Epigenetic modifications but not genetic polymorphisms regulate KEAP1 expression in colorectal cancer

Kelch-like ECH-associated protein 1 (KEAP1), as a negative regulator of nuclear factor erythroid 2 like 2 ( NRF2), plays a pivotal role in NRF2 signaling pathway and involves in tumorigenesis. Polymorphisms and methylation in gene promoter region may influence its expression and be related to cancer susceptibility. In this study, we examined the effect of the KEAP1-NRF2 interaction on the risk of colorectal cancer (CRC). The polymorphisms of NRF2 and KEAP1 were genotyped using the improved multiplex ligase detection reaction assay. KEAP1 promoter methylation and histone modification were analyzed using bisulfite genome sequencing and chromatin immunoprecipitation (ChIP) assay, respectively. The KEAP1 rs1048290 CC genotype and C allele were associated with increased risks of CRC (CC vs GG: odds ratio [OR] = 1.39; 95% confidence interval [CI], 1.08-1.78; CC vs GG/GC: OR = 1.29; 95% CI, 1.05-1.58; C vs G: OR = 1.18; 95% CI, 1.04-1.34). The rs1048290-rs11545829 GT haplotype was associated with a reduced risk of CRC. KEAP1-NRF2 interaction analysis revealed that the rs6721961, rs35652124, rs1048290, and rs11545829 conferred the susceptibility to CRC. The hypermethylation of KEAP1 promoter resulted in lower levels of KEAP1 messenger RNA (mRNA). After treatment with 5-aza-2'-deoxycytidine/trichostatin A, KEAP1 promoter methylation was decreased and KEAP1 mRNA levels were increased. ChIP-quantitative polymerase chain reaction results showed an enhanced enrichment of H3K4Me3 and H3K27Ac to the promoter of KEAP1. In vitro methylation analysis showed that the methylated plasmid decreased the transcriptional activity by 70%-84%. These findings suggest that the KEAP1- NRF2 pathway could potentially impact CRC risk and the downregulation of KEAP1 could be explained in part by epigenetic modifications.