Androgen Receptor Expression Research Articles

Predictive and Prognostic Values of Glycoprotein 96, Androgen Receptors, and Extranodal Extension in Sentinel Lymph Node-Positive Breast Cancer: An Immunohistochemical Retrospective Study

Objectives: In this paper, we investigate the association of glycoprotein 96 (GP96) and androgen receptor (AR) expression with clinicopathological factors, additional axillary lymph node burden, and their potential role in predicting 5-year overall survival (OS) and disease-free survival (DFS) in breast cancer (BC) patients with sentinel lymph node (SLN) involvement. We also explore the prognostic value of the presence of extranodal extension (ENE) in SLN. Methods: We retrospectively enrolled 107 female patients with cT1-T2 invasive BC and positive SLN biopsy. GP96 and AR expression were immunohistochemically evaluated on tissue microarrays constructed from two 2 mm diameter cores of formalin-fixed paraffin-embedded tumor tissues from each patient. ENE in SLN was measured in the highest (HD-ENE) and widest diameter (WD-ENE). Relative GP96 gene expression was determined using real-time quantitative PCR. Results: The analysis revealed ENE in SLN as the strongest predictive factor for non-SLN metastases. Patients with WD-ENE > HD-ENE had a higher risk of non-SLN metastases and worse DFS compared to those with WD-ENE ≤ HD-ENE. High GP96 expression was associated with a greater relative risk for locoregional recurrence but showed no significant impact on OS or DFS. Histological grade 3, extensive intraductal component (EIC), higher lymph node ratio (LNR), and negative AR were associated with worse DFS, while age, histological grade 3, EIC, and higher LNR were independent predictors of OS. GP96 mRNA levels were elevated in BC tissue compared to normal breast tissue. Conclusions: ENE in SLN is the strongest predictor of non-SLN involvement and could also have prognostic significance. While GP96 expression does not influence survival outcomes, AR expression could be used as a valuable biomarker in the follow-up of BC patients.

Expression of Androgen, Estrogen, and Progesterone Receptors in the Skin of Patients with Severe Acne and the Assessment of Their Predictive Potential Using Artificial Intelligence Methods

Expression of Androgen, Estrogen, and Progesterone Receptors in the Skin of Patients with Severe Acne and the Assessment of Their Predictive Potential Using Artificial Intelligence Methods

Prostatic androgen receptor signaling shows an age-related and lobe-specific alteration in mice

Benign prostatic hyperplasia (BPH) is an age-related disease that affects millions of aging males globally. While the pathogenesis of BPH remains incompletely understood, emerging evidence suggests a pivotal role for the androgen receptor (AR) in mediating prostate growth and function. Understanding age-related AR signaling alteration may inform novel BPH treatments. Here, we analyzed the prostatic protein expressions of AR, NKX3.1, and Ki-67 in young (2 months) and aged (24 months) mice. We also examined the potential mechanism of AR protein expression. Compared to young mice, decreased AR and NKX3.1 protein expression was observed in the anterior prostate (AP) and ventral prostate (VP) of aged mice, indicating reduced AR signaling in these prostate lobes. Additionally, we observed decreased protein expression of proliferation maker Ki-67 in aged AP, VP, and dorsal-lateral prostate (DLP), with no difference in apoptosis as compared to young counterparts. We conclude that prostatic androgen receptor signaling shows an age-related and lobe-specific alteration in mice.

Walthard Cell Nests/Transitional Cell Metaplasia in Distal Fallopian Tubes and Pelvic Peritoneum Derived From Reserve Cells.

