TGF-ß1-mediated repression of SLC7A11 drives vulnerability to GPX4 inhibition in hepatocellular carcinoma cells.

System xc- contributes to glutathione (GSH) synthesis and protects cells against ferroptosis by importing cystine and exchanging it with glutamate. Transforming growth factor ß1 (TGF-ß1) induces redox imbalance; however, its role in system xc- regulation remains poorly understood. The present study was the first to show that TGF-ß1 repressed the protein and mRNA levels of xCT, a catalytic subunit of system xc-, in PLC/PRF/5, Huh7, Huh6, and HepG2 cells with an early TGF-ß1 gene signature but not in SNU387, SNU449, SNU475, and SK-Hep1 cells with a late TGF-ß1 gene signature. TGF-ß1 treatment for 24 h reduced xCT expression in a dose-dependent manner but this TGF-ß1-induced repression was blunted by pretreatment with a TGF-ß1 receptor inhibitor. TGF-ß1-mediated xCT repression was prevented by Smad3, but not Smad2 or Smad4, knockdown, whereas it was enhanced by Smad3 overexpression. TGF-ß1 decreased GSH levels in control cells but not xCT-overexpressed cells. Furthermore, TGF-ß1 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-ß1 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-ß1 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-ß1 represses xCT expression via Smad3 activation and enhances lipid peroxidation in hepatocellular carcinoma cells with an early TGF-ß1 signature, which would benefit from the targeting of GPX4.