Abstract
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 the present study, we aimed 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. This model has been utilized in various studies to demonstrate chemopreventive efficacy of both natural products as well as pharmacological agents such as green and black tea or the COX‐2 inhibitor celecoxib.
For DNA methylation analyses, wildtype (wt) and transgenic (tg) mice were sacrificed at 4–24 weeks of age to collect mammary and tumor tissue, respectively. Initially, we selected eight candidate genes know to be silenced by hypermethlytion in human breast cancer: Cadherin1, Cyclin D2, RASSF1a, Timp3, Twist, GSTπ, SMPD3, and GHSR1a. DNA methylation of CpG islands associated with these genes was quantified by EpiTYPER MassARRAY technology based on mass spectrometry. Interestingly, none of these genes was hypermethylated in murine tumors. On average, we detected less than 16% methylation, which was not significantly different between wt and tg tissue and did not change with age.
Consequently, we next decided to analyze 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, five genes, Mab21l2, Lyl1, Atp6v1b1, Espn, A930037G, 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 detected. 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 investigated in relation to human breast cancer so far, may serve as interesting targets for chemoprevention studies and will be further characterized.
Citation Information: Cancer Prev Res 2010;3(1 Suppl):B49.
