Androgen Receptor Target Research Articles

Metformin regulates multiple signaling pathways within castration-resistant human prostate cancer cells

BackgroundThe biguanide metformin has been shown to not only reduce circulating glucose levels but also suppress in vitro and in vivo growth of prostate cancer. However, the mechanisms underlying the anti-tumor effects of metformin in advanced prostate cancers are not fully understood. The goal of the present study was to define the signaling pathways regulated by metformin in androgen-receptor (AR) positive, castration-resistant prostate cancers.MethodsOur group used RNA sequencing (RNA-seq) to examine genes regulated by metformin within the C4–2 human prostate cancer cell line. Western blot analysis and quantitative RT-PCR were used to confirm alterations in gene expression and further explore regulation of protein expression by metformin.ResultsData from the RNA-seq analysis revealed that metformin alters the expression of genes products involved in metabolic pathways, the spliceosome, RNA transport, and protein processing within the endoplasmic reticulum. Gene products involved in ErbB, insulin, mTOR, TGF-β, MAPK, and Wnt signaling pathways are also regulated by metformin. A subset of metformin-regulated gene products were genes known to be direct transcriptional targets of p53 or AR. Western blot analyses and quantitative RT-PCR indicated these alterations in gene expression are due in part to metformin-induced reductions in AR mRNA and protein levels.ConclusionsTogether, our results suggest metformin regulates multiple pathways linked to tumor growth and progression within advanced prostate cancer cells.

Hypoxia-Inducible Expression of Annexin A6 Enhances the Resistance of Triple-Negative Breast Cancer Cells to EGFR and AR Antagonists.

Physiological changes such as hypoxia in the tumor microenvironment (TME) endow cancer cells with malignant properties, leading to tumor recurrence and rapid progression. Here, we assessed the effect of hypoxia (1% Oxygen) on the tumor suppressor Annexin A6 (AnxA6) and the response of triple-negative breast cancer (TNBC) cells to epidermal growth factor receptor (EGFR) and androgen receptor (AR) targeted therapies. We demonstrate that brief exposure of TNBC cells to hypoxia (within 24 h) is associated with down regulation of AnxA6 while > 24 h exposure cell type dependently stimulated the expression of AnxA6. Hypoxia depicted by the expression and stability of HIF-1/2α led to up regulation of the HIF target genes SLC2A1, PGK1 as well as AR and the AR target genes FABP-4 and PPAR-γ, but the cellular levels of AnxA6 protein decreased under prolonged hypoxia. Down regulation of AnxA6 in TNBC cells inhibited, while AnxA6 over expression enhanced the expression and cellular levels of HIF-1/2α, SLC2A1 and PGK1. RNAi mediated inhibition of hypoxia induced AnxA6 expression also strongly inhibited glucose uptake and ROS production in AnxA6 expressing TNBC cells. Using a luciferase reporter assay, we confirm that short-term exposure of cells to hypoxia inhibits while prolonged exposure of cells to hypoxia enhances AnxA6 promoter activity in HEK293T cells. Compared to cells cultured under normoxia, TNBC cells were more resistant to lapatinib under hypoxic conditions, and the downregulation of AnxA6 sensitized the cells to EGFR as well as AR antagonists. These data suggest that AnxA6 is a hypoxia inducible gene and that targeting AnxA6 upregulation may be beneficial in overcoming TNBC resistance to EGFR and/or AR targeted therapies.

Glucocorticoid receptor and androgen receptor-targeting therapy in patients with castration-resistant prostate cancer.

