the impacts of PMEPA1 isoforms (a, b and d) on the development of hormone and radiation therapies resistance in prostate cancer. Methods: RNA-seq data of unmatched 50 benign and 499 malignant prostate samples from The Cancer Genome Atlas (TCGA) dataset, as well as 101 metastatic castration resistant prostate cancer (mCRPC) biopsy samples from Quigley lab at UCSF (WCDT dataset) were used to study the transcript levels of PMEPA1 isoforms (a, b and d) and their correlations to disease progression. Hormone dependent LNCaP cells and androgen independent PC-3, DU-145 cells were utilized to investigate the impacts of PMEPA1 isoforms (a, b and d) on the responses to of androgen inhibitors (enzalutamide and bicalutamide, 0, 5.0 and 25.0 nM) and radiation (0, 1.0 and 5.0 Gy). The vectors of pcDNA3.1-PMEPA1-a, -b and -d were transfected into prostate cancer cells via Lipofectamine, and endogenous PMEPA1 isoforms were knocked down by isoforms specific siRNAs. The cell growth was assessed by cell counting, BrdU incorporation and cell plating efficiency assays. The protein levels of AR and PSA were evaluated by immunoblotting. The transcript levels of PSA, NKX3.1, NEDD9 and THBS1 were assessed by RT-PCR technology. Results: The reduced transcript levels of PMEPA1 isoforms (a, b and d) were detected in mCRPC group. Lower expression of PMEPA1-b was significantly associated with earlier biochemical recurrence. The growth inhibition and androgen signaling blockage mediated by androgen inhibitors were explicitly reversed by depletion of PMEPA1-b in LNCaP cells. Ectopic PMEPA1-b in isoform b depleted LNCaP cells reinstituted the growth inhibition induced by androgen inhibitors. Knockdown of isoforms a and d conspicuously rescued the decreased cell proliferation and suppressed TGF-β signaling resulted from radiation treatment in PC-3 and DU-145 cells. Similarly, over-expression of PMEPA1-a and -d in siRNA treated cells recapitulated the growth inhibitory effects of radiation treatments. Conclusion: Loss of PMEPA1 gene isoforms (a, b and d) induced the development of resistance to hormone ablation and radiation treatments in prostate cancer cells in vitro through activating androgen and TGF-β signaling. Our research provided a new insight into treatment resistance mechanism of prostate cancer cells. Citation Format: Shashwat Sharad, Hua Li. Loss of PMEPA1 gene isoform facilitates the development of hormone and radiation therapies resistance in prostate cancer cells [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 A006.

Abstract B066: Genome-wide CRISPR screens identify PTGES3 as a druggable AR modulator

Abstract BACKGROUND The androgen receptor (AR) is the central therapeutic target in advanced prostate cancer (PCa). While treatments that directly target AR are effective in prolonging patient survival, aggressive prostate cancers invariably find a way to evade these therapies. Frequently, evasion mechanisms reestablish AR expression and therefore AR-driven oncogenic pathways are restored. Comprehensively understanding the protein regulation machinery of AR and identifying alternative therapeutic targets are crucial for effective treatment of this disease. METHODS To study regulators of AR, we utilized CRISPR-Cas9 technology to knock-in a split neon green fluorescent protein onto AR in prostate cancer cells. We evaluated the AR expression and activity in these AR reporter cell lines by multiple approaches including ARE-luciferase assay, ChIP, and RNA-seq. We further built a unique loss-of-function CRISPRi screening system in this AR reporter cell line where we performed a genome-wide fluorescence-activated cell sorting based screen and functionally interrogated hits that modulate AR protein levels. RESULTS We established the first prostate cancer cell line models harboring an endogenous AR reporter. Extensive genotypic and phenotypic characterization of this system demonstrated that the reporter is effective and does not alter AR activity. These models have enabled us to precisely measure the dynamic endogenous AR protein levels by its fluorescence signal. By utilizing a genome-wide CRISPRi screening method in this system, we identified classic AR regulators such as HOXB13, GATA2 and GRHL2, as well as many novel genes that have not been characterized in the context of prostate cancer. Among these novel hits, Prostaglandin E synthase 3 (PTGES3) is the highest ranked druggable target. We validated this finding by establishing PTGES3 knockdown cell lines and demonstrated that loss of PTGES3 downregulated AR and its downstream genes. Follow-up studies show that beyond the canonical chaperone function, PTGES3 physically interacts with AR in the nucleus, where it promotes AR activity. PTGES3 is required for cell proliferation in vitro and in vivo in multiple AR-driven PCa models but not in AR-independent PCa models, such as PC3 cells. From our preliminary data, this unique interaction further suggests that high tumor PTGES3 expression is associated with resistance to first-line androgen deprivation therapies in a retrospective clinical cohort. These results collectively confirm that PTGES3 regulates AR activity and is a potential therapeutic target. Lastly, using a disulfide tethering fragment screen, we developed a PTGES3 inhibitor that blocks the PTGES3/AR interaction and represses AR signaling in PCa cells. We aim to further optimize this PTGES3-specific inhibitor as a therapeutic alternative for patients with aggressive prostate cancer. CONCLUSIONS Together, these genetic, biochemical and chemical biology experiments point to a new class of AR-directed PTGES3 inhibitor therapies that may overcome known mechanisms of ARSI resistance in mCRPC. Citation Format: Haolong Li, James E. Melnyk, Becky Xu Hua Fu, Siyu Feng, Martin Sjöström, Raunak Shrestha, Meng Zhang, Lisa N. Chesner, Hyun Jin Shin, Marsha Calvert, Jonathan Chou, Rajdeep Das, Emily A. Egusa, Aidan Winters, Jun Zhu, Ashutosh Maheshwari, Junjie T. Hua, Mohammed Alshalalfa, William S. Chen, Bradley A. Stohr, Javed Siddiqui, Bo Huang, Eric J. Small, David A. Quigley, Kevan M. Shokat, Luke A. Gilbert, Felix Y. Feng. Genome-wide CRISPR screens identify PTGES3 as a druggable AR modulator [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 B066.

