A Dual Role for DGAT-mediated Lipid Droplet Biogenesis in Ferroptosis Regulation

ABSTRACT Lipid droplets (LDs) are dynamic fat storage organelles involved in fatty acid metabolism, signalling and trafficking. By storing polyunsaturated fatty acids (PUFAs) in the form of neutral lipids, LDs can either mitigate or exacerbate lipotoxic damage. However, the role of LDs in regulating cellular fatty acid distribution, membrane unsaturation and ferroptosis susceptibility remains poorly understood. Here, we show that inhibition of diacylglycerol acyltransferase (DGAT)-mediated LD biogenesis in PUFA-supplemented triple-negative breast cancer cells induces widespread lipidome rearrangements and membrane phospholipid acyl-chain remodelling, promoting lipid peroxidation and ferroptosis sensitivity. Lipidomic analyses reveal that LDs efficiently sequester exogenous PUFAs within triacylglycerols and cholesteryl esters, significantly altering the unsaturation profiles of these neutral lipids. When LD formation is impaired by DGAT inhibition, PUFAs are redistributed into membrane ester and ether glycerophospholipids, enhancing overall membrane unsaturation, lipid peroxidation, and increasing ferroptosis susceptibility, even in the absence of additional ferroptosis inducers. In contrast, in ferroptosis- and PUFA-resistant lung cancer cells, LDs exhibit a dual role, whereby the mode of ferroptosis induction and PUFA loading determined whether DGAT inhibition promoted or protected against ferroptosis. The pro-ferroptotic function of LDs predominates in these cells, particularly under conditions of ferroptosis suppressor protein 1 (FSP1) deficiency. This study highlights LDs as multifaceted regulators of ferroptosis sensitivity, integrating metabolic and redox quality control pathways.