Transitional cell metaplasia (TCM) resembling benign urothelium is commonly seen around the distal fallopian tube and/or neighboring mesothelial surface; however, its histogenesis remains largely unknown. We observed the emergence of a cytokeratin (CK) 17-positive reserve cell layer in early TCM foci beneath the tubal epithelium, leading us to hypothesize that TCM could be derived from reserve cells. To elucidate the histogenetic process of TCM, we analyzed the histomorphologic features and immunoprofiles for CK17, CK5/6, p63, GATA-3, estrogen receptor (ER), and androgen receptor (AR) in TCM foci arising in the tubal epithelium (31 foci) and pelvic mesothelium (35 foci). Overall, the histologic features and immunoprofiles of TCM in the tubal epithelium and pelvic mesothelium were similar, but distinct differences appeared during TCM development. A single-layered CK17-expressing reserve cells became apparent beneath the tubal epithelium, and the CK17 expression disappeared as these cells multiplied. In contrast, a short segment of normal mesothelium next to the tubo-peritoneal junction expressed CK17 even before the emergence of a single-layered reserve cells beneath the mesothelium, suggesting a potential reserve/stem cell function within the mesothelium itself. Then, the single-layered cells in both areas multiplied and differentiated to display urothelial characteristics, including nuclear grooves and clear cytoplasm. Strong CK5/6, p63, and GATA-3 expression appeared in the single-layered reserve cell stage and was maintained thereafter to the fully differentiated TCM. AR was expressed in both normal tubal epithelium and pelvic mesothelium, and the intensity of AR and ER were reciprocal during the entire histogenetic process of TCM in most reserve cell-derived populations (98.5%), AR expression being significantly stronger than ER. The histogenesis of TCM was initiated from the emergence of reserve cells beneath the tubal epithelium and pelvic mesothelium, which then multiplied and differentiated into urothelium. AR might have an important role during the histogenesis of TCM.

MiR-449, identified through antiandrogen exposure, mitigates functional biomarkers associated with ovarian cancer risk

The involvement of the androgen receptor (AR) pathway in developing epithelial ovarian cancer is increasingly acknowledged. However, the specific mechanisms by which anti-androgen agents, such as flutamide, may prevent ovarian cancer and their efficacy remain unknown. This study was initiated by investigating the impact of flutamide on miRNA expression in women at high risk (HR) for ovarian cancer. Ovarian and tubal tissues, free from ovarian, tubal, peritoneal cancers, and serous tubal intraepithelial carcinoma (STIC), were collected from untreated and flutamide-treated HR women as well as low-risk (LR) women controls. We performed miRNA sequencing on these 3 sample cohorts and observed that flutamide normalized miRNA levels in HR tissues, notably upregulating the miR-449 family to levels seen in LR tissues. In subsequent tests in primary ovarian epithelial cells and ovarian cancer cell lines (SKOV3 and Hey), flutamide also increased miR-449a and miR-449b-5p levels. Introducing mimics of these miRNAs reduced the mRNA and protein levels of AR and colony-stimulating factor 1 receptor (CSF1R, also known as c-fms), both of which are known contributors to ovarian cancer progression, with emerging evidence also supporting their roles in ovarian cancer initiation. Ovarian cancer cell migration was inhibited upon introducing miR-449a and miR-449b-5p mimics. Together, our study suggests a novel dual-inhibitory mechanism of flutamide on the AR pathway (AR expression suppression in addition to direct androgen antagonism) and supports its chemopreventive potential in ovarian cancer, especially for HR patients with low miR-449 expression.

Evaluating the Clinico-Pathological Relationship Between Stromal Tumor-Infiltrating Lymphocytes and Androgen Receptor Expression Across Molecular Subtypes of Invasive Breast Carcinoma.

Breast cancer remains a significant cause of mortality globally, necessitating effective treatment strategies. Neoadjuvant chemotherapy (NAC) is widely employed to minimize tumor burden and prevent local spread, with treatment efficacy varying based on molecular subtypes. Despite advancements, resistance to conventional therapies persists, prompting the exploration of alternative approaches, including immune cell therapy. Tumor-infiltrating lymphocytes (TILs) have emerged as immunological biomarkers in breast cancer, exhibiting associations with molecular subtypes and treatment response. This retrospective study assessed the clinico-pathological relationship between stromal TILs and AR expression across molecular subtypes of invasive breast carcinoma in an Indian cohort. Thirty-seven patients receiving NAC followed by modified radical mastectomy were analyzed for TILs and molecular subtyping. Immunohistochemistry was used to determine hormone receptor status and AR expression. A higher AR positivity was observed in hormone receptor-positive/Her2neu-negative and hormone receptor-positive/Her2neu-positive tumors compared to triple-negative breast cancers (TNBCs). Significant associations were observed between AR expression and tumor grade, but not with age or Her2neu status. Although no significant correlation was found between AR and complete response to NAC, a weak negative correlation between AR and TILs was noted. Notably, TNBCs with negative AR and Ki67 index exhibited poorer responses to NAC, emphasizing the need for adjuvant therapy. These findings underscore the complex interplay between AR, TILs, and treatment response in breast cancer, highlighting the potential of personalized therapeutic approaches. Further research is warranted to elucidate the prognostic significance of AR and its implications for tailored treatment strategies in breast cancer management.