Abstract B004: Genomic loss of TP53 impairs AR-targeted activity in metastatic prostate cancer

Abstract Background: Metastatic prostate cancer (mPC) is notable for a spectrum of recurrent genomic alterations, some associated with overall prognosis and others predicting response to specific therapeutics. Standard mPC interventions are designed to suppress the signaling program regulated by the androgen receptor (AR). These include androgen deprivation therapy (ADT) to reduce AR ligand levels and AR signaling inhibitors (ARSI) that directly impair AR function. While ADT and ARSI produce clinical responses in most patients, the duration and depth of response vary greatly. In addition, specific recurring genomic alterations are associated with shorter responses after ADT and ARSI therapy. Among these are mutations and/or genomic copy loss involving key tumor suppressor genes, including PTEN, RB1 and TP53. However, the mechanistic contribution of TP53 loss to AR signaling and resistance to AR pathway-directed therapy remains unresolved. Methods: Isogenic prostate cancer cell lines with intact and TP53 deletions were constructed using CRISPR/Cas9. The transcriptome profiles were determined by RNA-seq, and ChIP-seq was used to determine the chromatin landscapes of AR localization. In addition, we performed ATAC-seq to assess chromatin accessibility. Finally, to further validate the findings, we comprehensively analyzed AR-regulated gene expression and AR cistromes across a panel of patient-derived xenograft (PDX) models and clinical cohorts of mPC patient samples with respect to TP53 status. Results: Compared to TP53-wild-type (WT) cells, the transcriptional results of isogenic knockout TP53 lines showed impaired AR canonical activity. However, the downregulated AR response pathway is not caused by reduced levels of AR transcript or protein levels. Additionally, a similar trend was also found while comparing TP53-WT PDX models with TP53-loss models. We found that the loss of TP53 can shift the chromatin binding of AR, and the altered AR binding sites correlated with genes involved in neurogenesis pathways, which provide a potential mechanism of how the loss of TP53 might impact AR signaling and promote ADT and ARSI treatment resistance. We also identified additional AR chromatin binding sites that were enriched due to the loss of TP53. The targets potentially regulated by these sites are involved in multiple pathways, including cell morphogenesis involved in differentiation and response to the endogenous stimulus. Conclusions and Future Directions: Overall, the results indicate that in addition to altering DNA damage and repair pathways and contributing to cancer progression, genetic loss of TP53 in mPC may directly impair ADT and ARSI therapies, partially through alteration of AR chromatin activity. In addition, the AR cistrome reprogramming resulting from TP53 loss could also affect other pathways that could be exploited to overcome ARSI treatment resistance. Citation Format: Wanting Han, Michael D. Nyquist, Ilsa Coleman, Navonil De Sarkar, Lisa Ang, Eva Corey, Peter S. Nelson. Genomic loss of TP53 impairs AR-targeted activity in metastatic prostate cancer [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 B004.