ObjectiveThe glucocorticoid receptor (GR) promotes resistance to androgen receptor (AR)-targeting therapies in castration-resistant prostate cancer (CRPC) by bypassing AR blockade. However, the clinical relevance of evaluating GR expression in patients with CRPC has not been determined. The present study investigated the association of relative GR expression in CRPC tissue samples with treatment response to AR-targeting therapy.MethodsLevels of GR, AR-FL, and AR-V7 mRNAs were measured in prostate cancer tissue from prospectively enrolled CRPC patients who were starting treatment. Patients were divided into groups with high and low AR-V7/AR-FL ratios and with high and low GR/AR-FL ratios. The primary endpoint was prostate-specific antigen (PSA) response rate to treatment.ResultsEvaluation of 38 patients treated with AR-targeting therapies showed that the PSA response rate was significantly higher in patients with low than high AR-V7/AR-FL ratios (77.8% vs. 25.0%, p=0.003) and in patients with low than high GR/AR-FL ratios (81.3% vs. 27.3%, p=0.003). Patients with low GR/AR-FL ratios had higher rates of PSA progression-free survival (46.0% vs. 22.4%, p=0.006), radiologic progression-free survival (28.9% vs. 10.0%, p=0.02), and overall survival (75.2% vs. 48.0%, p=0.037) than patients with high GR/AR-FL ratios. The association of GR/AR-FL ratio with PSA response to AR-targeting therapy remained significant in multivariable models. Evaluation of the 14 patients who received taxane chemotherapy showed that PSA response rates did not differ significantly in those with low and high AR-V7/AR-FL and GR/AR-FL ratios, although no definitive conclusions can be drawn due to the small number of patients.ConclusionRelative GR expression is associated with sensitivity to AR-targeting therapy and survival in patients with CRPC. Large-scale prospective validation and liquid biopsy-based studies are warranted.

AR-regulated ZIC5 contributes to the aggressiveness of prostate cancer

The mechanisms by which prostate cancer (PCa) progresses to the aggressive castration-resistant stage remain uncertain. Zinc finger of the cerebellum 5 (ZIC5), a transcription factor belonging to the ZIC family, is involved in the pathology of various cancers. However, the potential effect of ZIC5 on PCa malignant progression has not been fully defined. Here, we show that ZIC5 is upregulated in PCa, particularly in metastatic lesions, in positive association with poor prognosis. Genetic inhibition of ZIC5 in PCa cells obviously attenuated invasion and metastasis and blunted the oncogenic properties of colony formation. Mechanistically, ZIC5 functioned as a transcription factor to promote TWIST1-mediated EMT progression or as a cofactor to strengthen the β-catenin-TCF4 association and stimulate Wnt/β-catenin signaling. Importantly, ZIC5 and the androgen receptor (AR) form a positive feed-forward loop to mutually stimulate each other’s expression. AR, in cooperation with its steroid receptor coactivator 3 (SRC-3), increased ZIC5 expression through binding to the miR-27b-3p promoter and repressing miR-27b-3p transcription. In turn, ZIC5 potentiated AR, AR-V7, and AR targets’ expression. Besides, ZIC5 inhibition reduced AR and AR-V7 protein expression and enhanced the sensitivity of PCa to enzalutamide (Enz) treatment, both in vitro and in vivo. These findings indicate that the reciprocal activation between AR and ZIC5 promotes metastasis and Enz resistance of PCa and suggest the therapeutic value of cotargeting ZIC5 and AR for the treatment of advanced PCa.

1274P Performance status (PS) and end-of-life (EOL) care in patients (pts) with metastatic prostate cancer (mPC) treated with androgen receptor targeted (ART) therapy

1274P Performance status (PS) and end-of-life (EOL) care in patients (pts) with metastatic prostate cancer (mPC) treated with androgen receptor targeted (ART) therapy

Lipid nanoparticles to silence androgen receptor variants for prostate cancer therapy.

Advanced-stage prostate cancer remains an incurable disease with poor patient prognosis. There is an unmet clinical need to target androgen receptor (AR) splice variants, which are key drivers of the disease. Some AR splice variants are insensitive to conventional hormonal or androgen deprivation therapy due to loss of the androgen ligand binding domain at the C-terminus and are constitutively active. Here we explore the use of RNA interference (RNAi) to target a universally conserved region of all AR splice variants for cleavage and degradation, thereby eliminating protein level resistance mechanisms. To this end, we tested five siRNA sequences designed against exon 1 of the AR mRNA and identified several that induced potent knockdown of full-length and truncated variant ARs in the 22Rv1 human prostate cancer cell line. We then demonstrated that 2'O methyl modification of the top candidate siRNA (siARvm) enhanced AR and AR-V7 mRNA silencing potency in both 22Rv1 and LNCaP cells, which represent two different prostate cancer models. For downstream in vivo delivery, we formulated siARvm-LNPs and functionally validated these in vitro by demonstrating knockdown of AR and AR-V7 mRNA in prostate cancer cells and loss of AR-mediated transcriptional activation of the PSA gene in both cell lines following treatment. We also observed that siARvm-LNP induced cell viability inhibition was more potent compared to LNP containing siRNA targeting full-length AR mRNA (siARfl-LNP) in 22Rv1 cells as their proliferation is more dependent on AR splice variants than LNCaP and PC3 cells. The in vivo biodistribution of siARvm-LNPs was determined in 22Rv1 tumor-bearing mice by incorporating 14C-radiolabelled DSPC in LNP formulation, and we observed a 4.4% ID/g tumor accumulation following intravenous administration. Finally, treatment of 22Rv1 tumor bearing mice with siARvm-LNP resulted in significant tumor growth inhibition and survival benefit compared to siARfl-LNP or the siLUC-LNP control. To best of our knowledge, this is the first report demonstrating therapeutic effects of LNP-siRNA targeting AR splice variants in prostate cancer.