Molecular response of canine testis to GnRH agonist: Insights into AR, HIF-1α, and HSPs expression during arrest and recovery of spermatogenesis.

Slow release gonadotropin releasing hormone (GnRH) agonist implants are frequently used for contraception in male dogs. Although the effects are fully reversible, there is still concern about the safety of the implant's mode of action. Addressing this, we investigated cellular stress and androgen receptor signaling during downregulation and recovery. Testicular tissues were sampled from dogs castrated at different time points after GnRH implant removal and compared with untreated controls. Androgen receptor (AR), hypoxia-inducible factor 1 (HIF1A) and heat shock proteins HSP72, HSP73, HSPA2, HSP90AA1 and HSP90AB1 were investigated by qPCR, and AR, HSP72, HSP73 and HSP90 immunohistochemically. While AR, HIF1A and HSP70 were upregulated at mRNA level, HSPA8, HSPA2 and HSP90AA1 expression were downregulated during spermatogenic arrest; HSP90AB1 expression did not change. Immunohistochemistry verified AR-expression in Sertoli, peritubular and Leydig cells, occasionally also in spermatogonia. Stress-inducible HSP72 was occasionally detected, while constitutive HSP73 and HSP90 were abundantly expressed by germ cells. Our results were similar with studies on seasonal breeders such as pine voles, geese, fish and soft-shelled turtles. Accordingly, GnRH implants did not impose additional cellular stress on testicular cells when compared with natural recrudescence. Since comparative data on HIF1α is scarce, we cannot draw conclusions about hypoxic conditions.

Stress in pregnancy alters hepatic unfolded protein responses in male adult offspring

Stress is considered an independent risk factor for the development of cardiometabolic disorders, especially when it occurs during pregnancy, and it may play an important role in stress-induced fetal programming on the protein-folding homeostasis in hepatic endoplasmic reticulum. The study aimed to determine the effects of prenatal stress on the unfolded protein responses in the liver of male and female offspring of Wistar rats. Pregnant Wistar rats at 90 days old were divided into control and stress groups. The unpredictable stress protocol was performed from the 14th to the 21st day of pregnancy. The offspring of each group were divided into four groups according to sex and intervention. After the lactation period, the dams were anesthetized and euthanized for blood collection to determine plasma corticosterone levels. At 90 days old, the offspring were anesthetized and euthanized for liver tissue collection to measure protein expression of the endoplasmic reticulum stress. Dams submitted to prenatal stress showed an increase in corticosterone levels when compared to the control group. In the male offspring, prenatal stress induced lower body mass at birth and at 90 days compared to control, while females presented lower body mass only at birth. Prenatal stress reduced eIF2α expression in males, while increased p-eIF2α expression similarly in both sexes. Furthermore, only males had a greater p-eIF2α/eIF2α ratio and androgen receptor expression when compared to its respective control group and females. Prenatal stress induced a hepatic programming in the reticulum endoplasmic responses only in males at 90 days old by increasing androgen receptor, eIF2α phosphorylation and activity, while in females stress during pregnancy reduced cHDL and had little impact on hepatic unfolded protein response.

Framework for the Pathology Workup of Metastatic Castration-Resistant Prostate Cancer Biopsies.

Lineage plasticity and histologic transformation from prostate adenocarcinoma to neuroendocrine prostate cancer (NEPC) occurs in up to 15-20% of patients with castration-resistant prostate cancer (CRPC) as mechanism of treatment resistance and is associated with aggressive disease and poor prognosis. NEPC tumors typically display small cell carcinoma morphology with loss of androgen receptor (AR) expression and gain of neuroendocrine (NE) lineage markers. However, there is a spectrum of phenotypes that are observed during the lineage plasticity process, and the clinical significance of mixed histologies or those that co-express AR and NE markers or lack all markers is not well defined. Translational research studies investigating NEPC have used variable definitions making clinical trial design challenging. Here we discuss the diagnostic workup of metastatic biopsies to help guide the reproducible classification of phenotypic CRPC subtypes. We recommend classifying CRPC tumors based on histomorphology (adenocarcinoma, small cell carcinoma, poorly differentiated carcinoma, other morphologic variant, or mixed morphology) and immunohistochemical markers with a priority for AR, NKX3.1, INSM1, synaptophysin and cell proliferation based on Ki-67 positivity, with additional markers to be considered based on the clinical context. Ultimately, a unified workup of metastatic CRPC biopsies can improve clinical trial design and eventually practice.

