Inhibition of the RORC/GPX4 mediated ferroptosis regulatory axis suppresses tumor growth and alleviates enzalutamide resistance in prostate cancer

Previously, we found that the expression of long non-coding RNA (lncRNA) small nucleolar RNA host gene 17 (SNHG17) was up-regulated in castration-resistant prostate cancer (CRPC) cells compared to that in hormone sensitive prostate cancer (HSPC) cells. Moreover, we found that CD51 was up-regulated in prostate cancer cells and promoted the carcinogenesis and progression of prostate cancer. However, the regulatory mechanism of SNHG17 and CD51 in the development of CRPC remains unclear. In the current study, we aimed to elucidate the expressions, functions, and underlying mechanism of SNHG17 and CD51 in CRPC. Our results further confirmed that both SNHG17 and CD51 were up-regulated in CRPC tissues and cells. In addition, we found that SNHG17 expression was positively correlated with CD51 expression in prostate cancer. Mechanically, SNHG17 functioned as a competing endogenous RNA (ceRNA) to up-regulate CD51 expression through competitively sponging microRNA-144 (miR-144), and CD51 was identified as a direct downstream target of miR-144 in CRPC. Functionally, down-regulation of SNHG17 or up-regulation of miR-144 inhibited the proliferation, migration, and invasion of CRPC cells, whereas up-regulation of SNHG17 and down-regulation of miR-144 promoted the proliferation, migration and invasion of CRPC cells in vitro and in vivo. Using gain and loss-of function assay and rescue assay, we showed that miR-144 inhibited cell proliferation, migration and invasion by directly inhibiting CD51 expression, and SNHG17 promoted cell proliferation, migration and invasion by directly enhancing CD51 expression in CRPC cells. Taken together, our study reveals the role of the SNHG17/miR-144/CD51 axis in accelerating CRPC cell proliferation and invasion, and suggests that SNHG17 may serve as a novel therapeutic target for CRPC.

Prostate cancer is characterized by aberrant lipid metabolism, including elevated fatty acid oxidation. Carnitine palmitoyltransferase 1B (CPT1B) catalyzes the rate-limiting step of fatty acid oxidation. This study aimed to determine if CPT1B has a critical role in prostate cancer progression and to identify its regulatory mechanism. CPT1B expression data from The Cancer Genome Atlas and Gene Expression Omnibus databases was compared with patient survival data. A tissue microarray was constructed with 60 samples of prostate cancer and immunohistochemically stained for CPT1B. Castration-resistant prostate cancer (CRPC) cell lines 22RV1 and C4-2 in which CPT1B expression had been stably knocked down were established; and cell proliferation, cell cycle distribution, and invasion were investigated by Cell Counting Kit-8 (CCK-8) and colony formation assays, flow cytometry, and Transwell assays, respectively. To examine the impact of androgen receptor (AR) inhibition on CPT1B expression, JASPAR CORE was searched to identify AR-binding sites in CPT1B. Dual luciferase and ChIP assays were performed to confirm CPT1B activity and AR binding, respectively. Differentially expressed genes (DEGs) in prostate cancer underwent gene set enrichment analysis (GSEA). Enzalutamide-resistant C4-2 cells were generated and the mechanism of enzalutamide resistance and downstream signaling pathway changes of CPT1B to C4-2 was explored through CCK-8 test. CPT1B expression was upregulated in human prostate cancer compared with normal prostate tissue and was associated with poor disease-free survival and overall survival. Silencing of CPT1B resulted in downregulated cell proliferation, reduced S-phase distribution, and lower invasive ability, whereas the opposite was observed in CRPC cells overexpressing CPTB1. DEGS in prostate cancer were correlated with G-protein-coupled receptor signaling, molecular transducer activity, and calcium ion binding. AR may regulate CPT1B expression and activity via specific binding sites, as confirmed by dual luciferase and ChIP assays. The CCK-8 experiment demonstrated that CPT1B overexpression in C4-2 cells did not significantly increase the ability of enzalutamide resistance. However, overexpression of CPT1B in C4-2R cells significantly increased the enzalutamide resistance. Upregulation of CPT1B expression increased AKT expression and phosphorylation. CPT1B is upregulated in prostate cancer and is correlated with poor prognosis, indicating its potential as a biomarker. AR inhibits the transcription of CPT1B. In the CRPC cell line, overexpression of CPT1B alone cannot promote enzalutamide resistance, but in the drug-resistant line C4-2R, overexpression of CPT1B can promote the resistance of C4-2R to enzalutamide.