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

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

Seleno-aspirin compound AS-10 promotes histone acetylation ahead of suppressing androgen receptor transcription, G1 arrest, and apoptosis of prostate cancer cells.

The novel selenium-aspirin compound AS-10 was recently reported by us with a cancer cell killing potency three orders of magnitude greater than aspirin in pancreatic cancer cell lines with caspase-mediated apoptosis and a reasonable selectivity against malignant cells. Although we also observed its cytocidal activity against PC-3 and DU145 androgen receptor (AR)-negative and P53-null/mutant aggressive human prostate cancer (PCa) cell lines in NCI-60 screen, the potential involvement and targeting of AR and P53 pathways that are intact in early-stage prostate carcinogenesis has not been examined, nor its primary molecular signaling after exposure. Human LNCaP PCa cells with functional AR and intact P53 were used to examine their cell cycle and cell fate responses to AS-10 exposure and upstream molecular signaling events including histone acetylation as a known aspirin effect. The AR-positive 22Rv1 human PCa cells were used to validate key findings. In addition to confirming AS-10's superior cytocidal potency than aspirin against all four PCa cell lines, we report a rapid (within 5 min) promotion of histone acetylation several hours ahead of the suppression of AR and prostate-specific antigen (PSA, coded by KLK3 gene) in LNCaP and 22Rv1 cells. AS-10 decreased AR and KLK3 mRNA levels without impacting pre-existing AR protein degradation or nuclear translocation in LNCaP cells. Sustained exposure to AS-10 arrested cells predominantly in G1 , and induced caspase-mediated apoptosis without necrosis. The death induced by AS-10 in LNCaP cells was attenuated by nontranscriptional activation of P53 protein or Jun N-terminal Kinase cellular stress signaling and was mitigated modestly by glutathione-boosting antioxidant N-acetylcysteine. AS-10 synergized with histone deacetylase inhibitor SAHA to suppress AR/PSA abundance and kill LNCaP cells. RNA-seq confirmed AR suppression at the transcriptional level and suggested multiple oncogene, cyclin, and CDK/CKI transcriptional actions to contribute to the cellular consequences. AS-10 promotes histone acetylation as its probable primary mechanism of action to induce PCa cell-cycle arrest and apoptosis, regardless of AR and P53 status. Nevertheless, the inhibition of AR signaling through mechanisms distinct from canonical AR antagonists may hold promise for combinatorial use with androgen deprivation therapy regimens or AR-axis targeting drugs.

α-Terthienyl induces prostate cancer cell death through inhibiting androgen receptor expression.

Prostate cancer is a disease that often occurs in elderly men. Androgen receptor signaling pathway runs through the occurrence and development of prostate cancer. Thereby, targeting androgen receptor is a crucial strategy for the treatment of prostate cancer. α-Terthienyl, which has been used as photosensitive activator and insecticide, is a natural compound rich in marigold. In the present study, we found α- terthienyl could inhibit the cell viability of four prostate cancer cell lines, especially on LNCaP and 22Rv1 cells which endogenously express androgen receptor. Then we proved that it could inhibit the proliferation of prostate cancer cells and induce apoptosis of prostate cancer cells by plate clone formation assay and flow cytometry respectively. Furthermore, we found α-terthienyl could inhibit androgen receptor nuclear translocation, reduce androgen receptor expression, reduce the mRNA and protein expression of androgen receptor target genes (KLK3, TMPRSS2, PCA3) and nuclear proliferation antigen Ki67 and PCNA. In addition, it inhibited the expression and phosphorylation of Akt protein while increasing the expression of tumor suppressor p27. Besides, we constructed a mouse xenograft prostate cancer model and confirmed that α-terthienyl also inhibited the growth of prostate cancer in vivo. In conclusively, α-terthienyl played an anti-prostate cancer role by inhibiting both the expression of androgen receptor and the transduction of its signal pathway, suggesting that it is a promising natural small molecule for the treatment of prostate cancer.

