Abstract
System xc− 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 xc− 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 xc−, 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.
Highlights
System xc−, a cystine/glutamate exchange transporter, uptakes cystine driven by higher concentrations of glutamate in the cytoplasm[1]
System xc− comprises the SLC7A11 catalytic subunit, which is called xCT, and the SLC3A2 non-catalytic heavy subunit, which is known as 4F2hc1. xCT expression is correlated with system xc− activity and poor prognoses for various types of tumors, including hepatocellular carcinoma (HCC), colorectal cancer, and glioblastoma[8,9,10]
A previous comparative genomics study discriminated two subtypes of human HCC cell lines based on Transforming growth factor β1 (TGF-β1) gene signatures[24]: PLC/PRF/5, Huh[7], Huh[6], and HepG2 cells were characterized by an early TGF-β1 gene signature associated with TGF-β1-induced cytostasis and apoptosis; and SNU387, SNU449, SNU475, and SK-Hep[1] cells were defined by a late TGF-β1 gene signature lacking TGF-β receptors, and exhibited high levels of epithelialto-mesenchymal transition (EMT)-associated proteins
Summary
System xc−, a cystine/glutamate exchange transporter, uptakes cystine driven by higher concentrations of glutamate in the cytoplasm[1]. Some populations of cancer cells are highly dependent on system xc− or GPX4 to cope with the redox imbalance caused by their rapid growth and the subsequent limited availabilities of oxygen and nutrients[5]. Triple-negative breast tumors are highly dependent on system xc− for survival and become glutamate auxotrophs[6]. XCT expression is correlated with system xc− activity and poor prognoses for various types of tumors, including hepatocellular carcinoma (HCC), colorectal cancer, and glioblastoma[8,9,10]. XCT expression is tightly regulated by signaling pathways that control cancer hallmarks. Epidermal growth factor receptor[11] and CD44 variants[12] directly interact with xCT and stabilize its expression at the cell surface, which enhances antioxidant capacity. Oncogenic PI3KCA inhibits xCT and promotes methionine dependency in mammary epithelial tumors[13], and 5′
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