TGF-β1-mediated Repression of SLC7A11 Drives Vulnerability to GPX4 Inhibition in Hepatocellular Carcinoma Cells

System x contributes to glutathione (GSH) synthesis and protects cells against ferroptosis by importing cystine and exchanging it with glutamate. Transforming growth factor β1 (TGF-β1) induces redox imbalance; however, its role in system x regulation remains poorly understood. The present study was the first to show that TGF-β1 repressed the protein and mRNA levels of xCT, a catalytic subunit of system x, in PLC/PRF/5, Huh7, Huh6, and HepG2 cells with an early TGF-β1 gene signature but not in SNU387, SNU449, SNU475, and SK-Hep1 cells with a late TGF-β1 gene signature. TGF-β1 treatment for 24 h reduced xCT expression in a dose-dependent manner but this TGF-β1-induced repression was blunted by pretreatment with a TGF-β1 receptor inhibitor. TGF-β1-mediated xCT repression was prevented by Smad3, but not Smad2 or Smad4, knockdown, whereas it was enhanced by Smad3 overexpression. TGF-β1 decreased GSH levels in control cells but not xCT-overexpressed cells. Furthermore, TGF-β1 increased reactive oxygen species (ROS) levels in PLC/PRF/5 cells and enhanced tert-butyl hydroperoxide-induced ROS levels in Huh7 cells; these changes were reversed by xCT overexpression. TGF-β1 treatment ultimately induced the ferrostatin-1- and deferoxamine-dependent lipid peroxidation after 2 days and 8 days in PLC/PRF/5 and Huh7 cells but not in SNU475 and SK-Hep1 cells. Pre-treatment of TGF-β1 for 2 days enhanced the reduction of cell viability induced by RSL3, a GSH peroxidase 4 (GPX4) inhibitor, in PLC/PRF/5 and Huh7 cells. In conclusion, TGF-β1 represses xCT expression via Smad3 activation and enhances lipid peroxidation in hepatocellular carcinoma cells with an early TGF-β1 signature, which would benefit from the targeting of GPX4.