Mapping of DNA Hypermethylation and Hypomethylation induced by Folate Deficiency in Sporadic Colorectal Cancer and Clinical Implication Analysis of Hypermethylation Pattern in CBS Promoter.

Background: Aberrant DNA methylation patterns play a major role in tumorigenesis and the effects of nutrients, especially folate in the diet, on methylation changes is of great importance in colorectal cancer (CRC). Folate deficiency would disrupt methylation patterns; however, its exact effects on DNA methylation patterns in CRC are unclear. This study was performed to gain insight into the methylation changes induced by folate deficiency and the putative role of methylation pattern diversities of related genes in the clinical outcome of CRC.

Methods: The NimbleGen MeDIP chip (Methylated DNA Immunoprecipitation chip) assay was used in high-resolution mapping of DNA methylation patterns in the normal human colon mucosal epithelial cell line, NCM460 cultured with or without folate. Aberrant CpG island methylation patterns in the promoter of genes were identified by chip assay and then were confirmed in paired colorectal tissues and corresponding non-malignant tissues obtained from patients by bisulfate sequencing PCR (BSP). Of the total, the expression of cystathionine-beta-synthase (CBS) involved in methyl metabolism and its important substrate, homocysteine, were all detected by realtime RT-PCR and immunostaining. We also analyzed the data of its hypermethylation level statistically correlated with pathological parameters and the clinical outcome in malignant tissues.

Results: The chip assay showed that there are 17 genes with hyper or hypomethylation in CpG islands of promoter on chromosome 21, and 8 of them seemed to be associated with tumorigenesis. Among the total, a hypermethylation patterns existed in the promoter of CBS in CRC (p < 0.001), and the hypermethylation is related with the down-regulation of CBS and the accumulation of homocysteine in vitro and vivo (p < 0.001). Univariate analysis showed CBS hypermethylation level is correlated with age (p < 0.001), pT stage (p = 0.008), pN stage (p = 0.038), liver metastasis (p = 0.017), pTNM stage (p = 0.032), Dukes' stage (p = 0.022), recurrence (p = 0.041), five-year survival (p = 0.034), recurrence-free probability (p = 0.011), and overall survival (p = 0.018). Multivariate analysis showed that CBS hypermethylation level significantly correlated with recurrence rate (p = 0.039) and overall survival (p = 0.012) independent of pT stage, pN stage, and liver metastasis.

Conclusions: Folate deficiency could induce aberrant DNA methylation patterns and gene expressions in CRC. CBS plays a critical role in tumorigenesis and could serve as a prognostic marker for tumor progression.