Role of Olaparib in the Management of Metastatic Castration-Resistant Prostate Cancer: A Japanese Clinician's Perspective.

Several studies have identified various targetable genomic alterations in prostate cancer, which accumulate during carcinogenesis and cancer progression. Genomic alterations in genes involved in DNA damage repair by homologous recombination repair may predict increased sensitivity to poly-ADP ribose polymerase (PARP) inhibitors. The Phase 3 PROfound trial has shown that treatment with the PARP inhibitor olaparib was associated with an improved radiographic progression-free survival and overall survival among patients with homologous recombination repair-deficient metastatic castration-resistant prostate cancer (mCRPC) after the treatment with androgen receptor targeting therapy, especially in men with BRCA1 or BRCA2 mutation. In Japan, olaparib was approved in December 2020 for the treatment of mCRPC with BRCA1 or BRCA2 mutation. In addition, genetic tests to detect BRCA1 or BRCA2 mutation to select patients who are likely to benefit from olaparib were also approved. This review summarizes the status of olaparib treatment for mCRPC, focusing on the situation in Japan.

The STAT3 inhibitor GPB730 enhances the sensitivity to enzalutamide in prostate cancer cells

Enzalutamide is a second-generation anti-androgen which has shown increased survival in patients with metastatic prostate cancer. However, some patients do not respond to this therapy or will develop resistance to treatment over time. Signal Transducer and Activator of Transcription 3 (STAT3) is known to be involved in castration-resistant prostate cancer and to interact with androgen receptor (AR)-signaling. This study aims to investigate the combination enzalutamide and the small molecule STAT3 inhibitor GPB730 for enhanced therapeutic effect in advanced prostate cancer in vitro. The prostate cancer cell lines LNCaP (androgen dependent) and C4–2 (androgen insensitive) were used. The effect of enzalutamide and GPB730, alone and in combination, was investigated on viability and IC50 values calculated. Enzalutamide and GPB730 treated LNCaP and C4–2 cells were subjected to western blot and QPCR analyses in order to investigate the expression of AR, STAT3 and down-stream targets. C4–2 were less sensitive to growth inhibition by enzalutamide than LNCaP cells. GPB730 enhanced the growth inhibitory effect of enzalutamide in LNCaP and C4–2 cells. The addition of GPB730 to enzalutamide decreased the IC50 values for enzalutamide by 3.3-fold for LNCaP and by 12-fold for C4–2. In C4–2 cells, GPB730 alone decreased PSA expression and enhanced the enzalutamide induced decrease in NKX3.1 expression. GPB730 and enzalutamide in combination enhanced inhibition of c-myc and survivin expression. This study suggests that enzalutamide may be combined with the STAT3 inhibitor GPB730 in order to enhance the efficacy of enzalutamide, offering a new therapeutic approach in advanced prostate cancer.

Androgen-Induced MIG6 Regulates Phosphorylation of Retinoblastoma Protein and AKT to Counteract Non-Genomic AR Signaling in Prostate Cancer Cells

