Methylation of the KEAP1 gene promoter region in human colorectal cancer

Abstract

BackgroundThe Keap1-Nrf2 pathway has been reported to be impaired in several cancers. However, the status of Keap1-Nrf2 system in human colorectal cancer (CRC) has not been elucidated.MethodsWe used colorectal cancer (CRC) cell lines and surgical specimens to investigate the methylation status of the KEAP1 promoter region as well as expression of Nrf2 and its downstream antioxidative stress genes, NQO-1 and AKR1C1.ResultsDNA sequencing analysis indicated that all mutations detected were synonymous, with no amino acid substitutions. We showed by bisulfite genomic sequencing and methylation-specific PCR that eight of 10 CRC cell lines had hypermethylated CpG islands in the KEAP1 promoter region. HT29 cells with a hypermethylated KEAP1 promoter resulted in decreased mRNA and protein expression but unmethylated Colo320DM cells showed higher expression levels. In addition, treatment with the DNA methyltransferase inhibitor 5-Aza-dC combined with the histone deacetylase inhibitor trichostatin A (TSA) increased KEAP1 mRNA expression. These result suggested that methylation of the KEAP1 promoter regulates its mRNA level. Time course analysis with the Nrf2-antioxidant response element (ARE) pathway activator t-BHQ treatment showed a rapid response within 24 h. HT29 cells had higher basal expression levels of NQO-1 and AKR1C1 mRNA than Colo320DM cells. Aberrant promoter methylation of KEAP1 was detected in 53% of tumor tissues and 25% of normal mucosae from 40 surgical CRC specimens, indicating that cancerous tissue showed increased methylation of the KEAP1 promoter region, conferring a protective effect against cytotoxic anticancer drugs.ConclusionHypermethylation of the KEAP1 promoter region suppressed its mRNA expression and increased nuclear Nrf2 and downstream ARE gene expression in CRC cells and tissues.

NRF2-driven miR-125B1 and miR-29B1 transcriptional regulation controls a novel anti-apoptotic miRNA regulatory network for AML survival

“…[3][4][5] In cancer, NRF2 activation is pro-tumoural in a spectrum of malignancies through mutations in NRF2 or its cytosolic inhibitor KEAP1. [6][7][8][9] However, we have previously shown that in human AML constitutive activation of the NRF2 signalling pathway is not through somatic mutations of NRF2/Keap1 but as a consequence of upstream constitutive activation by NF-κB. 10,11 In addition to contributing to the malignant phenotype of AML, we also found that NRF2 contributes to intrinsic leukaemia cell resistance to standard front-line chemotherapy agents.…”

AberrantKeap1methylation in breast cancer and association with clinicopathological features

“…18,26 The presence of two genetic or epigenetic abnormalities was associated with worst progression free survival in patients affected by NSCLC, 18 whereas concomitant methylation of MGMT and KEAP1 genes was able to better predict response to treatment with radiotherapy and temozolomide in malignant gliomas. 26 Hanada et al 27 recently reported aberrant promoter methylation in 53% of colorectal cancer without finding correlation with patients' survival.…”

The emerging role of the Nrf2–Keap1 signaling pathway in cancer

“…The promoter region of the KEAP1 gene is hypermethylated in lung (Wang et al 2008a;Muscarella et al 2011), prostate , malignant glioma (Muscarella et al 2011), and colorectal cancers (Hanada et al 2012). The methylation of specific CpG sites within the promoter region of KEAP1 affects KEAP1 expression by inducing local chromatin remodeling and restricting the ability of the transcriptional machinery to bind to the necessary DNA sequences (for review, see Copple 2012).…”