Background: Therapy resistance in cancer is often linked to cancer cell state or lineage changes. In prostate cancer (PCa), a subset of castration-resistant prostate cancer (CRPC) loses reliance on the androgen receptor (AR) signaling and gains neuroendocrine (NE) traits through a lineage switch along disease progression. However, the molecular mechanisms driving such cellular lineage plasticity remain unclear. This study investigated the functional and mechanistic role of monoamine oxidase A (MAOA) in driving adenocarcinoma (adeno)-to-NE lineage conversion in CRPC cells upon enzalutamide (ENZ), a FDA-approved newer-generation antiandrogen drug, and evaluated the efficacy of MAOA inhibitors for treating CRPC and overcoming ENZ resistance.Methods: ENZ-resistant C4-2B and CWR-R1 cells (C4-2BENZR and CWR-R1ENZR) CRPC cells were transfected with a PSA promoter-driven GFP expression construct for isolating a PSAlo/− cell population distinct from PSAhi cells, which was used for manipulation of MAOA expression levels and subsequent functional assays in vitro and in vivo. The MAOA expression levels were determined and correlated with NE parameters in PCa clinical samples by immunohistochemistry and data mining of multiple publicly available clinical datasets. Results: MAOA protein expression was significantly upregulated in the PSAlo/− ENZ-resistant cell population, accompanied by concomitant activation of NE markers and downregulation of luminal markers including AR and AR targets, compared to the PSAhi counterpart. Elevated MAOA expression was also found in PCa patient samples associated with NE features from multiple independent cohorts. Silencing MAOA suppressed the proliferation, migration, invasion, stem-like properties and NE plasticity of both PSAlo/- C4-2BENZR and CWR-R1ENZR cells. Moreover, co-culturing adeno LNCaP or C4-2B cells with MAOA-silenced PSAlo/- cells exhibiting a repressed secretory phenotype attenuated acquired induction of NE markers and proliferation of adeno cells as compared to controls. Mechanistically, MAOA induces Twist1, which directly activates the transcription and expression of BRN2, a master neural transcription factor known to confer NE differentiation and anti-AR therapy resistance in CRPC. Further, pharmacological inhibition of MAOA greatly enhanced the growth-inhibitory efficacy of ENZ with ENZ sensitivity restored in ENZ-resistant LuCaP PC PDX-derived organoids as well as a PSAlo/− CWR-R1ENZR xenograft mouse model.Conclusion: Our results suggest MAOA as a potential therapeutic target for reversing ENZ resistance in CRPC treatment by blockade of NE transdifferentiation.Funding Acknowledgements: This work was supported by the NIH/NCI grants R37CA233658 and R01CA258634 (to B. Wu) and the DOD Department of Defense EIRA award W81XWH-21-1-0218 (to J. Wei) Citation Format: Jing Wei, Jing Wang, Chia-hui Chen, Wen Guan, Boyang Wu. MAOA Drives Anti Androgen Therapy Resistance in Advanced Prostate Cancer by Conferring Neuroendocrine Plasticity via a Twist1 BRN2 Signaling Pathway [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 2011.

The highly aggressive prostate cancer (PC) cells utilize androgen receptor (AR) signaling to facilitate their growth and metastasis. Despite androgen deprivation therapy (ADT), recurrence of castration-resistant prostate cancer (CRPC) often occur due to constitutive AR signaling via both full-length AR (AR-FL) and AR splice variants, primarily AR-V7. Therefore, safe strategies to suppress both AR-FL & AR-V7 expression in CRPC cells will be of significant benefit. Several plant-derived organosulfur compounds, e.g. sulforaphane and diallyl-trisulfide, can potently suppress AR expression in CRPC cells, but are yet to be clinically approved. We investigated whether dimethyl sulfoxide (DMSO) an approved organosulfur compound often used as a solvent for pharmaceutical agents, can similarly suppress AR levels in the CRPC cell lines, C4-2B and 22Rv1. Exposure to low dose DMSO (0.1-1%) was not cytotoxic to these CRPC cells, and less than 20% cytotoxicity was seen with 2.5% DMSO even at 96 h post-exposure. Interestingly however, DMSO dose-dependently suppressed AR-FL in C4-2B cells and both AR-FL and AR-V7 in 22Rv1 cells within 24 h post-exposure. Exposure to DMSO also downregulated the expression of hetero-nuclear ribonucleoprotein H1 (hnRNPH1), which is known to regulate AR expression and splicing. Although DMSO exposure showed a dose- and time-dependent effect on reactive oxygen species (ROS) production by PC cells, the AR-suppressive effect of DMSO was not altered by the antioxidant n-acetyl cysteine (NAC). Furthermore, although DMSO did not affect cell viability at these clinically-achievable concentrations, a significant (p<0.01) decrease in the migratory ability of both CRPC cell lines was clearly evident. Our novel findings indicate that safe doses of DMSO may be used to decrease AR expression and metastatic ability in CRPC cells. Citation Format: Namrata Khurana, Hogyoung Kim, Talal Khan, Shohreh Kahhal, Amar Bukvic, Asim B. Abdel-Mageed, Suresh C. Sikka, Debasis Mondal. Low dose dimethyl sulfoxide (DMSO) downregulates the expression of androgen receptor (AR) and AR-variant 7 (AR-v7) in castration resistant prostate cancer (CRPC) cells [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 2446.

