CpG island methylator phenotype in colorectal cancer

Abstract

Aberrant methylation of promoter region CpG islands is associated with transcriptional inactivation of tumor-suppressor genes in neoplasia. To understand global patterns of CpG island methylation in colorectal cancer, we have used a recently developed technique called methylated CpG island amplification to examine 30 newly cloned differentially methylated DNA sequences. Of these 30 clones, 19 (63%) were progressively methylated in an age-dependent manner in normal colon, 7 (23%) were methylated in a cancer-specific manner, and 4 (13%) were methylated only in cell lines. Thus, a majority of CpG islands methylated in colon cancer are also methylated in a subset of normal colonic cells during the process of aging. In contrast, methylation of the cancer-specific clones was found exclusively in a subset of colorectal cancers, which appear to display a CpG island methylator phenotype (CIMP). CIMP+ tumors also have a high incidence of p16 and THBS1 methylation, and they include the majority of sporadic colorectal cancers with microsatellite instability related to hMLH1 methylation. We thus define a pathway in colorectal cancer that appears to be responsible for the majority of sporadic tumors with mismatch repair deficiency.

Figure 1

Comparison of detection of aberrant methylation by using Southern blotting, MCA, or bisulfite-PCR analysis. (A) Methylation of MINT2 detected by Southern blot analysis. Genomic DNA from normal colon mucosa of an 18-year-old person and the CRC cell line Caco2 was digested with restriction endonucleases (H, HindIII; HS, HindIII + SmaI; HX, HindIII + XmaI), electrophoresed, blotted, and hybridized with MINT2 probe. Caco2 DNA fails to digest completely with SmaI, indicating methylation of one or both SmaI sites, but cuts down with XmaI, ruling out polymorphisms at the SmaI sites. (B) Semiquantitative feature of MCA. DNAs from the Caco2 cell line and normal colon were mixed in various proportions before MCA, and methylation of MINT2 was analyzed by dot-blot hybridization (Upper). % refers to the relative proportion of Caco2 DNA. The signal intensity of each dot was determined by PhosphorImager densitometry, and it increased linearly relative to the proportion of tumor cells in the mix. (C) Detection of MINT2 methylation by MCA. We blotted 0.1 μg of MCA products from colorectal tumors and corresponding normal colon mucosa in duplicate onto a nylon filter and hybridized them with a MINT2 probe. Tumors 391, 467, 874, 709, and 351 are methylated at this site. Sample numbers are shown above each lane. N, normal colon; T, colon tumor. (D) Detection of methylation by bisulfite-PCR. Genomic DNA was treated with bisulfite and amplified with primers specific to MINT2. Twenty percent of the PCR products were digested with BstUI and electrophoresed in 3% agarose gels. Arrows indicate bands reflecting methylation of the BstUI site. BstUI cleaves only the methylated alleles, yielding 115- and 88-bp bands (seen in a CRC cell line, RKO, and tumors 391, 467, 874, and 709). The coexistence of methylated and unmethylated bands reflects the fact that all these tumors are not microdissected and contain various amounts of contaminating normal tissues. Sample numbers are shown above. N, normal colon; T, colon tumor.

Figure 2

Methylation of the MINT clones in CRC and mucosa. (A) Examples of MINT clone methylation in colorectal cancer. MCA products from colon tumors (T) and corresponding normal colon mucosa (N) were blotted on a nylon membrane and hybridized with one of the MINT clones (indicated on the left), as well as a p16 probe. MINT6, 24, and 32 are examples of type A methylation, whereas MINT2, 12, and p16 are examples of type C methylation. Tumors 850 and 874 are CIMP+, whereas tumors 835, 872, and 733 are CIMP− (see text). Also shown is the Caco2 cell line (right). (B) Examples of age-related methylation in normal colon as detected by MCA using MINT5, 8, and 21 as probes. MCA products from normal colon mucosa from patients of various ages (indicated at the top in years) were hybridized with the probe indicated on the left. The signal intensity for each sample was determined by densitometry, and a ratio of mucosa/Caco2 (right) is indicated below each sample as a percentage. In each case, methylation is more prominent in DNA from older individuals. (C) Southern blot analysis of clones showing type A methylation in normal colon. DNA from normal colon mucosa from patients of various ages (top) was digested with HindIII and the methylation-sensitive enzyme SmaI and probed with MINT 5 (Left) and MINT22 (Right). Arrows indicate the methylated alleles. The relative proportion of methylated alleles was determined by densitometry and is indicated below each lane. (D) Bisulfite-PCR analysis of clones showing type A and type C methylation in normal colon. DNA from normal colon mucosa from patients of various ages (top) was bisulfite treated, amplified by PCR, and digested with restriction enzymes specific for sites that are created after bisulfite treatment (if the CpG sites are methylated). The arrows indicate the methylated alleles. Percentage methylation was determined by densitometry and is indicated below each lane. In type A loci, the percentage of methylated alleles from older people (>70) was significantly higher than that of younger people (<30) (6.4 ± 1.1 vs. 22.9 ± 1.2, P < 0.000001 for MINT23; 1.3 ± 0.3 vs. 16.1 ± 4.0, P < 0.01 for MINT32). In type C loci no methylation was detected in normal colon mucosa. The loci analyzed and restriction enzymes used are indicated below each gel.

Figure 3

Methylation analysis of CpG islands in CRC by using bisulfite-PCR. Bisulfite-treated DNA from CRC with (Left) or without (Right) CIMP and paired normal colon mucosa were amplified and digested with restriction enzymes that cleave only the methylated CpG sites. The arrows indicate the methylated alleles. The loci analyzed and restriction enzymes used are indicated below each gel. MINT6 is an example of type A methylation, whereas hMLH1 exemplifies type C methylation.

Figure 4

Methylation profile of colorectal cancer. The methylation status of all type C MINT clones, as well as p16, THBS1, and hMLH1, was determined in a panel of primary CRC and adjacent mucosa. None of the normal tissues studied showed any degree of methylation for these genes. Also shown are representative type A clones (Right). Each column represents a separate gene locus (indicated on top). Each row is a primary CRC (samples above the bold solid line) or adenomatous polyp (below the bold solid line). Black rectangles, methylated tumor; white rectangles, unmethylated tumor. MSI denotes the presence of microsatellite instability and was not determined for the adenomas. In the Center are known genes methylated in CRC (p16, THBS1, hMLH1).

Figure 5

A model integrating CGI methylation in colorectal carcinogenesis. See text for details.