The bipolar androgen therapy (BAT) includes the treatment of prostate cancer (PCa) patients with supraphysiological androgen level (SAL). Interestingly, SAL induces cell senescence in PCa cell lines as well as ex vivo in tumor samples of patients. The SAL-mediated cell senescence was shown to be androgen receptor (AR)-dependent and mediated in part by non-genomic AKT signaling. RNA-seq analyses compared with and without SAL treatment as well as by AKT inhibition (AKTi) revealed a specific transcriptome landscape. Comparing the top 100 genes similarly regulated by SAL in two human PCa cell lines that undergo cell senescence and being counteracted by AKTi revealed 33 commonly regulated genes. One gene, ERBB receptor feedback inhibitor 1 (ERRFI1), encodes the mitogen-inducible gene 6 (MIG6) that is potently upregulated by SAL, whereas the combinatory treatment of SAL with AKTi reverses the SAL-mediated upregulation. Functionally, knockdown of ERRFI1 enhances the pro-survival AKT pathway by enhancing phosphorylation of AKT and the downstream AKT target S6, whereas the phospho-retinoblastoma (pRb) protein levels were decreased. Further, the expression of the cell cycle inhibitor p15INK4b is enhanced by SAL and ERRFI1 knockdown. In line with this, cell senescence is induced by ERRFI1 knockdown and is enhanced slightly further by SAL. Treatment of SAL in the ERRFI1 knockdown background enhances phosphorylation of both AKT and S6 whereas pRb becomes hypophosphorylated. Interestingly, the ERRFI1 knockdown does not reduce AR protein levels or AR target gene expression, suggesting that MIG6 does not interfere with genomic signaling of AR but represses androgen-induced cell senescence and might therefore counteract SAL-induced signaling. The findings indicate that SAL treatment, used in BAT, upregulates MIG6, which inactivates both pRb and the pro-survival AKT signaling. This indicates a novel negative feedback loop integrating genomic and non-genomic AR signaling.

Glucocorticoids are induced while dihydrotestosterone levels are suppressed in 5-alpha reductase inhibitor treated human benign prostate hyperplasia patients.

The development of benign prostatic hyperplasia (BPH) and medication-refractory lower urinary tract symptoms (LUTS) remain poorly understood. This study attempted to characterize the pathways associated with failure of medical therapy for BPH/LUTS. Transitional zone tissue levels of cholesterol and steroids were measured in patients who failed medical therapy for BPH/LUTS and controls. Prostatic gene expression was measured using qPCR and BPH cells were used in organoid culture to study prostatic branching. BPH patients on 5-α-reductase inhibitor (5ARI) showed low levels of tissue dihydrotestosterone(DHT), increased levels of steroid 5-α-reductase type II (SRD5A2), and diminished levels of androgen receptor (AR) target genes, prostate-specific antigen (PSA), and transmembrane serine protease 2 (TMPRSS2). 5ARI raised prostatic tissue levels of glucocorticoids (GC), whereas alpha-adrenergic receptor antagonists (α-blockers) did not. Nuclear localization of GR in prostatic epithelium and stroma appeared in all patient samples. Treatment of four BPH organoid cell lines with dexamethasone, a synthetic GC, resulted in budding and branching. After failure of medical therapy for BPH/LUTS, 5ARI therapy continued to inhibit androgenesis but a 5ARI-induced pathway increased tissue levels of GC not seen in patients on α-blockers. GC stimulation of organoids indicated that the GC receptors are a trigger for controlling growth of prostate glands. A 5ARI-induced pathway revealed GC activation can serve as a master regulator of prostatic branching and growth.

Dynamic plasticity of prostate cancer intermediate cells during androgen receptor-targeted therapy.

Treatment-emergent small cell neuroendocrine prostate cancer (t-SCNC) is associated with an epithelial lineage switch from an androgen receptor (AR)-positive to neuroendocrine (NE)-marker-positive status. Understanding the potential for reversibility of this aggressive disease state has been hampered by the paucity of models suitable for studying rate-limiting, transitional, or intermediate tumor cell subpopulations. We define a dual reporter model that measures acute transcriptional changes in response to castration or AR targeting agents. We identify steady-state transcriptional heterogeneity in AR and NE biomarkers, including intermediate subpopulations that are coordinately high for prostate-specific antigen (PSA) and neuron-specific enoclase (NSE) promoter activity. In the presence of castration or AR inhibitors, intermediate cells were necessary and sufficient for therapy-induced conversion of human PC cells to an NSE-high transcriptional status. Using hormone add-back studies, treatment-induced PSA-NSE transcriptional plasticity was reversible in PTEN-deficient PC cells but not in the presence of secondary genetic driver genes, including MYCN.

