A Mouse Skin Multistage Carcinogenesis Model Reflects the Aberrant DNA Methylation Patterns of Human Tumors

Abstract

Whereas accepted models of tumorigenesis exist for genetic lesions, the timing of epigenetic alterations in cancer is not clearly understood. We have analyzed the profile of aberrations in DNA methylation occurring in cells lines and primary tumors of one of the best-characterized mouse carcinogenesis systems, the multistage skin cancer progression model. Initial analysis using high-performance capillary electrophoresis and immunolocalization revealed a loss of genomic 5-methylcytosine associated with the degree of tumor aggressiveness. Paradoxically, this occurs in the context of a growing number of hypermethylated CpG islands of tumor suppressor genes at the most malignant stages of carcinogenesis. We have observed this last phenomenon using two approaches, a candidate gene approach, studying genes with well-known methylation-associated silencing in human tumors, and a mouse cDNA microarray expression analysis after treatment with DNA demethylating drugs. The transition from epithelial to spindle cell morphology is particularly associated with major epigenetic alterations, such as E-cadherin methylation, demethylation of the Snail promoter, and a decrease of the global DNA methylation. Analysis of data obtained from the cDNA microarray strategy led to the identification of new genes that undergo methylation-associated silencing and have growth-inhibitory effects, such as the insulin-like growth factor binding protein-3. Most importantly, all of the above genes were also hypermethylated in human cancer cell lines and primary tumors, underlining the value of the mouse skin carcinogenesis model for the study of aberrant DNA methylation events in cancer cells.

Methylation of the O6‐methylguanine‐DNA methyltransferase promoter suppresses expression in mouse skin tumors and varies with the tumor induction protocol

“…MGMT promoter methylation in tumors was not associated generally with initiating mutations in either Ha-ras or Ki-ras, but was found more frequently in MNNG-initiated, mezerein-promoted tumors with Haras mutations. As reported for human cancer 5 and for mouse keratinocyte cell lines, 14,17 methylation of the MGMT promoter in mouse skin tumors reduced the expression of MGMT.…”

“…Our findings utilizing tumors from the multistage mouse skin tumor induction model are in close agreement with those found for MGMT in human tumors. A recent report, 14 in which promoter methylation of MGMT as well as other critical genes were studied, has also demonstrated the general agreement between mouse and human tumors, and emphasized the value of the mouse skin carcinogenesis model for the study of aberrant DNA methylation in cancer cells.…”

“…Fraga et al 14 have reported recently that hypermethylation of the MGMT promoter is an early event in the carcinogenesis process because all 10 keratinocyte cell lines studied (with non-tumorigenic, benign and malignant phenotypes) displayed this methylation. In our studies of tumors induced in mice, however, more tumors showed methylation at later times, and carcinomas that appeared at later times were more likely to be methylated, suggesting a role for methylation in tumor progression.…”

“…13 More recently, the MGMT gene was shown to be silenced in 10 keratinocyte lines with defined phenotypes varying from nontumorigenic to benign papilloma, squamous cell carcinoma, and spindle cell carcinoma. 14 In transgenic mice overexpressing MGMT in the epidermis, the skin tumor yield in initiation-promotion studies with MNU as initiator was 0.9 papillomas per mouse, compared to 5.2 in non-transgenic controls. 15 Initiation by DMBA, which does not induce O 6 -methylguanine adducts, was not affected in the MGMT transgenic mice.…”

Repression of Ah receptor and induction of transforming growth factor-β genes in DEN-induced mouse liver tumors

“…In tumors and in cancer cell lines, aberrant methylation usually occurs at CpG islands, unmethylated in normal somatic cells and hypermethylated in tumor suppressor gene promoters, representing a major mechanism of gene inactivation in primary human tumors (Fraga et al, 2004;Esteller, 2007). A CpG island in the promoter of the human AHR gene has recently been found to be methylated in a panel of 19 tumor cell lines and in lymphocytes of one-third of acute lymphoblastic leukemia patients (Mulero-Navarro et al, 2006).…”