The mechanism of E3 ubiquitin ligase HERC1 regulating ferroptosis in lung adenocarcinoma cells

Abstract

Objective

Lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer. Herein, we probed into the role of E3 ubiquitin protein ligase family member 1 (HERC1) in promoting ferroptosis and inhibiting LUAD cell proliferation by regulating RAF proto-oncogene serine/threonine-protein kinase (C-RAF).

Methods

In cultured human normal lung epithelial cells and non-small cell lung adenocarcinoma cell lines, HERC1 expression was determined by RT-qPCR and Western blot tests. PC-9 and Calu-3 cells were transfected with oe-HERC1, oe-C-RAF or their negative controls. Reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), and Fe²⁺ levels were assessed by biochemical assays. Cell viability, death, and proliferation were evaluated by CCK-8, LDH and colony formation assays, followed by assessments of HERC1-C-RAF interaction, C-RAF ubiquitin level, and C-RAF protein stability.

Results

HERC1 was poorly expressed in LUAD cells. HERC1 promoted LUAD cell ferroptosis and repressed their proliferation and migration, corresponding to reduced levels of system xc-, GPX4, and GSH, as well as elevated levels of ROS, MDA, Fe²⁺, and ACSL4. LUAD cells overexpressing HERC1 displayed decreased C-RAF protein level, HERC1-C-RAF interaction, elevated C-RAF ubiquitin level, and accelerated C-RAF protein degradation, indicating that HERC1 facilitated C-RAF ubiquitin degradation and attenuated C-RAF protein stability via interaction with C-RAF. C-RAF overexpression partially abrogated the regulatory impact of HERC1 on LUAD cell ferroptosis and proliferation.

Conclusion

HERC1 expedites C-RAF ubiquitin degradation by interacting with C-RAF, which consequently promotes ferroptosis, thereby inhibiting LUAD cell proliferation.