MO20-3 Caffeic acid phenethyl ester suppresses drug resistance of enzalutamide or abiraterone in prostate cancer cells

Enzalutamide (ENZ) and Abiraterone acetate (AA) are drugs targeting androgen receptor axis for treatment of metastatic castration-resistant prostate cancer (mCRPC). However, a majority of patients receiving these treatments will eventually acquire drug resistance, which is caused by multiple mechanisms including the activation of AR splice variant 7 (AR-V7). We determined if caffeic acid phenethyl ester (CAPE) can suppress the signaling of AR-V7 and tumor growth of ENZ-resistant or AA-resistant mCRPC cells.

The Potential Tumor-Suppressor DHRS7 Inversely Correlates with EGFR Expression in Prostate Cancer Cells and Tumor Samples.

Simple SummaryProstate cancer is one of the most common malignancies in men. Current therapies are initially effective but resistance often develops, leading to tumor recurrence and death. Further research on new players, mechanisms involved in prostate cancer, and therapy resistance is needed. We studied the role of DHRS7, a potential tumor suppressor with currently unknown physiological function, in prostate cancer cells using proteome and gene expression analyses. Despite the fact that DHRS7 can inactivate 5α-dihydrotestosterone, its effect on prostate cancer cells seems to be unrelated to androgen metabolism. When comparing three widely studied prostate cancer cell lines, we observed a negative correlation between DHRS7 and EGFR expression. DHRS7 knockdown enhanced EGFR expression, while knockdown of EGFR tended to increase DHRS7 expression. Importantly, DHRS7 expression negatively correlates with EGFR expression and positively with survival rates in prostate cancer patients. This study suggests a tumor-suppressor role for DHRS7 by modulating EGFR expression in prostate cancer.Prostate cancer (PCa), one of the most common malignancies in men, typically responds to initial treatment, but resistance to therapy often leads to metastases and death. The dehydrogenase/reductase 7 (DHRS7, SDR34C1) is an “orphan” enzyme without known physiological function. DHRS7 was previously found to be decreased in higher-stage PCa, and siRNA-mediated knockdown increased the aggressiveness of LNCaP cells. To further explore the role of DHRS7 in PCa, we analyzed the proteome of LNCaP cells following DHRS7 knockdown to assess potentially altered pathways. Although DHRS7 is able to inactivate 5α-dihydrotestosterone, DHRS7 knockdown did not affect androgen receptor (AR) target gene expression, and its effect on PCa cells seems to be androgen-independent. Importantly, proteome analyses revealed increased expression of epidermal growth factor receptor (EGFR), which was confirmed by RT-qPCR and Western blotting. Comparison of AR-positive LNCaP with AR-negative PC-3 and DU145 PCa cell lines revealed a negative correlation between DHRS7 and EGFR expression. Conversely, EGFR knockdown enhanced DHRS7 expression in these cells. Importantly, analysis of patient samples revealed a negative correlation between DHRS7 and EGFR expression, both at the mRNA and protein levels, and DHRS7 expression correlated positively with patient survival rates. These results suggest a protective role for DHRS7 in PCa.

Abstract 5670: TRPM8 ion channel role in prostate cancer: Actions as a rapid testosterone signaling receptor

Abstract The transient receptor potential melastatin 8 (TRPM8) is well-known for its role in cold sensation in somatosensory neurons. TRPM8 was first identified as a prostate epithelial cell-specific gene, however, its role was unclear due to the absence of its endogenous agonist. We are the first to discover the novel role of TRPM8 as a rapid testosterone receptor. The TRPM8 mRNA is highly expressed in prostate cancer (PC) and is lost during the transition to androgen-independent prostate cancer (AIPC). Although emerging studies have shed light regarding androgen regulation of TRPM8 mRNA expression, we found that the addition of androgen receptor (AR) on the lipid bilayers inhibited testosterone-TRPM8 induced Ca2+ uptake. Additionally, our IHC revealed increased internalization of the TRPM8 protein in high-grade PC and that TRPM8 protein was targeted for proteasomal degradation in PC. We observed that inhibition of AR and UBA1 promoted the stabilization of TRPM8 on the plasma membrane, triggering Ca2+-induced cytotoxicity and apoptosis in both androgen dependent and androgen independent PC cells. Furthermore, in line with previous studies, we showed that testosterone-induced TRPM8 activation on the planar lipid bilayers also required phosphatidylinositol 4,5-bisphosphate (PIP2). Loss of PTEN is associated with tumor recurrence and the transition to AIPC. Thus, PTEN loss mediated PIP2 deficiency may be an important mechanism of TRPM8 desensitization in PC. Therefore, we propose that the rescue of TRPM8 activity on the plasma membrane combined with AR targeting may be an effective therapy for PC. Citation Format: Kiran Velpula, Katherine Shishido, Susovon Bayen, Swapna Asuthkar. TRPM8 ion channel role in prostate cancer: Actions as a rapid testosterone signaling receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5670.

