Epigenetic changes during disease progression in a murine model of human breast cancer

It is now well accepted that epigenetic alterations contribute to tumorigenesis. DNA methylation has been recognized as an early and possibly initiating event in the development of breast cancer and thus represents a potentially valuable marker for early detection and chemoprevention.

In an ongoing project, we aim to characterize DNA methylation changes associated with the development of breast cancer in the C3(1)/SV40 large T antigen transgenic mouse model of mammary carcinogenesis. In various studies both natural products as well as pharmacological agents such as green and black tea or the COX-2 inhibitor celecoxib have been shown to prevent mammary tumor growth in this model.

Preliminary data:
We have established a breed of C3(1)/SV40 large T antigen transgenic mice at the German Cancer Research Center. For DNA methylation analyses, we have collected >250 samples of mammary and tumor tissue from both wildtype (wt) and transgenic (tg) mice at 4-24 weeks of age. We have analyzed methylation changes at a genome-wide level, using methylation-specific DNA array analysis after methyl-CpG immunoprecipitation (MCIp). Hypermethylated DNA of age-matched wt and tg animals was enriched by binding to recombinant methyl-CpG-binding domain protein MBD2. Dual-color labeled samples were then hybridized to Agilent murine CpG island microarrays containing oligonucleotide probes for >16.000 CpG islands. Based on data obtained with tissue derived of animals aged 16-24 weeks, we selected genes commonly hypermethylated in tg mice for subsequent validation by MassARRAY analysis. As an example, a series of five genes showed significant hypermethylation in a range of 52 to 69% in tumor tissue of mice aged 24 weeks, whereas an average methylation of 6 to 15% was detected in mammary tissue of age-matched wt animals. Interestingly, when we analyzed mammary tissue of mice at increasing age, we observed a gradual increase in hypermethylation in tg vs. wt animals for all selected genes, starting at 12-16 weeks of age even before tumors were detectable.

These data indicate a potential early role of deregulated DNA methylation in C3(1)/SV40 large T antigen-induced mammary carcinogenesis. The newly identified genes, which have rarely been mentioned in relation to human breast cancer so far, may serve as interesting targets for chemoprevention studies.

The project will define the role of DNA methylation deregulated during mammary carcinogenesis in the C3(1)/SV40 large T antigen transgenic mouse model. Identified genes/pathways will provide an important basis for future dietary intervention studies and prevention strategies.

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