Small-molecule disruption of androgen receptor–dependent chromatin clusters

Sustained androgen receptor (AR) signaling during relapse is a central driver of metastatic castration-resistant prostate cancer (mCRPC). Current AR antagonists, such as enzalutamide, fail to provide long-term benefit for the mCRPC patients who have dramatic increases in AR expression. Here, we report AR antagonists with efficacy in AR-overexpressing models. These molecules bind to the ligand-binding domain of the AR, promote AR localization to the nucleus, yet potently and selectively down-regulate AR-target genes. The molecules BG-15a and the pharmacokinetically optimized BG-15n elicit a decrease in cell and tumor growth in vitro and in vivo in models of mCRPC. BG-15a/n treatment causes the collapse of chromatin loops between enhancers and promoters at key genes in the AR-driven epigenome. AR binding in the promoter, as well as 3D chromatin clustering, is needed for genes to respond. BG-15a/n represent promising agents for treating patients with relapsed AR-driven mCRPC tumors.

MitoQ enhances CYP19A1 expression to stimulate WNT/β-catenin signaling pathway for promoting hair growth in androgenetic alopecia

Increased sensitivity to androgens and androgen receptors is the underlying cause of androgenetic alopecia (AGA), a hereditary disease. Our study investigated the preventive effects of MitoQ on dihydrotestosterone (DHT)-induced mitochondrial dysfunction and subsequent hair loss from three perspectives: in vivo, in vitro, and network pharmacology. A mouse model of AGA was used to assess the effectiveness of MitoQ intervention. Seventy-five drug targets and 367 disease targets were identified through network pharmacology analysis. Molecular docking analysis revealed that the androgen receptor (AR) and CYP19A1, which are key targets of MitoQ, may play a role in AGA treatment. CYP19A1 expression was downregulated in lesions from patients with AGA compared to healthy scalp tissue, while AR expression was upregulated. Cellular tests of human dermal papilla cells (DPCs) treated with MitoQ revealed that the mRNA and protein expression of AR remained unchanged, but the mRNA expression of CYP19A1 was upregulated. Our experiments also confirmed that CYP19A1 overexpression prevented DHT-induced apoptosis and upregulated the expression levels of WNT3A and β-catenin, whereas increased apoptosis levels and the downregulation of WNT3A and β-catenin due to CYP19A1 knockdown were reduced by MitoQ. We verified that MitoQ enhanced hair growth in DHT-induced hair loss model mice and reversed DHT-induced apoptosis by enhancing the expression of CYP19A1 in DPCs and that MitoQ may act by mediating the WNT/β-catenin pathway. These findings indicate that MitoQ could be a promising intervention for AGA and that CYP19A1 may serve as a valuable therapeutic target for AGA.

Prognostic role of Androgen Receptor splice variant 7 (AR-V7) in the pathogenesis of breast cancer

BackgroundThe Androgen Receptor (AR) has emerged as an endocrine therapy target in Breast Cancer, exhibiting up to 80% expression in clinical cases. AR-V7, a constitutively activated splice variant of AR with a truncated ligand-binding domain (LBD), demonstrates ligand-independent transcriptional activity and resistance to nonsteroidal antiandrogens like Bicalutamide or Enzalutamide, targeting the LBD. In metastatic prostate cancer, elevated AR-V7 levels lead to therapeutic resistance and increased metastasis.MethodsIn this study, we evaluated the expression of AR and AR-V7 in cell lines and a cohort of 89 patients undergoing surgical intervention for treatment-naïve breast cancer. Further clinicopathological correlations and survival analysis were performed to evaluate the relationship between the AR and AR-V7 expression and clinical outcomes.ResultsAR-V7/AR-FL ratio was elevated in the TNBC cell line and downregulation of AR-FL upon AR antagonists’ treatment led to a compensatory increase in AR-V7. Clinical samples showed significantly elevated expression of AR and AR-V7 in tumors compared to control cases. Further clinicopathological correlation revealed aggressive clinical traits, higher pathological grades, and poor survival with AR-V7 expression.ConclusionsOur study unravels AR-V7 as a marker for poor clinical outcomes, predicting breast cancer aggressiveness, and encourages consideration of AR-V7 as a probable target for therapeutic intervention.

