Methotrexate-triggered ferroptosis suppresses oral cancer progression by phosphorylated KEAP1-mediated NRF2 degradation to inhibit SLC7A11/GPX4 signaling pathway.

BACKGROUND: Oral cancer (OC) is the most common type of head and neck cancer, with a high mortality rate, and is a leading cause of cancer-related deaths worldwide. Drug-induced ferroptosis is a novel form of non-apoptotic cell death that offers a promising strategy for cancer therapy. Accumulating evidence has emphasized the significant role of methotrexate (MTX) in the treatment of many malignancies; however, its role in the ferroptosis pathway in OCs and its underlying mechanisms remain poorly understood. METHODS: After treating the OC cells with MTX, several cellular function assays were performed, including cell proliferation, apoptosis, colony formation, and wound healing assays. Distinctive features of ferroptosis were detected, and qPCR and western blot (WB) assays were performed to validate the expression of genes and proteins related to ferroptosis pathways in MTX-treated cells. In vitro experiments were conducted to further explore the mechanisms by which MTX regulates the stability of nuclear factor erythroid 2-related factor 2 (NRF2) in OC cells. Finally, in a mouse model using MOC1 cells, some experiments were performed to demonstrate MTX-induced ferroptosis and tumor suppression. RESULTS: In this study, based on in vitro and in vivo experiments, we found that MTX significantly reduced OC cell viability by inducing ferroptosis. Mechanistically, MTX administration increased the phosphorylation of Kelch-like ECH-associated protein 1 (KEAP1) at threonine 43 via activation of the ERK/MAPK signaling pathway, thereby maintaining the protein complex formed by KEAP1 and NRF2. As result of the decreased NRF2 expression, the levels of SLC7A11 and GPX4 proteins were markedly suppressed in MTX-treated OC cells, ultimately leading to the induction of ferroptosis in OC. CONCLUSIONS: Our data demonstrated that MTX-mediated activation of the ERK/KEAP1 signaling pathway significantly induced ferroptosis by inhibiting the NRF2/HO-1/SLC7A11/GPX4 axis, thereby suppressing OC progression. These findings suggest that MTX is a promising candidate for OC treatment, offering a meaningful and effective therapeutic-strategy.