Abstract 3587: NRF2-driven KEAP1 transcription in human lung cancers
Authors: Tian Yijun , Liu Qian , Yu Shengnan , Chu Qian , Chen Yuan , Wu Kongming , Wang Liang 
Published: September 2020

Introduction: NRF2/KEAP1 copy number variations and somatic mutations lead to NRF2 activation in a variety of human cancers. Though NRF2 plays a role in activating KEAP1 expression in turn, exact mechanisms and clinical implications remain unknown.

Methods: We examined expression correlation between NRF2 and KEAP1 in RNA profiling databases (cBioportal, GEO) with focus on lung cancer. We performed qPCR and western blotting to measure KEAP1 mRNA and protein alterations during NRF2 activator or knockdown treatments. We also queried ENCODE and SRA for ChIP-seq datasets to identify NRF2 binding within KEAP1 promoters in human cells. To demonstrate the regulatory role of NRF2 binding on KEAP1 expression, we applied luciferase reporter assay and CRISPR editing to assess the ARE-related KEAP1 promoter activity and endogenous mRNA abundance change. To determine specimen-level implication of the feedback pattern, we tested gene expression ratio for predicting NRF2/KEAP1 copy number variations and somatic mutations.

Results: RNA profiling datasets analysis showed that mRNA expression of KEAP1 was consistently in positive correlation with NRF2 in multiple primary squamous cell cancers, including LUSC (Pearson r=0.50, p&lt;1e-15), ESCA: squamous (r=0.49, p=7.1e-7), HNSC (r=0.33, p=5e-15), and CESC: squamous (r=0.2, p=0.0014). The positive correlations were consistent across all squamous cell lung cancer datasets (Pooled r=0.5, p&lt;1e-15, n=1409), but not in lung adenocarcinoma (Pooled r=0.06, p=0.11, n=2428). To determine regulatory role of NRF2 on KEAP1, we first treated multiple lung cell lines with NRF2 activator and observed significantly increased KEAP1 mRNA and protein levels. Correspondingly, we observed KEAP1 reduction in NRF2 knockdown A549 cells. ChIP-seq dataset analysis revealed highly consistent and variable NRF2 occupancy in KEAP1 promoter regions. This occupancy was drastically increased in lymphocytes and Bease2B cells after NRF2 activation. We then deleted NRF2 binding site in KEAP1 promoter and observed significant reduction of baseline and inducible luciferase activity. The ARE deletion in H292 genome reduced KEAP1 mRNA expression and increased NRF2 downstream gene expression. In one ARE homozygous deletion clone, we observed an elimination of inducible KEAP1 mRNA expression. To see if the feedback pattern can be applied to predict per-sample NRF2 signaling disruption, we calculated gene expression ratios of NRF2/KEAP1 and TXN (NRF2 target gene) /KEAP1. We found significant associations of increased TXN/KEAP1 ratio with KEAP1 deletion, and decreased NRF2/KEAP1 ratio with somatic mutation. From this result, we further developed a linear regression model that can reliably predict functional mutations in NRF2 pathway.

Conclusion: NRF2-driven KEAP1 transcription suggested a calibrated NRF2 signaling transduction. Novel cancer prevention and therapy targeting NRF2 signaling will benefit from understanding this unique pattern.

Citation Format: Yijun Tian, Qian Liu, Shengnan Yu, Qian Chu, Yuan Chen, Kongming Wu, Liang Wang. NRF2-driven KEAP1 transcription in human lung cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3587.