Abstract 1530: Elucidating the role of exogenous lipids in ferroptosis in castration resistant prostate cancer

Abstract Prostate Cancer (PCa) is the leading cause of cancer cases in U.S. men and the second leading cause of male cancer death. An advanced form of the disease, termed castration-resistant PCa (CRPC), is not curable, and accounts for most PCa mortality. Several studies have observed that the use of statins, cholesterol-lowering drugs, leads to a reduced risk of PCa advancing to the metastatic CRPC stage. Additionally, in combination with abiraterone or enzalutamide, statins have been able to prolong time to progression and overall survival in patients with metastatic CRPC. The purpose of this study is to understand the mechanism of how a systemic reduction of cholesterol leads to these outcomes. Ferroptosis is a lipid peroxidation-dependent form of cell death. Several metabolites in the cholesterol synthesis pathway have been observed to protect cells against ferroptosis. We hypothesize that extracellular cholesterol, in conjunction with its precursors, protect CRPC cells from ferroptosis.We used the isogenic LNCaP and LN95 cell line models of CRPC progression as well as PC3 cells, another model of CRPC. We analyzed relative cell number using crystal violet assays and used flow cytometry to assess proliferation, cell death, apoptosis, and lipid peroxidation. Additionally, RNA sequencing and mass spectrometry-based metabolomics were used to analyze changes to the transcriptome and metabolome. We observed that all cell line models exhibit dependence on extracellular lipids for their growth and proliferation. Additionally, removal of extracellular lipids (EL) led to an increase in non-apoptotic forms of cell death. Further experiments showed that the growth reduction caused by removal of EL was rescued in the CRPC models by Ferrostatin-1, a ferroptosis inhibitor, but not the apoptosis inhibitor Q-VD-OpH. Removal of EL also led to an increase in lipid peroxides. Upon gene set enrichment analysis of RNA sequencing data, we identified the Hallmark Cholesterol Homeostasis gene set as the most upregulated across all models cultured in lipid free medium. Additionally, we observed a reduction in cholesteryl ester levels upon the removal of EL across all models. Interestingly, the growth reduction caused by removal of EL was rescued by cholesterol only in the CRPC models but not in the LNCaP model. Cholesterol supplementation after lipid depletion also led to a protection of cells from lipid peroxides and a reduced sensitivity to the ferroptosis inducing agent JKE-1674. Collectively, these data indicate the dependence of PCa models, particularly CRPC models, on extracellular cholesterol for their growth and proliferation. Additionally, CRPC models showed susceptibility to ferroptosis upon removal of EL, which was prevented by adding back cholesterol to the growth medium. These results show cholesterol as an antiferroptotic molecule in cell line models of CRPC. Citation Format: José C. Valentín López, Scott M. Dehm. Elucidating the role of exogenous lipids in ferroptosis in castration resistant 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 1530.