NRF2 Upregulates GPX2 to Inhibit Ferroptosis and Enhance Immune Escape of Melanoma

Abstract

Background: Melanoma is the deadliest form of skin malignant tumor, with NFE2 like bZIP transcription factor 2 (NRF2) and glutathione peroxidase 2 (GPX2) implicated in its progression. In this report, we explored the interplay of NRF2 and GPX2 in melanoma using both in vitro and in vivo approaches. Methods: B16 cells were transfected with NRF2 overexpression plasmid and/or small interfering RNA against GPX2 (Si-GPX2) plasmid and treated with erastin to induce ferroptosis. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine transfection efficiency. To investigate immune escape, B16 cells were co-cultured with CD8⁺ T cells, and mice bearing subcutaneous xenograft tumor were established and injected with programmed cell death 1 (PD-1) or CD274 molecule (PD-L1) antibody. Cell viability, colony formation, and expressions of GPX2 and PD-L1 were analyzed by Cell Counting Kit-8, colony formation, and western blot assays. Levels of ferrous iron (Fe²⁺), malondialdehyde (MDA), lipid peroxidation, and reactive oxygen species (ROS) were measured. CD8⁺ T cell apoptosis and infiltration were determined by flow cytometry and immunohistochemistry. Results: NRF2 overexpression increased viability, colony formation, and GPX2/PD-L1 expression (p < 0.05), but reduced levels of Fe²⁺, MDA, lipid peroxidation, and ROS in erastin-treated B16 cells, while GPX2 knockdown decreased colony formation and PD-L1 expression, but increased levels of Fe²⁺ and lipid peroxidation (p < 0.01). Following co-culture, CD8⁺ T cell apoptosis was promoted by NRF2 overexpression, but inhibited by GPX2 knockdown (p < 0.01). GPX2 knockdown reversed the effects of NRF2 overexpression on the above indices (p < 0.01). In mice with subcutaneous xenograft tumor, NRF2 overexpression decreased CD8⁺ T cell infiltration, which was restored by blocking PD-1 and PD-L1 (p < 0.01). Conclusion: NRF2 upregulates GPX2 to inhibit ferroptosis and enhance immune escape of melanoma, unveiling a previously unknown therapeutic target to improve the efficacy of melanoma immunotherapy.