NRF2-Driven KEAP1 Transcription in Human Lung Cancer

Constitutive NRF2 activation by disrupted KEAP1-NRF2 interaction has been reported in a variety of human cancers. However, studies focusing on NRF2-driven KEAP1 expression under human cancer contexts are still uncommon. We examined mRNA expression correlation between NRF2 and KEAP1 in multiple human cancers. We measured KEAP1 mRNA and protein alterations in response to the activation or silencing of NRF2. We queried chromatin immunoprecipitation sequencing (ChIP-seq) datasets to identify NRF2 binding to KEAP1 promoters in human cells. We used reporter assay and CRISPR editing to assess KEAP1 promoter activity and mRNA abundance change. To determine specimen implication of the feedback pattern, we used gene expression ratio to predict NRF2 signal disruption as well as patients' prognosis. Correlation analysis showed KEAP1 mRNA expression was in positive association with NRF2 in multiple squamous cell cancers. The positive correlations were consistent across all squamous cell lung cancer cohorts, but not in adenocarcinomas. In human lung cells, NRF2 interventions significantly altered KEAP1 mRNA and protein expressions. ChIP-quantitative PCR (ChIP-qPCR) and sequencing data demonstrated consistent NRF2 occupancy to KEAP1 promoter. Deleting NRF2 binding site significantly reduced baseline and inducible KEAP1 promoter activity and KEAP1 mRNA expression. By incorporating tumor tissue KEAP1 mRNA expressions in estimating NRF2 signaling disruptions, we found increased TXN/KEAP1 mRNA ratio in cases with NRF2 gain or KEAP1 loss and decreased NRF2/KEAP1 mRNA ratio in cases with NRF2-KEAP1 somatic mutations. In TCGA PanCancer datasets, we also identified that cases with loss-of-function mutations in NRF2 pathway recurrently appeared above the NRF2-KEAP1 mRNA expression regression lines. Moreover, compared with previous NRF2 signatures, the ratio-based strategy showed better predictive performance in survival analysis with multiple squamous cell lung cancer cohort validations. IMPLICATIONS: NRF2-driven KEAP1 transcription is a crucial component of NRF2 signaling modulation. This hidden circuit will provide in-depth insight into novel cancer prevention and therapeutic strategies.