Abstract 1527: Developing a multifaceted approach to target androgen receptor (AR) in castration resistant prostate cancer

Abstract Androgen receptor (AR) signaling is also regulated epigenetically by histone-modifying enzymes (histone deacetylases-HDACs) that result in the development and progression of prostate cancer (CaP). It has been established that the inhibition of HDACs suppresses CaP proliferation by inhibiting AR signaling via multiple mechanisms, including AR degradation. Hence, the goal of this study was to develop small molecules that employ multiple mechanisms to inhibit AR expression and promote its degradation, which would effectively control the emergence and treatment of castration-resistant prostate cancer (CRPC). Several rationally designed antiandrogen equipped HDAC inhibitors (HDACis) were synthesized, and their efficacy on CRPC growth was examined both in vitro and in vivo models. Results demonstrated that these compounds' target (HDAC and AR) binding properties translated into potent anticancer activity against AR+ CRPC cell lines and to a lesser extent in AR- CRPC cell lines. In addition, among these compounds, we found that SB-146, a nontoxic agent, not only inhibited the growth of AR+ CRPC cells at nanomolar IC50s without sparing toxicity to healthy prostate epithelial cells. Molecular analysis confirmed SB-146 downregulated the expressions of both AR and AR-spice variants (AR-SV) in CRPC cells. Further analyses revealed downregulation of AR downstream (PSA, HOXB13 and FOXA1) targets as well as AR-SV’s (NUP210 and SLC3A2) in CRPC cells. SB-146 was also able to overcome DHT-induced AR and AR-SV signaling in C4-2B and 22Rv1 cells. Assessment of SB-146’s therapeutic efficacy on in vivo models revealed that oral administration of this compound abrogated AR+ and AR-SV (C4-2B and 22Rv1) tumor growth in xenotransplanted nude mice models. Finally, immunohistochemical analysis confirmed SB-146’s target of both AR and AR-SVs on xenografted tumor tissues. Together these results confirm that SB-146 is a potent agent that inhibits AR signaling in CRPC cells. Citation Format: Uttara Saran, Balaji Chandrasekaran, Ashish Tyagi, Subhasish Tapadar, Bocheng Wu, Oyelere Adegboyega, Chendil Damodaran. Developing a multifaceted approach to target androgen receptor (AR) in castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1527.

Abstract 2510: An atlas of single-cell and spatial transcriptomics reveals alterations that correlate with human prostate cancer progression

