Distinct genetic profiles in colorectal tumors with or without the CpG island methylator phenotype

Abstract

Colorectal cancers (CRCs) are characterized by multiple genetic (mutations) and epigenetic (CpG island methylation) alterations, but it is not known whether these evolve independently through stochastic processes. We have recently described a novel pathway termed CpG island methylator phenotype (CIMP) in CRC, which is characterized by the simultaneous methylation of multiple CpG islands, including several known genes, such as p16, hMLH1, and THBS1. We have now studied mutations in K-RAS, p53, DPC4, and TGFbetaRII in a panel of colorectal tumors with or without CIMP. We find that CIMP defines two groups of tumors with significantly different genetic lesions: frequent K-RAS mutations were found in CIMP(+) CRCs (28/41, 68%) compared with CIMP(-) cases (14/47, 30%, P = 0.0005). By contrast, p53 mutations were found in 24% (10/41) of CIMP(+) CRCs vs. 60% (30/46) of CIMP(-) cases (P = 0.002). Both of these differences were independent of microsatellite instability. These interactions between CIMP, K-RAS mutations, and p53 mutations were preserved in colorectal adenomas, suggesting that they occur early in carcinogenesis. The distinct combinations of epigenetic and genetic alterations in each group suggest that activation of oncogenes and inactivation of tumor suppressor genes is related to the underlying mechanism of generating molecular diversity in cancer, rather than simply accumulate stochastically during cancer development.

Figure 1

Mutational analysis of K-RAS in colorectal tumors. Mutations were detected by mutant allele-specific amplification. (A). PCR reactions were first performed by using a primer mixture to detect six different mutations of K-RAS codon 12 (Upper). A second PCR reaction was then performed by using specific primers to detect the exact mutation in each case (Lower). For example, p30 was found to have a mutation by using the primer mix (Upper), and the mutation was determined to be a GGT to GAT change by using specific primers (Lower). P30, P31, P36, and P37, colorectal adenomas; H157, a human lung cancer cell line that has a mutation in codon 12 of K-RAS. “Normal” refers to normal colon (a negative control). (B) Frequencies of K-RAS mutations in colorectal tumors with or without CIMP. The frequencies of K-RAS mutations in each population were expressed as percentages. The number of tumors examined is shown at the bottom. The adenomas were divided into <1.5 cm and ≥1.5 cm.

Figure 2

Mutations of p53 and methylation of p16 in colorectal tumors. (A) Detection of p53 mutations by SSCP and direct sequencing. Aberrantly migrating SSCP bands (reflecting base pair changes) are indicated by arrows (left part of each panel). Shifted bands were excised from gels and were reamplified by using the same set of primers, and direct sequencing was performed by using automated sequencers (right part of each panel). Tumor 703 has a band shift in exon 8 caused by a mutation in codon 282 (CGG to CAG). Tumor 547 has a band shift in exon 7 caused by a mutation in codon 245 (GGC to AGC). Tumor 1290 has a band shift in exon 5 caused by a 10-bp duplication that creates a premature stop codon. N, normal tissue; T, colorectal cancer. (B) Frequencies of alterations of p53 and p16 in CRCs. Forty-one CRCs are divided into four categories based on the presence of alterations of p53 (p53+) and/or p16 (p16+). Alterations of p16 and p53 were inversely correlated (P = 0.0007, Fisher's Exact test). (C) Summary of alterations of p53 and p16 in colorectal tumors with or without CIMP. The frequencies of alterations in p53 and p16 in cancers and adenomas are expressed as percentages. n, number of tumors analyzed.

Figure 3

Methylation analysis of multiple CpG islands in colorectal adenomas. (A) Methylation of multiple CpG islands detected by methylated CpG island amplification. Methylated CpG island amplification products from colon adenomas (P2–P48) were blotted on a nylon membrane and were hybridized with one of the MINT clones (indicated on the top), as well as a p16 probe. M2, MINT2; M1, MINT1; M12, MINT12; and M31, MINT31. Tumors P46, P47, and P48 showed methylation of multiple loci, indicating the presence of CIMP. Tumor P23 showed methylation of MINT31 only. (B) Methylation analysis of MINT2 by bisulfite-PCR. PCR products were digested with BstUI, which cleaves CGCG sequences that are retained after bisulfite treatment only when both cytosines are methylated. Methylated alleles are shown by arrows. Tumors P46, P47, and P48 are methylated at this locus. No methylated alleles were detected in PCR products from normal colon. RKO is a colon cancer cell line used as a positive control. (C) Methylation analysis of p16 in colorectal adenomas by MSP. U, unmethylated allele-specific primers; M, methylated allele-specific primers. Tumor P28 and P33 showed methylated alleles.