Rooibos suppresses proliferation of castration-resistant prostate cancer cells via inhibition of Akt signaling

Our previously study shown that Lysophosphatidylcholine Acyltransferase1 (LPCAT1) is overexpressed in castration resistant prostate cancer (CRPC) relative to primary prostate cancer (PCa), and androgen controls its expression via the Wnt signaling pathway. While highly expressed in CRPC, the role of LPCAT1 remains unclear. In vitro cell experiments referred to cell transfection, mutagenesis, proliferation, migration, invasion, cell cycle progression and apoptosis, Western blotting, Pulse-chase RNA labeling. BALB/c nude mice were used for in vivo experiments. We found that LPCAT1 overexpression enhanced the proliferation, migration, and invasion of CRPC cells both in vitro and in vivo. Silencing of LPCAT1 reduced the proliferation and the invasive capabilities of CRPC cells. Providing exogenous PAF to LPCAT1 knockdown cells increased their invasive capabilities; however platelet activating factor acetylhydrolase (PAF-AH) and the PAFR antagonist ABT-491 both reversed this phenotype; proliferation of CRPC cells was not affected in either model. LPCAT1 was found to mediate CRPC growth via nuclear re-localization and Histone H4 palmitoylation in an androgen-dependent fashion, increasing mRNA synthesis rates. We also found that LPCAT1 overexpression led to CRPC cell resistance to treatment with paclitaxel. LPCAT1 overexpression in CRPC cells drives tumor progression via increased mRNA synthesis and PAF production. Our results highlight LPCAT1 as a viable therapeutic target in the context of CRPC.

Our previously study shown that Lysophosphatidylcholine Acyltransferase1 (LPCAT1) is overexpressed in castration resistant prostate cancer (CRPC) relative to primary prostate cancer (PCa), and androgen controls its expression via the Wnt signaling pathway. While highly expressed in CRPC, the role of LPCAT1 remains unclear. In vitro cell experiments referred to cell transfection, mutagenesis, proliferation, migration, invasion, cell cycle progression and apoptosis, Western blotting, Pulse-chase RNA labeling. BALB/c nude mice were used for in vivo experiments. We found that LPCAT1 overexpression enhanced the proliferation, migration, and invasion of CRPC cells both in vitro and in vivo. Silencing of LPCAT1 reduced the proliferation and the invasive capabilities of CRPC cells. Providing exogenous PAF to LPCAT1 knockdown cells increased their invasive capabilities; however platelet activating factor acetylhydrolase (PAF-AH) and the PAFR antagonist ABT-491 both reversed this phenotype; proliferation of CRPC cells was not affected in either model. LPCAT1 was found to mediate CRPC growth via nuclear re-localization and Histone H4 palmitoylation in an androgen-dependent fashion, increasing mRNA synthesis rates. We also found that LPCAT1 overexpression led to CRPC cell resistance to treatment with paclitaxel. LPCAT1 overexpression in CRPC cells drives tumor progression via increased mRNA synthesis and PAF production. Our results highlight LPCAT1 as a viable therapeutic target in the context of CRPC.

