[Dual inhibition of PSME2 and STAT3 induces ferroptosis in esophageal squamous cell carcinoma cells by regulating the SLC7A11/GPX4 axis].

Abstract

Objectives: To investigate the form of cell death induced by PSME2 silencing combined with the STAT3 inhibitor WP1066 in esophageal squamous cell carcinoma (ESCC) cells and explore the underlying mechanism.

Methods: ESCC cell lines KYSE30 and NE6-T with PSME2 silencing were treated with WP1066. Phenotypic rescue experiments were performed to determine the predominant form of cell death using different cell death-related inhibitors. Western blotting was used to detect the expressions of the protein markers of ferroptosis, apoptosis, pyroptosis and necroptosis. Intracellular reactive oxygen species (ROS) levels and lipid peroxidation were assessed using DCFH-DA and C11-BODIPY fluorescent probes, respectively. Mitochondrial ultrastructural changes were observed by transmission electron microscopy (TEM). mRNA transcriptomic analysis was performed to identify differentially expressed genes in cells treated with PR171 or siPSME2 combinedwith WP1066. Gene set enrichment analysis to analyze the enriched biological functions.

Results: Dual inhibition of PSME2 and STAT3 induced obvious ESCC cell death. This lethal effect was markedly reversed by the ferroptosis inhibitor ferrostatin-1 and attenuated by the apoptosis inhibitor Z-VAD-FMK, whereas 2-bromohexadecanoic acid or necrosulfonamide did not show significant inhibitory effects. The combined treatment with siPSME2 and WP1066 significantly downregulated SLC7A11 and GPX4 protein expressions and increased intracellular ROS levels and lipid peroxide accumulation in ESCC cells. The ESCC cells with dual inhibition showed obvious mitochondrial shrinkage, reduced cristae, and increased membrane density. mRNA transcriptomic analysis identified 45 overlapping differentially expressed genes between the PR171 plus WP1066 group and the siPSME2 plus WP1066 group, enriched in pathways related to stress response, redox imbalance, protein translation/ribosomal function, metabolism, and cell cycle regulation.

Conclusions: Dual inhibition of PSME2 and STAT3 promotes lipid peroxide accumulation and induces ferroptosis in ESCC cells possibly by regulating the SLC7A11/GPX4 antioxidant axis, indicating a therapeutic strategy targeting both PSME2 and STAT3 for ESCC.