Palmitoyl acyltransferase ZDHHC7 inhibits androgen receptor and suppresses prostate cancer.

The hormonal transcription factor androgen receptor (AR) is a master regulator of prostate cancer (PCa). Protein palmitoylation, which attaches a palmitate fatty acid to a substrate protein, is mediated by a class of 23 ZDHHC (Zinc-Finger DHHC motif)-family palmitoyltransferases. Although palmitoylation has been shown to modify many proteins and regulate diverse cellular processes, little is known about ZDHHC genes in cancer. Here we examined ZDHHC family gene expression in human tissue panels and identified ZDHHC7 as a PCa-relevant member. RNA-seq analyses of PCa cells with ZDHHC7 de-regulation revealed global alterations in androgen response and cell cycle pathways. Mechanistically, ZDHHC7 inhibits AR gene transcription and therefore reduces AR protein levels and abolishes AR signaling in PCa cells. Accordingly, ZDHHC7 depletion increased the oncogenic properties of PCa cells, whereas restoring ZDHHC7 is sufficient to suppress PCa cell proliferation and invasion in vitro and mitigate xenograft tumor growth in vivo. Lastly, we demonstrated that ZDHHC7 is downregulated in human PCa compared to benign-adjacent tissues, and its loss is associated with worse clinical outcomes. In summary, our study reveals a global role of ZDHHC7 in inhibiting androgen response and suppressing PCa progression and identifies ZDHHC7 loss as a biomarker for aggressive PCa and a target for therapeutic intervention.

Design, Synthesis, and Biological Evaluation of Androgen Receptor (AR) Antagonist-Heat Shock Protein 90 (Hsp90) Inhibitor Conjugates for Targeted Therapy of Castration-Resistant Prostate Cancer.

Androgen deprivation in cases of castration-resistant prostate cancer (CRPC) leads to adverse effects, including loss of muscle and bone mass and gain of subcutaneous fat. The tumor-specific suppression of androgen receptor (AR) signaling, while not global, may reduce side effects. We present a class of small-molecular conjugates consisting of an AR antagonist linked to a heat shock protein 90 (Hsp90) inhibitor. We demonstrate that the high accumulation of Hsp90 on the surface of CRPC cells allows uptake of conjugates and increases the enrichment of drugs in the tumor cells. After penetrating prostate cancer cells, the conjugates not only inhibit AR function by the antagonist component but also bind to Hsp90 and suppress the AR protein level. Compared to AR antagonists, these conjugates showed improved tumor-targeting ability and enhanced potency against Enzalutamide-resistant 22Rv1 cells.

Association of Androgen-Receptor Gene Mutations with the Copy Number of Androgen-Receptor Silk Protein A Complex and Glutathione-S-Transferases T1 and M1 in Prostate Cancer Patients.

The purpose of our work was to explore the association of mutations in the androgen receptor gene and copy numbers of the androgen-receptor silk protein A complex with glutathione-S-transferases T1 and M1 in prostate cancer patients. Eighty-five patients with PC and 85 healthy controls were included in the study. Fasting peripheral venous blood was collected, whole blood genomic DNA was extracted, and AR gene-receptor genotype was detected by a high-resolution melting curve analysis detection technology. Expression levels of androgen receptor (AR) and filamin protein A (FlnA) were detected by Western blotting. RT-PCR was used to detect the copy number of T1 and M1 glutathione-S-transferases. The wild-type androgen receptor gene rs5918762 is of TT type. The frequencies of CC and TC genes in the prostate cancer group were significantly higher than those in the normal control group (P < 0.05). Compared with TT-type PC patients, PC patients with TC-type and CC-type had higher expression levels of sex hormone receptor silk protein A complex and higher copy numbers of GSTT1 and GSTM1 (P < 0.05). Androgen-receptor gene mutation (T ⟶ C) was significantly positively correlated with the expression level of androgen-receptor silk protein A complex and the copy number of GSTT1 and GSTM1. Androgen-receptor gene polymorphisms were significantly associated with expression levels of androgen receptor complex A and silk proteins, and copy numbers of T1 and M1 glutathione-S-transferases. A combination of four factors can be used to identify prostate cancer susceptibility and disease progression.