Background: Cancer drug resistance arises not only from selection of resistant clones, but also through rapid activation of adaptive transcriptional programs. One mechanism of transcriptional regulation involves N6-methyladenosine (m6A) RNA modification, which dynamically regulates mRNA processing and alternative splicing, ultimately impacting cell fate and differentiation. In metastatic prostate cancer (PC), alternative splicing of the androgen receptor (AR) generates a truncated variant (ARV7) that is resistant to inhibition with AR antagonists like enzalutamide. Therefore, we hypothesized that m6A modifications are associated with gene expression changes that promote enzalutamide resistance in PC. Methods: We used methyl-RNA-immunoprecipitation followed by sequencing (MeRIP-seq) in parallel with RNA-seq to identify gene transcripts that were both differentially methylated and differentially expressed in enzalutamide-sensitive (C4-2B) vs. enzalutamide-resistant (MDVR) PC cell lines. Differentially methylated and expressed transcripts were further filtered and prioritized using clinical and functional database tools, including Gene Ontology (GO) enrichment analysis, Gene Set Enrichment Analysis (GSEA), The Cancer Genome Atlas (TCGA) and the Oncology Research Information Network (ORIEN) avatar. Top ranked candidates were validated by targeted MeRIP-PCR and qPCR, then tested functionally using cell viability assays combined with siRNA-depletion and enzalutamide treatment. Results: We identified 487 transcripts that were both differentially methylated and differentially expressed, then we functionally prioritized 46 genes based on PC relevance and validated 8 of the top 12 via targeted qPCR and MeRIP-PCR. In a stable enzalutamide-resistant cell line (MDVR), one such gene, THBS1, had significantly increased mRNA m6A level associated with a 41-fold decrease in transcript level. Consistent with this, short term enzalutamide treatment of C4-2B and LNCaP PC cell lines induced a 2-fold reduction in THBS1 transcript levels, accompanied by significant increases in AR and MALAT1 mRNA levels. Similarly, siRNA-mediated THBS1 depletion potentiated enzalutamide resistance in these cell lines, but it did not increase AR, ARV7, or MALAT1 levels, suggesting that alternate mechanisms mediate its effects. Conclusions: Unbiased transcriptome-wide profiling revealed a large set of differentially methylated and expressed gene transcripts associated with enzalutamide resistance in PC. One such gene, THBS1, has previously been implicated in aggressive PC phenotypes like neuroendocrine transdifferentiation. We now show that THBS1 downregulation directly contributes to a rapid transition to enzalutamide resistance, suggesting a novel role for this gene in PC hormonal therapy resistance. Citation Format: Emmanuelle Hodara, Lisa M. Swartz, Aubree Mades, Tong Xu, Suhn Rhie, Amir Goldkorn. Combined transcriptomic and epitranscriptomic profiling identifies THBS1 as a regulator of enzalutamide resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4413.

Prostate cancer is the second leading cause of cancer death in men in western countries. Advanced prostate cancer is often resistant to hormonal treatment and systemic chemotherapy has limited efficacy. Androgen receptor (AR), a ligand dependent transcription factor plays pivotal role in the development and progression of prostate cancer. While majority of prostate cancers are initially androgen dependent and respond to androgen ablation therapy, most patients eventually recur with more aggressive castration-resistant prostate cancer (CRPC) where AR signaling is reactivated even in the absence of androgen stimulation. Therefore developing novel chemotherapeutic agents for castrate resistant prostate cancer (CRPC) treatment is critical to improve survival in men with CRPC. Triptolide, a diterpene triepoxide isolated from a chinese herb, is extremely effective against several cancers like pancreatic cancer, colorectal cancer and liver cancer both in vivo and in vitro. The water-soluble pro-drug of triptolide, Minnelide, downregulates HSP70 via inhibition of the activity of transcription factor Sp1. Since both Sp1 and HSP70 have been reported to be critical in functionality of AR, we assessed therapeutic potential of Minnelide on androgen dependent, CRPC in vitro and in vivo. Triptolide treatment resulted in dose- and time-dependent cell death in an androgen dependent cell line LNCaP, CRPC cell line C4-2 and enzalutamide resistant CRPC tumor cell line 22RV1. Triptolide treatment decreased expression of AR full length, AR splice variants and its downstream targets (PSA, NKX3.1) at the mRNA and protein levels. Further, reporter assay with AR responsive elements showed that triptolide decreased transcriptional activity of AR. Expression levels of Sp1 and HSP70 were also reduced following treatment with triptolide these cell lines. To test the efficacy of Minnelide in vivo, male athymic nude mice were castrated 7 days prior to implantation of enzalutamide resistant CRPC (22RV1) cells subcutaneously. The animals received daily intraperitoneal injection of Minnelide and tumor volume was measured weekly until tumor size reached 2cm3.Mice receiving daily injection of Minnelide had significantly smaller tumors than controls as early as two week of treatment (p = 0.008). Triptolide therapy inhibited enzalutamide resistant CRPC growth both in vitro and in vivo. Further, our studies for the first time showed that triptolide induces prostate tumor cell death by reducing expression of both full length AR and AR splice variants in a similar manner. Citation Format: Sumit Isharwal, Shrey Modi, Usman Barlass, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee, Badrinath Konety. Minnelide reduces castration-resistant and enzalutamide-resistant prostate cancer via downregulation of androgen receptor-mediated signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-017. doi:10.1158/1538-7445.AM2015-LB-017

