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DOI: 10.1055/s-0031-1278498
DNA methylation contributes to the colonic adenoma-dysplasia-carcinoma development in a non-sequential line
Background: The adenoma-dysplasia-carcinoma sequence (ADCS) driven by genetic mutations is a widely accepted concept in colorectal carcinogenesis. In some proximal cancer arising from different precursor lesions DNA methylation has been shown to be an important mechanism and a distinct phenotype of colorectal cancer (CRC), CpG island methylator phenotype (CIMP) was described. In distal CRC DNA methylation has scarcely been studied. Aims: To study the role of DNA methylation during ADCS in the left side of the colon and find a set of DNA methylation markers that distinguishes between the different stages. Our further aim was to correlate our DNA methylation profiles with whole-genome mRNA expression microarray results and find genes which show underexpression in line with DNA hypermethylation.
Methods: Endoscopically obtained colonic biopsy samples were taken from the left side of the colon. For DNA methylation analysis 5 healthy colonic, 5 low-grade dysplasia (LGD), 5 high-grade dysplasia (HGD) and 5 CRC (UICC stage I-III) samples were applied for Methyl-Profiler DNA Methylation PCR array system. Primers of 96 genes were lyophilized to the appropriate wells of a 384-well plate. Using methylation restriction enzyme digestion followed by real-time PCR enabled us to define the methylation status in our samples. For mRNA expression analysis 33 healthy colonic and 22 CRC (UICC stage I-III) samples were applied for Whole-Genome Expression Profile Microarray provided by Affymetrix. Data was analyzed with Significance Analysis of Microarrays.
Results: The analysis of 96 genes revealed the hypermethylation of 30 genes in LGD, 51 genes in HGD and 18 genes in CRC as compared to normal. All but one (ID4) hypermethylated genes in CRC were affected in the dysplasia groups. All groups could be distinguished from normal tissue by their different methylation pattern. ALDH1A3 was identified as a novel hypermethylated gene in CRC. DNA hypermethylation inversely correlated with mRNA expression in SFRP1, SLIT2 and MAL.
Conclusions: In summary DNA methylation can contribute to the development of distal, left-sided, CIMP-negative CRC. Although sequential methylation pattern was not observed, the higher number of hypermethylated genes in precancerous lesions suggests a potential role for DNA methylation in the development and the early detection of precursor lesions.