Vaccination Against Androgen Receptor Splice Variants to Immunologically Target Prostate Cancer.

Background/Objectives: Androgen receptor (AR) expression and signaling are critical for the progression of prostate cancer and have been the therapeutic focus of prostate cancer for over 50 years. While a variety of agents have been developed to target this axis, many of these fail due to the emergent expression of AR RNA splice variants, such as AR-V7, that can signal independently of ligand binding. Other therapies, such as vaccination against prostate-specific antigens, have achieved FDA approvals but have fallen short of being incorporated as standard-of-care therapies for advanced prostate cancer. This may be due to the elevated level of immunosuppression observed in prostate cancer, which remains largely refractory to immune checkpoint blockade. Methods: We developed a vaccine targeting AR-V7, a common isoform associated with treatment resistance, and demonstrated its ability to elicit AR-V7-specific immunity and enable anti-tumor responses against AR-V7+ cancers in subcutaneous tumor models. Results: Our studies also revealed that AR-V7 expression conferred an immune suppressive phenotype that was significant in a non-AR-dependent prostate cancer model. Notably, in this model, we found that vaccination in combination with enzalutamide, an AR antagonist, suppressed these aggressive immune suppressive cancers and resulted in enhanced survival in comparison to control vaccinated and enzalutamide-treated mice. While anti-PD-1 immune checkpoint inhibition (ICI) alone slowed tumor growth, the majority of vaccinated mice that received anti-PD-1 therapy showed complete tumor elimination. Conclusions: Collectively, these results validate the importance of AR signaling in prostate cancer immune suppression and suggest the potential of AR-V7-specific vaccines as therapeutic strategies against prostate cancer, offering significant protective and therapeutic anti-tumor responses, even in the presence of androgen signaling inhibitors.

Unveiling RACK1: a key regulator of the PI3K/AKT pathway in prostate cancer development.

The dysregulated PI3K/AKT pathway is pivotal in the onset and progression of various cancers, including prostate cancer. However, targeting this pathway directly poses challenges due to compensatory upregulation of alternative oncogenic pathways. This study focuses on the novel regulatory activity of the Receptor for Activated Protein Kinase (RACK1), a scaffolding/adaptor protein, in governing the PI3K/AKT pathway within prostate cancer. Through a genetic mouse model, our research unveils RACK1's pivotal role in orchestrating AKT activation and the genesis of prostate cancer. RACK1 deficiency hampers AKT activation, effectively impeding prostate tumor formation induced by PTEN and p53 deficiency. Mechanistically, RACK1 facilitates AKT membrane translocation and fosters its interaction with mTORC2, thereby promoting AKT activation and subsequent tumor cell proliferation and tumor formation. Notably, inhibiting AKT activation via RACK1 deficiency does not trigger feedback upregulation of HER3 and androgen receptor (AR) expression and activation, distinguishing it from direct PI3K or AKT targeting. These findings position RACK1 as a critical regulator of the PI3K/AKT pathway and a promising target for curtailing prostate cancer development arising from pathway aberrations.

Abstract PR011: Advance prostate cancer detection through epigenomic profiling of cell-free DNA

