Methylation of the KEAP1 gene promoter region in human colorectal cancer.
BACKGROUND
The 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.


METHODS
We 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.


RESULTS
DNA 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.


CONCLUSIONS
Hypermethylation of the KEAP1 promoter region suppressed its mRNA expression and increased nuclear Nrf2 and downstream ARE gene expression in CRC cells and tissues.
Department of Medical Oncology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.
Naoyuki
N
Hanada
Takenori
T
Takahata
Qiliang
Q
Zhou
Xulu
X
Ye
Ruowen
R
Sun
Jugoh
J
Itoh
Atsushi
A
Ishiguro
Hiroshi
H
Kijima
Junsei
J
Mimura
Ken
K
Itoh
Shinsaku
S
Fukuda
Yasuo
Y
Saijo
D015089:20-Hydroxysteroid Dehydrogenases / Q000378:metabolism; D015153:Blotting, Western; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D000072019:Kelch-Like ECH-Associated Protein 1; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics / Q000378:metabolism; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012313:RNA / Q000378:metabolism; D000077278:RNA, Mitochondrial; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D000077278:RNA, Mitochondrial; D012313:RNA; D015089:20-Hydroxysteroid Dehydrogenases; C115140:3 alpha-beta, 20 beta-hydroxysteroid dehydrogenase; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human