Aberrant DNA methylation and overexpression of NR0B1 are prognostic biomarkers in KEAP1-mutant lung adenocarcinomas

The KEAP1-NRF2 pathway regulates cytoprotective responses to endogenous and exogenous stresses induced by reactive oxygen species (ROS) and electrophiles. To maintain oxidative homeostasis, cancer cells increase the transcription of antioxidant genes by acquiring either stabilizing mutations in NRF2 or inactivating mutations in its negative regulator, KEAP1. NR0B1 is a recently identified NRF2 target that supports anchorage-independent growth in KEAP1-mutant non-small cell lung cancer (NSCLC) cells. In this study, we used multi-omics and wet-lab approaches to explore the correlation between KEAP1 mutations, the methylation status of the NR0B1 promoter, NR0B1 gene expression and survival of patients with lung adenocarcinoma (LUAD). We found that the NR0B1 promoter was highly hypomethylated and NR0B1 was significantly overexpressed in KEAP1-mutant LUAD cell lines and patient samples compared to their wild-type counterparts. Correlation analysis showed a strong negative correlation between the hypomethylated CpG sites (NR0B1 promoter) in KEAP1 mutant LUAD patients and NR0B1 gene expression. Moreover, hypomethylation of cg22696549 CpG site (NR0B1 promoter) was associated with poor survival in LUAD patients. Furthermore, the overexpression of NR0B1 was correlated with worse prognosis of LUAD patients. These findings suggest a potential synergy between KEAP1 mutation-associated DNA methylation changes and the transcription factor NRF2, which may drive NR0B1 overexpression. Additionally, this study may enhance the understanding of the molecular mechanisms associated with NR0B1 in lung cancer progression and drug resistance.