INTRODUCTION AND OBJECTIVES: Androgen receptor signaling is the key driving force in prostate cancer progression, even in castration-resistant prostate cancer (CRPC). SD70, a competitive inhibitor of KDM4 class of histone demethylases, was demonstrated to have significant inhibitory effect on AR transactivation function in LNCaP cells and androgen-insensitive basal activity in CWR22Rv1. In-vitro assessment of efficacy in CWR22Rv1 cells demonstrated cytotoxity at 5 μM. We aimed to demonstrate the efficacy of SD70 in CRPC cell lines, determine its effect in conjunction with currently approved therapies, and identify a useful biomarker for this novel drug. MATERIALS AND METHODS: Using MTT viability assays, we first assessed SD70 dosing against two CRPC lines (C42B and GRP), then against C42B drug-resistant strains (abiratarone, enzalutamide, abi+enza, docetaxel). For the resistant lines, cells were then treated with 1 μM SD70 in combination with 10 μM abiratarone, 10 μM enzalutamide or 1 nM docetaxel. In vivo drug study was conducted with mouse xenograft models (3 million CWR22Rv1 cells via s.c. injection) in athymic nude mice. When the tumor volume reached 100 mm3, animals were randomly assigned to receive 10 mg/kg SD70 or vehicle via i.p. injection daily. Tumor monitoring was continued until tumor burden exceeded humane endpoint limits indicated by IACUC. RESULTS: SD70 demonstrated complete cytotoxic response at 5 μM against C42B and GRP cell lines; IC50 was 2.1 μM and 2.9 μM, respectively. All drug-resistant C42B cell lines were responsive to SD70 treatment; IC50 ranged from 0.90-1.57 μM. SD70 was found to be synergistic with abiratarone, enzalutamide, and docetaxel against drug-resistant C42B and GRP cell lines. In the CWR22Rv1 mouse xenograft model, SD70-treated mice demonstrated significantly lower tumor volume at the end of treatment period (SD70: 507.31±203.72 mm3, control: 2388.01±543.58 mm3); no significant drug toxicity was observed. Expression of AR surrogate markers (PSA, KLK2, TMPRSS2) were diminished in C42B and enzalutamide-resistant C42B lines treated with SD70. No changes in autophagy reporter LC-3 or activation of Akt was observed. CONCLUSIONS: SD70 demonstrates promising cytotoxic efficacy against CRPC cells lines in vitro, and appears to have a synergistic effect with currently approved CRPC medications (docetaxel, enzalutamide, and abiratarone) in drug-resistant CRPC lines. Initial in vivo trial demonstrated excellent efficacy against CWR22Rv1 without significant toxicity. Ongoing evaluation into identifying biomarkers will allow for further evaluation of this promising new agent. Citation Format: Thenappan Chandrasekar, Joy C. Yang, Min Xie, Sheng Ding, Michael G. Rosenfeld, Christopher P. Evans. Effect of a histone DNA demethylase on castration resistant prostate cancer cell lines, a potential therapeutic application. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3453. doi:10.1158/1538-7445.AM2015-3453

