We conducted this study to examine the relationship between DNMT3B and the CIMP in colorectal cancer, and the relation between DNMT3B expression in cancer and matched normal colonic mucosa. Molecular correlates with DNMT3B activation may be important for better understanding of epigenetic and epigenomic aberrations during the carcinogenic process. We have shown that DNMT3B overexpression is associated with CIMP-high independent of other clinical and molecular variables, including age, sex, tumor location, stage, pathologic features, MSI, and BRAF mutation. Our data support the hypothesis that DNMT3B may contribute to CpG island methylation, which may eventually lead to the development of CIMP-high colorectal cancer. Considering that DNMT3B is a potential target of chemotherapy and chemoprevention, our findings may have considerable clinical implications.
To measure DNA methylation, we used real-time PCR (MethyLight technology) for DNA methylation at the eight CIMP-specific loci and in eight other CpG islands. We also used Pyrosequencing to measure LINE-1 methylation, which is a good indicator of cellular 5-methylcytosine level (i.e., genome-wide DNA methylation level; ref. 36
). Our resource of a large number of colorectal cancers derived from the two prospective cohort studies has enabled us to precisely estimate the frequency of colorectal cancers with a specific molecular feature (such as DNMT3B overexpression, CIMP-high, etc.). The large number of cases has also provided a sufficient power in our multivariate logistic regression analysis.
DNMT3B activation has been implicated in aberrant de novo
methylation of CpG islands in colorectal cancer (2
). Dnmt3b can induce colon tumor in mice, and those tumors exhibit DNA methylation in specific CpG islands (7
). In addition, a recent study using the MDA-MB231 breast cancer cell line has shown that Dnmt3b plays an important role in both methylation and demethylation (9
mRNA is overexpressed in human colorectal cancers compared with matched normal colonic mucosa (11
). In the current study, we have shown DNMT3B overexpression in 15% of colorectal cancers and the positive association between DNMT3B expression and CIMP-high, independent of other variables. DNMT3B overexpression has also been linked with the methylator phenotype in ovarian cancer cell lines (6
). Our data are likely important because no study has comprehensively examined the relationship between DNMT3B expression and CIMP in human colorectal cancer.
The molecular features of colorectal cancer have been extensively studied (40
). Although BRAF
mutation has been tightly linked to CIMP-high in colorectal cancer (25
), the exact mechanism of high propensity for widespread CpG island methylation leading to CIMP-high has remained uncertain. In the current study, we have shown the relation between DNMT3B and CIMP-high, independent of BRAF
mutation and other factors associated with CIMP-high. On the other hand, our data do not support a direct link between DNMT3B and high-level methylation in LINE-1, a surrogate of genome-wide DNA methylation level. A recent study using a murine colon cancer model has shown that Dnmt3b overexpression induces DNA methylation in some, but not all, CpG islands, suggesting that Dnmt3b may facilitate CpG island methylation in a non-random fashion (7
). DNMT3B depletion in cancer cells causes gene-specific demethylation, rather than genome-wide demethylation (14
). In addition, different splicing variants of DNMT3B seem to regulate gene-specific CpG island methylation patterns (10
). These experimental data are consistent with our data of the significant relation between DNMT3B and CIMP-high, but no significant relation between DNMT3B and genome-wide DNA methylation level. Based on our current results and data in the literature, represents hypothetical relations with CIMP-high in colorectal cancer. Further studies are necessary to elucidate the exact mechanism of DNMT3B activity on specific CpG islands without substantially affecting genome-wide DNA methylation level.
Regarding the different subtypes of CIMP in colorectal cancer, accumulating evidence suggests that CIMP-low is associated with KRAS
mutation and is molecularly different from CIMP-high and CIMP-negative (CIMP-0; refs. 32
). It has been suggested that KRAS
mutation is associated with a random CpG island methylation pattern that leads to CIMP-low tumors (35
). Together with the previous study suggesting nonrandom targeting of DNMT3B activity (7
), our data further support a molecular difference between CIMP-high (associated with DNMT3B up-regulation, a nonrandom CpG island methylation pattern and BRAF
mutation) and CIMP-low (associated with a random methylation pattern and KRAS
mutation). Further studies are necessary to elucidate the pathogenic mechanisms of these different epigenomic aberrations.
In conclusion, DNMT3B overexpression is associated with CIMP-high in colorectal cancer, independent of other clinical and molecular characteristics. Our data support the hypothesis that DNMT3B may play an important role in widespread promoter CpG island methylation during the carcinogenic process. Considering that DNMT3B is a potential target of chemotherapy and chemoprevention, our findings may have considerable clinical implications.