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

Background:
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.

Aims:
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.

Outlook:
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.

Publication:

Heilmann K, Toth R, Bossmann C, Klimo K, Plass C, Gerhauser C. Genome-wide Screen for Differentially Methylated Long Noncoding RNAs identifies Esrp2 and lncRNA Esrp2-as Regulated by Enhancer DNA Methylation with Prognostic Relevance for Human Breast Cancer. Oncogene 2017, 36(46): 6446-6461