Abstract Introduction: Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease which can be classified into clinically relevant subtypes based on the expression of genes, such as the androgen receptor (AR) and neuroendocrine markers. Neuroendocrine prostate cancer (NEPC), characterized by gain of stem-like and neuroendocrine features and lack of AR expression is a clinically aggressive variant. Due to the lack of adequate biomarkers, NEPC is usually detected at a very advanced stage. There is mounting evidence that molecular subtype changes seen in NEPC are enforced by widespread epigenetic alterations, in particular DNA methylation changes. In this study, we aim to devise a novel DNA methylation-based assay for molecular subtyping and disease monitoring from cell-free DNA (cfDNA). Methods: We analyzed genome wide methylation patterns in 56 prostate cancer patient-derived xenograft (PDX) and 128 mCRPC tumors using array- and sequencing-based assays. We integrated DNA methylation at promoters, gene bodies and transcription factor binding site (TFBS) to determine the landscape of methylation alterations at key lineage specific genes. Using whole genome methylation derived from tissue with matched expression data we developed a deep learning framework to predict gene expression directly from tissue or cfDNA. Using key marker genes, the model was used to discern tumor molecular phenotypes from tissue and cfDNA in three independent cohorts of mCRPC patients using whole genome bisulfite sequencing and low-pass Enzymatic Methyl-Seq (EM-seq). Results: We observed a tight association between promoter, gene body and TFBS methylation with gene expression. Inferring gene expression from methylation for lineage specific markers such as AR, KLK3, ASCL1, INSM1, SRRM4 and DLL3 we classified molecular subtypes from both tissue and cfDNA. Additionally, for AR and ASCL1, we identified core sets of TFBSs whose differential methylation allowed for accurate assay-independent molecular subtype quantification. Applying the optimized quantitative model to mCRPC patients who underwent comprehensive tissue sampling by rapid autopsy we observed accurate subtype classification from both tissue samples and cfDNA for all cases. A similar analytical performance was observed in additional clinical mCRPC cohorts with cfDNA. Conclusion: Whole-genome methylation analysis of cfDNA allows for the prediction of gene expression patterns in tumor tissues, enabling non-invasive tumor subclassification and assessment of therapeutic targets. Citation Format: Mohamed Adil, Brian Hanratty, Pallabi Mustafi, Chitvan Mittal, Helen Richards, Ilsa Coleman, Radhika Patel, Anna-Lisa Doebley, Robert Patton, Eden Cruikshank, Patricia Galipeau, Ruth Dumpit, Martine Roudier, Jin-Yih Low, Navonil Sarkar, Robert Montgomery, Eva Corey, Colm Morrissey, Peter Nelson, Gavin Ha, Michael Haffner. Advance prostate cancer detection through epigenomic profiling of cell-free DNA [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR011.

Abstract 4129520: The mechanism of cardiac hypertrophy induced by testosterone in postmenopausal women through the activation of the mTORC2/Atg9a

Objectives: The estrogen level of postmenopausal women decreased sharply, while the androgen level did not decrease significantly, which was characterized by the inversion of estrogen / androgen ratio. Therefore, it could be observed that relative excess androgen in the circulation of postmenopausal women, which may be one of the reasons for the increase of cardiovascular disease in postmenopausal women. In this study, the model of postmenopausal hypertension was established by bilateral ovariectomy in spontaneously hypertensive rats (SHR), and the specific mechanism of testosterone-induced myocardial hypertrophy in ovariectomized SHR was explored. Methods: 24 SHR were randomly divided into four groups: WKY group (n=6), sham operation group (Sham group, n=6), bilateral ovariectomized group (OVX group, n=6) and testosterone propionate intervention group (OVX+T group, 6mg/kg/Weekly, im for 4 weeks, n=6). Western Blot was used to detect the protein expression of β-MHC, ANP, Androgen receptor, Akt, p-Akt, mTORC2 and its subunits Rictor. Moreover, the expression of autophagy-related proteins LC3 was observed. Results: 1. The study revealed that the expression of β-MHC and ANP was significantly increased in both the OVX and OVX+T groups when compared to the WKY and Sham groups. Remarkably, testosterone induced a more significant cardiac hypertrophy response than observed in the OVX group alone. Additionally, the expression of androgen receptors was markedly upregulated in both the OVX and OVX+T groups. These findings strongly suggest that testosterone primarily induces myocardial hypertrophy in SHR after ovariectomy through androgen receptors. 2. Although no significant difference in mTORC2 expression was noted between the WKY, Sham, and OVX groups, testosterone exhibited a notable capacity to activate mTORC2 expression and the p-Rictor subunit in the OVX+T group. 3. The expression of LC3II/LC3I and mitochondrial fission protein DRP1 were increased in the OVX+T group when compared to the WKY, Sham, and OVX groups. While the expression of MFN2 in the four groups shows no significant difference. Meanwhile, the expression of Atg9a was increased in OVX+T group. The results indicate that testosterone might activate mitophagy via inducing the expression of Atg9a. Conclusions: In bilateral ovariectomized SHR, testosterone has the potential to activate mitophagy by activating the mTORC2/Atg9a pathway, which, in turn, leads to cardiac hypertrophy.

An Autophagy-Targeting Chimera Induces Degradation of Androgen Receptor Mutants and AR-v7 in Castration-Resistant Prostate Cancer.

