Abstract 365: Proteomic analysis of cytoplasmic lipid droplets and whole cell lysates reveal ferroptosis regulation by fatty acid synthase-derived lipid droplets in metastatic breast cancer

Abstract Breast cancer remains a serious public health concern, where metastasis accounts for the majority of patient mortality. Lipid accumulation in metastases is associated with reduced treatment response and poorer patient outcomes, although the mechanisms linking lipid accumulation and metastasis are not clear. We previously demonstrated that metastatic MCF10CA1a breast cancer cells have higher de novo lipogenesis and cytoplasmic lipid droplets (CLDs) compared to non-metastatic MCF10A-ras cells. Additionally, fatty acid synthase (FASN)-derived TAG stores sustained key metastatic processes in the MCF10CA1a cells. The proteome of CLDs is diverse and plastic, reflecting differing CLD functions; therefore, we sought to identify proteins differentially abundant on CLDs from vehicle-treated and FASN-inhibited MCF10CA1a breast cancer cells. We performed untargeted, global proteomics analysis on whole cell lysates (WCLs) and CLDs from vehicle-treated and FASN-inhibited (TVB-3166) MCF10CA1a cells to determine protein pathways differentially enriched on CLDs. Together, 2,416 and 3,539 proteins were identified within CLD fractions and WCLs, respectively. Of the proteins identified in CLD fractions, ~30% (717) were commonly identified within both conditions. Interestingly, 1,571 proteins were unique to the CLD fraction from vehicle-treated cells, whereas only 19 proteins were unique to the CLD fraction from FASN-inhibited cells. Proteins unique to CLDs from vehicle-treated cells include those known to reduce cancer cell survival and progression, such as proteins involved in cadherin-binding and cell cycle regulation. Proteins known to reduce cancer progression that associate with CLDs may prevent them from functioning at their normal site of action. Interestingly, an inhibitor of ferroptosis, glutathione peroxidase 4 (GPX4), was only detected in the vehicle-treated CLD and WCL samples, whereas proteins that promote ferroptosis were more abundant on CLDs of the more migratory cells, although their levels between WCLs were similar. Taken together, these data suggested that the machinery for ferroptosis may be mislocalized in TAG-rich cells, thereby protecting them from ferroptosis. Indeed, FASN-inhibited cells had nearly 30% more intracellular iron compared to the vehicle-treated cells, an important prerequisite for ferroptosis, thus indicating higher levels of ferroptotic stress. Functionally, the vehicle-treated cells were 25% more readily rescued by N-acetylcysteine from ferroptosis induced by erastin than were FASN-inhibited cells, despite comparable sensitivity to erastin. These preliminary findings suggest that FASN-derived CLDs in metastatic cells may aid in protecting against ferroptosis to promote breast cancer progression. Citation Format: Chaylen Andolino, Kimberly K. Buhman, Michael F. Coleman, Stephen D. Hursting, Marjorie Layosa, Michael K. Wendt, Dorothy Teegarden. Proteomic analysis of cytoplasmic lipid droplets and whole cell lysates reveal ferroptosis regulation by fatty acid synthase-derived lipid droplets in metastatic breast 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 365.