Methylation pattern of the O⁶‐methylguanine‐DNA methyltransferase gene in colon during progressive colorectal tumorigenesis

Abstract

O 6 -methylguanine-DNA methyltransferase (MGMT) is a DNA repair gene which is frequently methylated in colorectal cancer (CRC). However, it remains controversial whether methylation of specific CpG sequences within MGMT promoter leads to loss of its protein expression, and if MGMT methylation correlates with G to A transition mutations in KRAS. Two methylation sensitive regions (Mp and Eh region) of MGMT promoter were investigated in 593 specimens of colorectal tissue: 233 CRCs, 104 adenomatous polyps (AP), 220 normal colonic mucosa from CRC patients (N-C) and 36 normal colonic mucosa specimens obtained from subjects without colorectal neoplasia (N-N) by combined bisulfite restriction analysis (COBRA). The region-specific methylation data were compared to the MGMT protein expression, spectrum of KRAS mutations and other clinical features. Extensive (including both Mp and Eh) and partial (either Mp or Eh) MGMT methylation were found in 24.5% and 11.6% of CRCs, 3.8% and 27.9% of APs, 0.5% and 7.7% of C-Ns and 2.8% and 2.8% of N-Ns, respectively. Extensive methylation of MGMT promoter was primarily present in CRCs while partial methylation was common in APs. Extensive methylation of MGMT promoter was associated with loss/reduced protein expression (p < 0.0001), as well as with G to A mutations in KRAS (p 5 0.0017). We herein provide first evidence that extensive methylation of MGMT promoter region is essential for methylation-induced silencing of this gene. Our data suggest that MGMT methylation may evolve and spread throughout the promoter in a stepwise manner as the colonic epithelial cells progress through the classical-adenoma-cancer multistep cascade. ' 2008 Wiley-Liss, Inc.Key words: O 6 -methylguanine-DNA methyltransferase; KRAS mutations; promoter methylation; colorectal cancer; adenomatous polyps O 6 -methylguanine-DNA methyltransferase (MGMT) is a DNA repair gene which removes promutagenic O 6 -methylguanine (O 6 -MeG) residues from DNA and is considered an important predictive factors for chemoresistance in human cancers. [1][2][3][4][5] Loss of MGMT function permits the increased accumulation of O 6 -MeG in DNA, which promotes tumorigenesis through G:C to A:T transition mutations in growth regulating genes such as KRAS and p53. 6-9 Mutations in MGMT have rarely been found, and it has been suggested that MGMT inactivation is primarily manifested through hypermethylation-induced silencing of its promoter in human cancers, including those of the colon and rectum. 5,[9][10][11][12][13][14] However, the associations between MGMT methylation and G to A transition mutations has not been consistently reproduced in different studies. 15,16 Central to this controversy is the fact that there is a lack of clear understanding for precise relationships between methylation of specific MGMT promoter regions and its relationship with the loss of protein expression.It is becoming increasingly clear that the distribution of methylated cytosines in the CpG islands of promoters is not uniform, and the regions most importan...

MGMT methylation is associated primarily with the germline C>T SNP (rs16906252) in colorectal cancer and normal colonic mucosa

“…This is a slightly lower prevalence than the generally reported range, perhaps reflecting the analytical sensitivities of the various methylation assays. [9][10][11][12][13] The strong but incomplete concordance between MGMT methylation, transcriptional repression and protein loss is well established, [9][10][11] and our findings confirm that promoter methylation is the predominant cause of MGMT loss in sporadic colorectal cancer.…”

“…10 Thus, it is plausible that loss of MGMT precedes and induces G4A transitions in a subset of colorectal cancers, irrespective of the mechanism of MGMT loss. However, the results of our study coupled with the lack of consistency in the findings of other studies 11,12,15,17,[19][20][21] argue against this direct sequence of events as a generalised phenomenon in the development of colorectal cancer. The concurrence of these epigenetic and genetic lesions in a subset of colorectal cancers suggests a more complex relationship between these events, perhaps akin to the close association between MLH1 methylation and the BRAF V600E mutation.…”

“…[10][11][12] There is a general trend evident at other loci, for both women and the elderly, to show more frequent aberrant methylation in tumours. 46,47 The frequency and level of MGMT methylation in normal colonic mucosa was also age related, suggesting that additional factors influence the accumulation of methylation at the MGMT promoter.…”

“…8 Loss of expression of the O 6 -methylguanine DNA methyltransferase (MGMT) protein is a frequent occurrence in many types of cancer, including 30-46% of sporadic colorectal cancers. [9][10][11][12][13] This is almost invariably associated with methylation of the MGMT promoter. 9 MGMT is a ubiquitously expressed DNA repair protein that protects against mutagenesis by repairing mutagenic O 6 -methylguanines within DNA.…”

“…14 MGMT methylation or loss of MGMT protein expression is reportedly associated with an increased frequency of mutations in the KRAS oncogene and P53 tumour-suppressor gene in colorectal cancer, particularly G4A transitions, consistent with a causative function of G4A mutagenesis in tumourigenesis. 11,12,[15][16][17][18] However, other studies have found no specific associations with this sequence change, 19,20 or only a weak association with the presence of a G4A mutation within one of the four cancer-associated genes KRAS, P53, APC and b-catenin. 21 Association studies between MGMT methylation, CIMP and microsatellite stability (MSS) status in colorectal cancer have also reported inconsistent findings.…”