We found frequent hypermethylation of the KEAP1 promoter region in human CRC cell lines. This hypermethylation of KEAP1 resulted in reductions in KEAP1 mRNA and protein expression, upregulation of Nrf2 activity, and thus overexpression of downstream genes, such as NQO-1 and AKR1C1. We also observed aberrant methylation of KEAP1 in human CRC tissues. This is the first report discussing activation of Keap1/Nrf2 signaling by KEAP1 hypermethylation in CRC.
Loss of Keap1 function has been reported associated with KEAP1
gene mutations in tumor tissue samples from lung, gall bladder, breast, and prostate cancer [15
]. We found only synonymous mutations consisting of a C-to-T transition with G157G in exon 2, a T-to-C transition of L471L in the DGR4 domain, and a C-to-T transition with Y537Y in the DGR5 domain in CRC cell lines. However, these mutations were single-nucleotide polymorphisms. Frequent KEAP1
gene mutations were reported in human non-small cell lung cancer (NSCLC) [15
]. All mutations were within highly conserved amino acid residues located in the Kelch or intervening region domain of the Keap1 protein, suggesting that these mutations were likely to abolish Keap1 repressor activity against Nrf2. In addition, C23Y mutation in the N-terminal domain of Keap1 has been reported to have impaired ability to repress Nrf2 activity due to its inability to stimulate the ubiquitylation and degradation of Nrf2 in breast cancer [19
]. A C-to-T transition with T314M and a T-to-C transition with Y255H were detected in six prostatic cancer cell lines [21
]. Shibata et al
. also reported mutations of KEAP1
in biliary tract cancer tissue [18
]. These changes are in the central intervening region of Keap1 and alter highly conserved amino acids.
Another mechanism of impaired Keap1 activity is hypermethylation of KEAP1
. We found that 8 of 10 CRC cell lines had methylated CpG islands in the promoter region of the KEAP1
gene where methylation was found in other types of cancer [20
]. Hypermethylation of KEAP1
resulted in decreased mRNA expression, which was confirmed by the increase in KEAP1
mRNA expression by combined treatment with the DNA methyltransferase inhibitor 5-Aza-dC and the histone deacetylase inhibitor TSA (Figure ). Hypermethylation of KEAP1
caused final stimulation of Nrf2 target genes. However, the reason for the expression of KEAP1
mRNA being lower in unmethylated SW837 cells than in methylated HCT15 cells is unknown. Wang et al
. investigated three lung cancer cell lines and five tumor samples, and found frequent hypermethylation of the CpG islands in the promoter region of KEAP1
and reduced levels of KEAP1
mRNA expression. In contrast, a normal bronchial cell line had clearly less methylation of the KEAP1
promoter region and elevated mRNA expression [20
]. Hypermethylation of KEAP1
found in prostate cancer also stimulated the Nrf2 signal [21
Biological effects of constitutive Nrf2 activation by Keap1 dysfunction due to mutations or low-level expression by hypermethylation have been reported previously [18
]. Constitutive expression of the cytoprotective gene by Nrf2 activation in lung cancer cells led to chemotherapy resistance [23
]. Nrf2 activation also stimulated growth of lung cancer cells. Nrf2 activation by KEAP1
mutation or hypermethylation of promoter CpG islands causes radioresistance and promotes tumor growth in prostatic cancer [21
]. In the present study, we observed accumulation of Nrf2 protein in the nuclei in methylated HT29 cells, and overexpression of phase II detoxifying enzymes NQO-1 and AKR1C1 both at baseline and after t-BHQ stimulation. These reports indicate that KEAP1 functions as a tumor suppressor gene in human tumors. Although we did not evaluate the biological effects of activated Nrf2, we assume that CRC cells with KEAP1
gene hypermethylation may be resistant to chemotherapeutic agents and show upregulated cell growth, as reported in other types of cancer.
There have been only two previous reports regarding Keap1/Nrf2 l in CRC cells [24
]. Activation of the Keap1/Nrf2 signaling pathway mediates protective responses to mitigate nitric oxide (NO)-induced damage and may contribute to the resistance of CRC cells to NO-induced cytotoxicity [24
]. Arlt et al
. reported that Nrf2 activity is elevated in colon cancer, accounting for overexpression of the proteasome subunit proteins and thus for increased proteasome activity [25
]. Conversely, small interfering RNA-mediated Nrf2 knockdown decreased their expression and reduced proteasome activity, thus indicating that Nrf2 is related to colorectal carcinogenesis. This Nrf2 activation may be due to the low level of Keap1 expression due to hypermethylation, as found in the present study.
Biological effects that activate Nrf2 signaling prompted us to study the relationship between the status of Keap1/Nrf2 signaling and clinicopathological features of the tumors. Type II endometrial cancer, which is mostly malignant and is associated with a poor prognosis among gynecological malignancies, shows elevated Nrf2 protein expression, whereas benign tumors and type I endometrial cancer do not [26
]. On immunohistochemical analysis of human NSCLC, increased Nrf2 expression and low or absent Keap1 expression were associated with worse survival [27
]. In contrast, the prognosis of malignant glioma was better among patients with than among those without a methylated KEAP1
promoter region [22
]. Although we did not investigate the prognosis of patients with CRC, further studies are needed to understand the role of Keap1/Nrf2 signaling in human CRC.
In conclusion, the results of the present study revealed hypermethylation of the KEAP1 promoter region in human CRC, leading to downregulation of KEAP1 mRNA expression, thus activating Nrf2 and expression of its downstream target genes. Cancerous tissues exhibited more frequent methylation of KEAP1 than normal tissue in surgically resected CRC specimens.