Lipid metabolic mechanisms in EMT/ZEB1-dependent ferroptosis

Ferroptosis is a cell death program that alters the membrane architecture by causing oxidative damage to phospholipids. To keep ferroptosis at bay, polyunsaturated fatty acids (containing multiple double bonds) are balanced in cell membranes by fatty acids with a single double bond (monounsaturated fatty acids) that are less sensitive towards oxidative modification. Recent evidence suggests that cancer cells undergoing epithelial-to-mesenchymal transition (EMT) become more susceptible towards ferroptosis. This finding is of high therapeutic relevance because EMT is an embryonic program that is hijacked by cancer cells that undergo metastasis, escape immune surveillance, and gain therapy resistance. Induction of ferroptosis in cancer cells is therefore considered as a promising strategy to fight aggressive, metastatic cancer. How EMT determines ferroptosis sensitivity is largely unknown. We recently found that distinct EMT transcription factors reprogram fatty acid and phospholipid metabolism, essentially alter the membrane fatty acid composition, and sensitize cancer cells towards ferroptosis. By combining lipidome and transcriptome analyses, we recently identified six candidate metabolic pathways that we will here functionally investigate. These pathways will be genetically or pharmacologically manipulated and the flux of labeled metabolites through them monitored. We will i) study whether the EMT-dependent regulation of these pathways mediates ferroptosis sensitivity, ii) search for lipid species that bear the functional activity, and iii) explore whether an interference with their biosynthesis has the potential to overcome tumor resistance. We exploit recent advances in differential ion mobility spectrometry to distinguish between isobaric lipids (with the same nominal mass) and extend our studies on a broad spectrum of membrane lipids as well as tumor- and immunomodulatory signaling lipids. Kinetic aspects and the subcellular localization of lipids will be taken into account, and we will elucidate whether certain thresholds or pulses of specific lipid species are required to trigger ferroptotic cell death. To answer these questions, we will apply model systems of varying complexity and account for tumor heterogeneity by including i) cancer cell lines from four different origins, ii) breast cancer cell lines with diverse genetic background, ferroptosis sensitivity, and acquired resistance, iii) tumor-derived pancreatic cancer cell lines, iv) and a murine model of metastasizing pancreatic cancer. We expect from this project basic insights into the mechanisms of therapy resistance that might open the door towards new pharmacological strategies for tackling the aggressive fraction of mesenchyme-type metastatic cancer cells.