Abstract Most systemic therapies for prostate cancer target androgen receptor signaling. Given the ongoing disease burden and mortality from advanced prostate cancer that persist despite available therapies, novel therapeutic targets are clearly needed. To this end, we conducted a high-resolution analysis of primary prostate cancer samples across a spectrum of tumor stage using 10x Chromium scRNAseq. Within each patient, we compared the prostate tumor to its adjacent normal tissue. These samples were also compared to healthy prostate samples collected from patients whose prostates were removed in the setting of bladder cancer surgery. Joint analysis revealed a rich repertoire of immune and non-immune stroma cells. Significant alterations were apparent in the prostate tumor microenvironment compared to adjacent normal and healthy samples. One striking difference was an expansion of T-cell cluster characterized by decreased cytotoxic and an increased exhaustion gene signatures. Another difference was the significantly higher regulatory T cell (Treg) activity. There was a substantial population shift within the myeloid compartment. Specifically, tumor tissue showed a selective enrichment in M2-like macrophages which was validated by multiplex immunohistochemistry. In addition, tumor tissue showed a significant increase in the myeloid-derived suppressor cell scoring in tumor inflammatory monocytes that strongly correlated with Treg activity. The changes in both myeloid and T cell gene expression suggest an immune suppressive environment within the tumor. In addition, we performed Slide-seqV2 to generate high spatial resolution maps of cellular organization of prostate tissues. The highly detailed spatial configuration of the healthy prostate tissue showed well-organized prostate epithelial glands surrounded by immune and non-immune stromal cells. This tissue architecture was severely disrupted within the cancerous prostate. Endothelial and fibroblast populations were disorganized and dispersed throughout the invading tumor. Context-dependent differential expression gene analysis on endothelial cells showed significant upregulation of sprouting angiogenesis and vascular endothelial growth factor pathways in the tumor context. In addition, we developed an approach to characterize the coordination between tumor cells and stromal compartment via spatial ligand-receptor (L/R) interaction. Data revealed 405 significant potential communication channels with many of these interactions known to have a role in prostate tumor angiogenesis and progression. These data provide a high resolution landscape of localized prostate cancer via 10x and Slide-seq. Data showed an immunosuppressive milieu in addition to highly active endothelial angiogenic program. L/R interaction analysis revealed interactions that may serve as potential therapeutic targets. Citation Format: Taghreed Hirz, Shenglin Mei, Hirak Sarkar, Youmna Kfoury, Shulin Wu, Chin-Lee Wu, Fei Chen, Evan Z. Macosko, Douglas M. Dahl, Ninib Baryawno, David T. Scadden, Philip J. Saylor, Peter V. Kharchenko, David B. Sykes. An atlas of single-cell and spatial transcriptomics reveals alterations that correlate with human prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2510.

Abstract 1070: Dual inhibition of AR and β-catenin induces cell death in abiraterone-resistant prostate cancer

Abstract Abiraterone acetate has been clinically approved for treatment of patients with advanced-stage prostate cancer. It reduces testosterone production by blocking the enzyme cytochrome P450 17 alpha-hydroxylase. Despite exhibiting improved survival outcomes, almost all patients with advance-stage disease relapse, progressing to a more aggressive and lethal castration-resistant prostate cancer. Our pathway-related analysis and characterization of gene expression identified activation of the β-catenin pathway in abiraterone-resistant prostate cancer. Increased expression of androgen receptor (AR) and β-catenin and their crosstalk between distinct signaling pathways causes activation of AR target genes and regulatory networks for which overcoming innate or acquired resistance remains a major challenge. Here we show that co-targeting AR and β-catenin induces cell death in abiraterone-resistant prostate cancer. For these studies, we generated abiraterone-resistant C4-2B cells (C4-2B-Abi) by growing them in progressive concentration of abiraterone 5-20µM to develop resistance and maintained in 5µM abiraterone in the culture medium for >20 generations. Treatment of C4-2B-Abi cells individually with abiraterone (20µM) and ICG001 (2µM) for 72 h exhibited a partial suppressive effect in cell growth compared to C4-2B parental cell line. A combination of abiraterone and ICG001 (20µM + 2µM) exhibited marked cell growth inhibition with significant increase in G1-phase cell cycle arrest, inhibition of migration and invasion in C4-2B-Abi cells, compared to the parental counterpart. Furthermore, combination treatment led to significant reduction in cell proliferation as assessed by PCNA along with reduction in the protein expression of AR, AR-v7, CDC20, PSA, β-catenin and its downstream targets, cyclinD1 and c-Myc, which were more prominent in C4-2B-Abi cells, compared to parental C4-2B cells. Co-targeting AR and β-catenin potentiated cell death in C4-2B-Abi cells as evident by increased PARP cleavage. Together, our data identify activation of the β-catenin pathway as a major mechanism contributing to abiraterone resistance and demonstrate that combined treatment with abiraterone and ICG001 appears to be an effective regimen for treatment of abiraterone-resistant cancer cells. This opens new therapeutic modality for advance-stage castration-resistant prostate cancer patients. Citation Format: Ibrahim M. Atawia, Prem P. Kushwaha, Shiv S. Verma, Spencer Lin, Eswar Shankar, Osama Abdel-Gawad, Sanjay Gupta. Dual inhibition of AR and β-catenin induces cell death in abiraterone-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1070.