Regulation of Ferroptosis Sensitivity in Hepatocellular Carcinoma Cells by Lysosomal Ion Channels TPC2 and TRPML1

Ferroptosis is an iron-dependent, lipid peroxidation–driven form of regulated cell death that has emerged as a therapeutic vulnerability in hepatocellular carcinoma (HCC), yet the contribution of lysosomes to this process remains incompletely understood. In this study, we investigated whether lysosomal ion channels regulate ferroptosis sensitivity in HCC cells, focusing on the two-pore channel 2 (TPC2) and the transient receptor potential mucolipin 1 (TRPML1). Using pharmacological modulation, genetic knockout models, flow cytometry-based cell death and lipid peroxidation assays, lipidomics, calcium measurements, and molecular analyses across multiple HCC cell lines, we examined how these channels influence ferroptotic signaling. We show that NAADP-dependent TPC2 activity is required for efficient ferroptosis induction, whereas TPC2 loss renders HCC cells resistant to ferroptosis triggered by system Xc⁻ inhibition or glutathione peroxidase 4 (GPX4)blockade. This resistance is associated with reduced lipid peroxidation, altered calcium signaling, and selective depletion of polyunsaturated phosphatidylethanolamine species linked to decreased Acyl-CoA Synthetase Long-Chain Family Member 4 (ACSL4) expression. In contrast, TRPML1 deficiency sensitizes cells to ferroptosis and correlates with enhanced endoplasmic reticulum stress and oxidative imbalance rather than major lipid remodeling. Collectively, these findings identify lysosomal ion channels as key modulators of ferroptosis in HCC and highlight distinct mechanisms by which TPC2 and TRPML1 regulate cellular redox balance and death susceptibility.