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