While androgen deprivation therapy (ADT) continues to be a fundamental aspect of prostate cancer treatment, the development of castration-resistant prostate cancer (CRPC) emphasizes the necessity for a more profound understanding of the tumor microenvironment (TME). Normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs) were isolated and characterized from normal control and prostate cancer specimens, respectively. PC3 and DU145 cells, and the corresponding enzalutamide resistant counterparts, PC3-EnzR and DU145-EnzR, were co-cultured with NFs or CAFs to evaluate the effects of TME in driving enzalutamide resistance. Cell viability of prostate cancer cells was examined by MTT assay. The study also utilized recombinant human neuregulin-1 (NRG1) protein and siRNA to modulate NRG1 expression in CAFs. RT-qPCR, Western blot, and ELISA were employed to assess gene and protein expressions related to the NRG1-HER3 signaling pathway and its association with enzalutamide resistance. CAFs significantly promoted cell growth and enzalutamide resistance of PC3-EnzR and DU145-EnzR cells through substantial increased secretion of NRG1 by CAFs. Co-culturing enzalutamide-resistant prostate cancer cells (PC3-EnzR and DU145-EnzR) with CAFs further enhanced enzalutamide resistance, as evidenced by elevated IC50 values. Inhibition of NRG1 in CAFs attenuated their impact on enzalutamide resistance, providing insight into the role of NRG1 in mediating the crosstalk between CAFs and prostate cancer in the context of enzalutamide resistance. This study elucidates the pivotal role of CAF-secreted NRG1 in promoting enzalutamide resistance in prostate cancer, providing valuable insights for developing targeted therapeutic strategies to overcome resistance in advanced prostate cancer.

Background: Recent studies suggest that glucocorticoid receptor (GR) activation can cause enzalutamide resistance in advanced prostate cancer (PCa) via functional bypass of androgen receptor (AR) signaling. However, the specific molecular mechanism(s) driving this process remain unknown. In an effort to identify drivers of prostate cancer progression, in a previous study, we determined that TBX2, a developmental T-box transcription factor (TF) master regulator, is over-expressed in CRPC and drives bone metastatic progression. A recent report confirmed that TBX2 and GR are two of the four TFs that drive enzalutamide resistance in advanced PCa. Our current study demonstrates that TBX2 with known repressor and activator functions, is the molecular switch that represses AR levels while activating GR expression thereby resulting in the replacement of AR signaling to control tumor growth. Methods: We genetically modulated TBX2 using multiple approaches: a) dominant negative, DN, to block TBX2 (TBX2DN), and b) overexpression, OE, to increase TBX2 expression (TBX2OE), c) shRNA mediated knockdown (shTBX2). RNA-seq was performed, and qRT-PCR, Western blot and immunohistochemical (IHC) analyses were used for validation. Further, we used chromatin immunoprecipitation (ChIP) and site directed mutagenesis (SDM) to confirm TBX2 binding on the AR promoter. We also used co-immunoprecipitation (Co-IP) to determine protein partners of TBX2. Results: Mechanistically, our studies revealed that TBX2 bound to the promoters of both AR and GATA2, an AR coregulator, thereby resulting in a bimodal repression of AR expression. Conversely, TBX2 upregulated GR via direct GR promoter binding and TBX2-GR protein-protein interaction. Together, concurrent repression of the AR and activation of GR resulted in enzalutamide resistance. Importantly, we found that SP2509, an allosteric inhibitor of the demethylase-independent function of LSD1, a TBX2-interacting protein in the COREST complex, can disrupt both TBX2-LSD1 and TBX2-GR protein-protein interactions thereby uncovering a unique mode of SP2509 action in CRPC. Together, our study supports a molecular model of CRPC wherein: 1) TBX2, LSD1 and GR proteins interface with each other, and 2) pharmacological inhibition of LSD1 blocks the TBX2-driven AR-to-GR switch through disruption of TBX2-GR interaction. Conclusions: In summary, our study identifies TBX2 as the molecular switch that drives the AR to GR signaling bypass thereby conferring enzalutamide resistance. Further, our study provides key insights into a potential therapeutic modality for targeting the AR to GR signaling switch via disruption of the TBX2-LSD1 and TBX2-GR protein-protein interactions Citation Format: Sayanika Dutta, Girijesh Kumar Patel, Hamed Khedmatgozar, Daniel Latour, Jonathan Welsh, Manisha Tripathi, Srinivas Nandana. A TBX2-driven signaling switch from androgen receptor to glucocorticoid receptor confers enzalutamide resistance in prostate cancer [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 6603.