Genetic alterations play a pivotal role in various human diseases, particularly cancer. The androgen receptor (AR) is a crucial transcription factor driving prostate cancer (PCa) progression across all stages. Current AR-targeting therapies utilize competitive AR antagonists or pathway suppressors. However, therapy resistance often emerges due to AR mutations and AR splice variants, such as AR-v7. To overcome this, we developed ATC-324, an AR degrader using the innovative protein degradation technology platform AUTOphagy-TArgeting Chimera (AUTOTAC). ATC-324 was designed to comprise enzalutamide, an AR inhibitor, as a target-binding ligand and YT 6-2, a ligand of the autophagy receptor p62/SQSTM1, as an autophagy-targeting ligand. ATC-324 induces the formation of the AR/p62 complex, leading to autophagy-lysosomal degradation of AR. Importantly, ATC-324 effectively degrades AR mutants frequently detected in PCa and co-degrades AR-v7 as a heterodimer with full-length AR. ATC-324 reduces nuclear AR levels and downregulates the target gene expression of AR and AR-v7, leading to cytotoxicity in AR-positive PCa cells. We also provide evidence of the therapeutic potential of ATC-324 in vivo as well as ex vivo bone organ culture. Moreover, ATC-324 remains potent in enzalutamide-resistant PCa cells. These results demonstrate the potential of the AUTOTAC platform to target previously considered undruggable proteins and overcome certain drug resistance mechanisms.

EXTH-26. EXPLORING THE ROLE OF ANDROGEN RECEPTOR SIGNALING IN GLIOBLASTOMA IN AN ORTHOTOPIC ANIMAL MODEL

Abstract OBJECTIVE Gliomas, particularly glioblastoma which is the most common and deadly type, frequently express androgen receptors (AR). We systematically characterized AR expression in low- and high-grade gliomas to investigate whether disrupting AR signaling with readily available blood-brain-barrier penetrant drugs could improve treatment. METHODS Low- and high-grade glioma samples were stained for AR by immunohistochemistry, with AR-expression correlated to grade and sex. Localization of AR and the influence of dihydrotestosterone (DHT) and anti-androgens in glioblastoma cell line (U251) were determined by immunofluorescence and immunoblot. Building on previous in vitro data, we used an orthotopic AR-positive patient-derived model (RN1) to test the efficacy of seviteronel, a blood-brain-barrier penetrant anti-androgen. RESULTS Two thirds of biopsy samples were positive for AR expression by IHC (n=25/39). AR expression correlated with increasing tumor grade (p<0.05) but was similar between sexes (p=0.94). Anti-androgens enzalutamide and seviteronel prevented DHT-induced nuclear translocation of AR; however, seviteronel did so to a lesser extent, as measured by immunofluorescence and immunoblot. In the mouse model, seviteronel significantly improved survival compared to vehicle (n=12, 36.0 vs 27.5 days, p=0.03), and demonstrated reduced Ki67 expression in tumors at the endpoint (68.9% vs 85.0%, p<0.001). Combining seviteronel with temozolomide limited tumor growth as measured by bioluminescence imaging, but did not significantly extend survival compared to temozolomide alone (n=16, 63.5 vs 56.5 days, p=0.37). Adding seviteronel to radiotherapy did not extend survival compared to radiotherapy alone (n=16, 33.0 vs 39.0 days, p=0.19). CONCLUSIONS Targeting AR signaling with blood-brain-barrier penetrant anti-androgens may represent a promising biomarker-directed therapeutic strategy. The mechanisms underlying the effects of seviteronel are being further investigated through affinity-purification mass spectrometry analysis and CUT&RUN sequencing of untreated and treated glioma cells. Additional animal experiments are underway to examine the role of biological sex and the glioma immune microenvironment on the efficacy of anti-androgens in vivo.

Androgen Receptor Mediates Dopamine Agonist Resistance by Regulating Intracellular Reactive Oxygen Species in Prolactin-Secreting Pituitary Adenoma.

