Alterations of DNA Methylation during Multistage Carcinogenesis

---

Genetic alterations causing activation of oncogenes and inactivation of tumor suppressor genes have been considered to provide the molecular framework responsible for multistage carcinogenesis in humans. However, genetic events alone may not explain the histological heterogeneity that reflects the complexity of the biological characteristics of tumors. Therefore, we have focused on epigenetic events such as alterations of DNA methylation, which can be reversible and thus explain why cancers show such histological heterogeneity (Fig. 1).

In order to clarify the mechanism responsible for reduced E-cadherin expression in poorly differentiated cancers that have lost their cell-cell adhesion and show a strong invasive tendency, we cloned the promoter region of the human E-cadherin gene in 1995 and demonstrated that it showed DNA methylation in human cancer cell lines lacking E-cadherin expression (Ref. 1). We also observed induction of E-cadherin expression after treatment with the DNA methyltransferase inhibitor 5-azacytidine in such cell lines (Ref. 1). Thus, following the RB and VHL genes, the E-cadherin gene became the third example of a tumor suppressor gene that is silenced by DNA hypermethylation.

We found alterations of DNA methylation in precancerous conditions or lesions, such as chronic hepatitis or liver cirrhosis preceding hepatocellular carcinomas (Ref. 2). This was one of the earliest reports of alterations of DNA methylation at the precancerous stage. Alterations of DNA methylation are frequently observed in cancers associated with chronic inflammation and/or persistent infection with viruses or other pathogenic microorganisms, such as hepatitis B or C virus (Refs. 3, 4), Epstein-Barr virus (Ref. 5) and human papillomavirus, or with cigarette smoking (Ref. 6). Alterations of DNA methylation precede genomic instability (Fig. 2, Ref. 4) and are involved even at the early and precancerous stage. On the other hand, in patients with cancers, aberrant DNA methylation is significantly associated with tumor aggressiveness and poor prognosis. Therefore, we consider that precancerous conditions showing alterations of DNA methylation may progress rapidly and generate more malignant cancers (Ref. 7).

Overexpression of DNMT1, a major DNA methyltransferase, is not a secondary result of increased cell proliferative activity but is significantly correlated with accumulation of DNA methylation of tumor-related genes (Ref. 8), the CpG island methylator phenotype of cancers (Ref. 5), tumor aggressiveness and poor prognosis (Fig. 3, Ref. 9). Splicing alteration of DNMT3b, namely overexpression of an inactive variant, DNMT3b4, may result in chromosomal instability through DNA hypomethylation of pericentromeric satellite regions (Ref. 10).



Alterations of DNA methylation are associated with multistage carcinogenesis from precancerous conditions to malignant progression(Ref. 11). Therefore, we now employ array-based technology for accessing genome-wide DNA methylation status (Fig. 4). On the basis of genome-wide DNA methylation status, we are attempting to establish a new classification of cancers that may reflect the distinct epigenetic pathways of carcinogenesis, and to identify the DNA methylation profile that is the optimum indicator for carcinogenetic risk estimation, early diagnosis and prognostication. In order to apply correction of DNA methylation status to practical prevention and therapy of cancers, the full picture and molecular mechanisms of DNA methylation alterations corresponding to specific carcinogenetic factors should be further clarified in each organ.



[ Representative publications ]

1. Proc Natl Acad Sci USA, 92: 7416-7419, 1995.
2. Jpn J Cancer Res, 87: 1210-1217, 1996.
3. Hepatology, 29: 703-709, 1999.
4. Hepatology, 32: 970-979, 2000.
5. Am J Pathol, 164: 689-699, 2004.
6. Cancer Res, 57: 4913-4915, 1997.
7. Int J Cancer, 119: 288-296, 2006.
8. Carcinogenesis, 27: 1160-1168, 2006.
9. Int J Cancer, 105: 527-532, 2003.
10. Proc Natl Acad Sci USA, 99: 10060-10065, 2002.
11. Carcinogenesis, 28: 2434-2442, 2007.