Epigenetic-genetic interactions in the APC/WNT, RAS/RAF, and P53 pathways in colorectal carcinoma

Abstract

Purpose: Early events in colorectal tumorigenesis include mutation of the adenomatous polyposis coli (APC) gene and epigenetic hypermethylation with transcriptional silencing of the O(6)-methylguanine DNA methyltransferase (MGMT), human mut L homologue 1 (hMLH1), and P16/CDKN2A genes. Epigenetic alterations affect genetic events: Loss of MGMT via hypermethylation reportedly predisposes to guanine-to-adenine or cytosine-to-thymine (G:C-->A:T) transition mutations in KRAS and P53, and silencing of hMLH1 leads to high levels of microsatellite instability (MSI-H)/mutator phenotype, suggesting that epigenetic-genetic subtypes exist.

Experimental design: We evaluated the relationships of aberrant methylation of APC, MGMT, hMLH1, P16, N33, and five MINTs to mutations in APC, KRAS, BRAF, and P53 in 208 colorectal carcinomas.

Results: We found that APC hypermethylation was age related (P = 0.04), in contrast to the other genes, and did not cluster with CpG island methylator phenotype (CIMP) markers. Hypermethylation of APC concurrently with either MGMT or hMLH1 was strongly associated with occurrence of G-to-A transitions in APC [odds ratio (OR), 26.8; P < 0.0002 from multivariable logic regression model], but C-to-T transitions had no associations. There was no relationship of hypermethylation of any gene, including MGMT, with G-to-A or C-to-T transitions in KRAS or P53, although APC hypermethylation was associated with P53 mutation (P < 0.0002). CIMP with MSI-H due to hMLH1 hypermethylation, or CIMP with loss of MGMT expression in non-MSI-H tumors, was associated with BRAF mutation (OR, 4.5; P < 0.0002). CIMP was also associated with BRAF V600E T-to-A transversion (OR, 48.5; P < 0.0002).

Conclusions: Our findings suggest that the heterogeneous epigenetic dysregulation of promoter methylation in various genes is interrelated with the occurrence of mutations, as manifested in epigenetic-genetic subgroups of tumors.

Fig. 1

Examples of methylation assays of MGMT, APC, and hMLH1 gene promoters in CRCs. The presence of a visible PCR product from methylation-specific PCR in lanes labeled U indicates the presence of an unmethylated allele, and in lanes labeled M, the presence of a methylated allele. MGMT hypermethylation is evident in case 87; APC hypermethylation is evident in cases 60 and 64; and hMLH1 hypermethylation is evident in case 114. DNA from normal lymphocytes (NL) was used as a control for unmethylated MGMT and APC, and in vitro methylated DNA (IVD) from placenta was used as a control for the hypermethylated genes.

Fig. 2

Hierarchical cluster analysis of the 10 methylation makers used. P16, hMLH1, and MINTs1, 2, and 31 clustered together and were used to define CIMP, as in our previous studies (26, 32, 35). Hypermethylation of the APC, MGMT, and N33 genes and of MINTs 25 and 27 did not cluster with the CIMP markers.

Fig. 3

Diagram of epigenetic and genetic alterations in 208 CRCs arranged by methylation and mutation status. The CIMP markers from Fig. 2 are indicated by asterisk in the column headings. Shading, the presence of the molecular alteration; X, unavailable data; open cell, absence of the alteration. Numbers in the APC columns indicate when two or more mutations are present, and numbers in the BRAF column indicate the type of or nucleotide with mutation. The intertumoral heterogeneity of the alterations and the epigenetic-genetic associations are apparent. The strong relationship between hypermethylation of APC with hypermethylation of MGMT or hMLH1 and G-to-A transition mutation in APC is evident in cases 1 to 11.

Fig. 3

Diagram of epigenetic and genetic alterations in 208 CRCs arranged by methylation and mutation status. The CIMP markers from Fig. 2 are indicated by asterisk in the column headings. Shading, the presence of the molecular alteration; X, unavailable data; open cell, absence of the alteration. Numbers in the APC columns indicate when two or more mutations are present, and numbers in the BRAF column indicate the type of or nucleotide with mutation. The intertumoral heterogeneity of the alterations and the epigenetic-genetic associations are apparent. The strong relationship between hypermethylation of APC with hypermethylation of MGMT or hMLH1 and G-to-A transition mutation in APC is evident in cases 1 to 11.

Fig. 4

Diagram illustrating the subgroups of CRC with epigenetic-genetic interactions identified through multivariable logic regression model. Hypermethylation of APC concurrently with either MGMT or hMLH1 hypermethylation was associated with G-to-A transition mutation in APC, whereas APC hypermethylation or absence of CIMP in MSS/MSI-L tumors was associated with P53 mutation. CIMP and MSI-H or CIMP in the absence of MSI-H but with loss of MGMT expression was associated with BRAF mutation. CIMP was also associated with BRAF V600E. ORs were adjusted by tumor stage, and all P values are based on 5,000 permutations of the original data set.