IntroductionTo investigate the molecular mechanisms underlying enzalutamide resistance in castration-resistant prostate cancer (CRPC) and explore potential therapeutic strategies to overcome resistance.MethodsWe conducted comprehensive bioinformatic analysis using LNCaP/enzalutamide-resistant cells to identify key pathways associated with resistance. Functional validation was performed through targeted inhibition of the elongation of very-long chain fatty acid protein 2 (ELOVL2), followed by assays to assess cancer cell proliferation and enzalutamide sensitivity. Mechanistic studies were conducted to evaluate the impact of ELOVL2 on the ubiquitin-proteasome system and AR signaling pathways.ResultsBioinformatic analysis revealed that activation of fatty acid metabolism, particularly through upregulation of ELOVL2, plays a critical role in driving enzalutamide resistance in PCa. Functional studies demonstrated that targeted inhibition of ELOVL2 significantly suppressed cancer cell proliferation and restored enzalutamide sensitivity in resistant cells. Mechanistically, ELOVL2 facilitates enzalutamide resistance by impairing the ubiquitin-proteasome system, leading to the subsequent activation of AR signaling pathways.DiscussionOur findings demonstrate that ELOVL2 drives enzalutamide resistance in CRPC by stabilizing AR through inhibition of ubiquitin-proteasome-mediated degradation. Targeting ELOVL2 represents a promising therapeutic strategy to overcome resistance in CRPC, with potential to improve clinical outcomes for patients.

Androgen receptor (AR) is a ligand-induced transcription factor, which binds to thousands of genomic loci and activates a cell-type specific gene expression program. Androgen deprivation therapy has been the cornerstone of treatment for advanced prostate cancer for 70 years. Despite initial response, an incurable castration-resistant prostate cancer (CRPC) inevitably develops as a result of restored AR activity. Given the importance of AR signaling in CRPC, there has been a dedicated interest in dissecting the mechanisms of AR function after androgen deprivation. In CRPC, AR activity remains critical for tumor growth despite androgen deprivation. However, the gene expression program characteristic of AR signaling in androgen-dependent prostate cancer (ADPC) is attenuated, indicating AR signaling may be altered after androgen deprivation. Recent studies indicate that AR targets may be altered through direct reprogramming of ligand-induced AR binding (Wang et al, Cell 2009, 138: 245) or through reprogramming of FoxA1 (Wang et al, Nature 2011, 474:390) in the presence of androgen. In this study, we show that in the absence of ligand AR binds a distinct set of genomic loci that drive a gene expression program necessary for CRPC growth and may be an important alternative therapeutic target when androgen-deprivation therapies fail. We characterized AR action in the presence or absence of androgen (DHT) in LNCaP and C4-2B cells. Using chromatin immunoprecipitation sequencing (ChIP-seq) we identified thousands of distinct AR binding events in the absence of DHT in CRPC C4-2B cells, which direct a unique gene expression program in CRPC cells. Whole transcriptome sequencing (RNA-seq) revealed ∼ 450 genes were basally upregulated in the absence of DHT in C4-2B compared to the parental androgen-dependent LNCaP cell line. These genes are strongly correlated with androgen-independent AR binding sites. Quantitative chromosome conformation capture (3C) assays demonstrated a strong physical interaction between androgen-independent AR binding sites and nearby basally upregulated genes. Interestingly, in androgen-depleted conditions, the AR occupies genomic loci with constitutively open chromatin structures that lack the canonical androgen response element (ARE) and are not directed by FoxA1, a transcription factor involved in ligand-dependent AR targeting. After DHT treatment, androgen-independent AR binding was diminished as AR binding switched to canonical androgen-dependent AR binding sites. The novel androgen-independent AR target genes, which showed significant enrichment for cell-cycle pathways, are required for the survival and proliferation of CRPC cells after androgen withdrawal. RNA interference experiments showed significant inhibitory effects on proliferation, and a corresponding increase in apoptosis in the absence of or at low concentrations of androgen. Finally, we found that androgen-independent AR genes were to be significantly over-represented in castration-resistant metastatic patient samples exhibiting moderate AR expression. Current treatments for CRPC have focused on limiting androgen synthesis and blocking AR ligand binding based on the notion that persistent ligand-dependent AR signaling drives tumor growth. Our results show that androgen depletion results in a dramatic androgen-independent alteration of genome-wide AR occupancies and reprogramming of AR-mediated gene expression, which may be a critical alternative mechanism for CRPC growth. Citation Format: Keith F. Decker, Dali Zheng, John R. Edwards, Li Jia. Androgen receptor-mediated transcription is reprogrammed after hormone depletion [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A6.

Aspalathin-rich green Aspalathus linearis extract suppresses migration and invasion of human castration-resistant prostate cancer cells via inhibition of YAP signaling