Loss-of-function mutations in KEAP1 drive lung squamous cell carcinoma progression via KEAP1/NRF2 pathway activation

Abstract Background and Purpose Targeted therapy has led to dramatic change in the treatment of lung adenocarcinoma, but lung squamous cell carcinoma(LSCC) lacks targeted therapy options. High-frequency somatic mutations in KEAP1/NRF2 (27.9%) have been identified in LSCC. In this study, we explored the role of KEAP1 somatic mutations in the development of LSCC and whether a nuclear factor erythroid 2-related factor 2(NRF2) inhibitor be potential to target lung cancer carrying KEAP1/NRF2 mutations.Methods Lung cancer cell lines A549 and H460 with loss-of-function mutations in KEAP1 stably transfected with wild-type (WT) KEAP1 or somatic mutations in KEAP1 were used to investigate the functions of somatic mutations in KEAP1 .Flow cytometry,plate clone formation experiments,and scratch tests were used to examine reactive oxygen species, proliferation, and migration of these cell lines.Results The expression of NRF2 and its target genes increased, and tumor cell proliferation, migration, and tumor growth were accelerated in A549 and H460 cells stably transfected with KEAP1 mutants compared to control cells with a loss-of-function KEAP1 mutation and stably transfected with WT KEAP1 in both in vitro and in vivo studies. Inhibited proliferation was more apparent in the A549 cell line trasfected with the R320Q KEAP1 mutant than the A549 cell line trasfected with WT KEAP1 after treatment with NRF2 inhibitor ML385.Conclusion Somatic mutations in KEAP1 identified from patients with lung carcinoma likely promote tumorigenesis mediated by activation of the KEAP1/NRF2 antioxidant stress response pathway. NRF2 inhibition with ML385 could inhibit the proliferation of tumor cells with KEAP1 mutation.