PIAS2 Overexpression Attenuates Erastin-Induced Ferroptotic Changes and Is Associated With Increased GPX4 Protein and Enhanced GPX4-Related SUMO3 Signaling in Hepatocellular Carcinoma Cells

Background: Protein inhibitor of activated STAT 2 (PIAS2) is highly expressed in various solid tumors. Its precise function as a ferroptosis regulator in hepatocellular carcinoma (HCC) is still unknown. The purpose of this work was to determine whether PIAS2 overexpression attenuates erastin-induced ferroptotic changes and, in turn, is associated with the development of HCC in a manner that correlates with changes in Glutathione Peroxidase 4 (GPX4) protein levels and small ubiquitin-like modifier 3 (SUMO3) modification signals. Methods: Erastin (Era) was used to induce ferroptosis in HepG-2 and H22 cells, while Ferrostatin-1 (Fer-1) was employed for validation. Western blotting, qPCR, and co-immunoprecipitation (Co-IP) were used to examine the expression and interaction of PIAS2 and GPX4. Ferroptosis markers (lipid-reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), Fe2+), cell viability, migration, and invasion were all analyzed. Functional rescue experiments were performed using PIAS2 overexpression (oe-PIAS2) and GPX4 knockdown (sh-GPX4). In addition, a murine model of H22 cells in the right groin/lateral abdomen was established for in vivo validation. Tumor weight and volume were measured in these animals. Additionally, tumor tissues were collected and subjected to immunohistochemical staining for Ki-67 expression, as well as biochemical analysis of ferroptosis-related markers (GSH, MDA, Fe2+). Results: In HCC cells, Era-induced ferroptosis resulted in a significant downregulation of PIAS2 and GPX4 expression, as well as elevated levels of lipid ROS, MDA, and Fe2+, decreased GSH content, and decreased cell viability, migration, and invasion. Under Era-treated conditions, overexpression of PIAS2 effectively reversed these changes, suppressing ferroptosis and partially restoring malignant phenotypes within the context of Era challenge. Co-treatment with Fer-1 significantly reversed Era-induced ferroptotic changes and rescued cell viability, migration, and invasion, without affecting PIAS2 expression, while also restoring GPX4 levels. Mechanistic studies revealed that PIAS2 and GPX4 co-immunoprecipitate in HCC cells, and PIAS2 overexpression was associated with increased SUMO3 modification signals and higher remaining GPX4 protein levels at the examined 12- and 24-h CHX time points. GPX4 knockdown attenuated the phenotypes observed under PIAS2 overexpression, suggesting possible involvement of GPX4 in the effects of PIAS2. In Era-treated tumor-bearing mice, PIAS2 overexpression was associated with increased tumor growth, elevated Ki-67 and GPX4 levels, and reduced ferroptosis indicators. Conclusion: PIAS2 overexpression was associated with increased GPX4 SUMO3 modification signals and higher GPX4 protein levels at the examined CHX time points, together with reduced ferroptotic changes under Era-treated conditions.