Aims: Dopamine agonists (DAs) are the first-line treatment for patients with prolactin-secreting pituitary adenoma (PRL adenoma). However, a subset of individuals exhibits poor responses, known as DA resistance. Previous studies have reported that DA resistance is more prevalent in male patients. This study aims to investigate the relationship between androgen receptor (AR) expression and DA resistance, as well as to explore underlying mechanisms of AR-mediated DA resistance. Results: Our results demonstrated that patients with higher AR expression exhibit greater resistance to DA in our cohort of DA-resistant PRL adenoma. Furthermore, AR was found to be involved in cell proliferation, PRL secretion, and resistance to bromocriptine (BRC) both in vitro and invivo. Mechanistically, we demonstrated that intracellular reactive oxygen species (ROS) function as upstream mediators of apoptosis and ferroptosis following BRC treatment. As a ligand-dependent transcription factor, AR could translocate to the nucleus and transcriptionally promote NFE2-like bZIP transcription factor 2 (NRF2) expression, which regulates intracellular ROS levels, thereby enhancing cell viability and conferring DA resistance to pituitary adenoma (PA) cells. Finally, AR targeting agents were used to inhibit AR signaling, downregulate NRF2 transcription, and sensitize PA cells to BRC treatment. Conclusion and Innovation: We demonstrated that AR plays a crucial role in mediating DA resistance in PRL adenoma. Mechanistically, AR promotes cell proliferation and PRL secretion and confers drug resistance by transcriptionally regulating NRF2 expression to maintain redox homeostasis in PA cells. Finally, combining AR targeting agents with BRC shows promise as a therapeutic strategy for treating PRL adenomas. Antioxid. Redox Signal. 00, 000-000.

Danazol and Stanozolol Induce Expression of Pro-Apoptotic Genes

Androgens (danazol and stanozolol) have been used to treat bone marrow failure (BMF) syndromes due to their multifaceted effects on erythropoiesis, telomere maintenance and immune homeostasis. In low and middle-income countries, androgens are used to treat aplastic anaemia, particularly in individuals who cannot afford immunosuppressive therapy and HSCT. However, patient responses to these treatments vary, with previous studies showing a 50-70% response to inherited bone marrow failures and a 40-50% response to acquired AA and MDS. There is uncertainty surrounding how androgens work, with only a few elucidated effects on the stimulation of erythropoiesis, stem cell telomere elongations and immune cell modulation. This study aims to understand the mechanisms of androgen action in hepatic cell lines since they are metabolised there and extrapolate the findings to HSCs. HepG2 cell lines at passage 6 were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum. Mobilised peripheral blood from stem cell donors was used to enrich CD34+ cells cultured in StemSpan SFEM Hematopoietic cell culture medium. The purity and percentage of CD34+ cells were assessed by flow cytometry, and > 90% purity was maintained. MTT assays determined the drug concentration in HepG2 and CD34+positive cells. HepG2 and CD34 cells were treated with danazol and stanozolol at different concentrations (100nM, 1uM and 10uM) for 24, 48, and 72 hours. Cell proliferation rates were monitored by staining the cells with CFSE dye, incubating them with the respective concentrations for 24, 48 and 72 hours and analysing the proliferation in a flow cytometer. Further, the drug-treated cells were stained with propidium iodide to examine the effects of drugs in the cell cycle at 24 hours post-drug treatment. RNA was extracted from treated cells, and expression of apoptotic (CASP8, CASP9, Fas, and BAX), senescent genes (TERF1, SIRT1, ARID1a), and androgen receptor gene was analysed using quantitative real-time PCR using relative quantification. The influence of androgens on genes involved in iron metabolism is being investigated, and protein data is awaited. Based on the MTT assay on CD34+ and HepG2 cells, 100nM and 1uM danazol and stanozolol were used for further experiments due to increased viability at 24 and 48 hrs. HepG2 cells showed a maximum proliferation at 24 hours at varying concentrations of danazol and stanozolol. We further observed an arrest of cells at the G1 phase post-drug treatment at 24 hrs. Both danazol and stanozolol, the peak significant androgen receptor expression was observed at 6 hours (fold change > 3.3; p = 0.0004). Pro-apoptotic gene CASP8 increased significantly with danazol over 24 hours (p = 0.025 and 0.0009, and fold change >1.5 and 1.4, respectively), but stanozolol caused a minor increase in expression levels. Danazol and stanozolol (100 nM conc.) significantly enhanced the expression level of CASP9 (p = 0.0253, 0.0009, and 0.007; fold change > 1.5 and 1.8 respectively), while FAS9 levels were significantly upregulated with stanozolol (1 uM) and danazol (p = 0.0001, 0.0001, and 0.004; fold change 2.3, 2.39, and 1.7). The mechanism of action of androgens appears complex. Genes involved in apoptotic processes are upregulated with no difference in the expression of senescent genes with both Danazol and Stanozolol. Danazol is known to have immunosuppressive effects on immune cells in AA, and this may be due to the induction of apoptotic processes in activated cells. Results from CD34 cell experiments and protein analysis may shed light on these pathways and their mechanisms.