Epigenetic characterization of the C3(1) SV40T mouse model of human breast cancer

Abstract

Breast cancer is a heterogeneous disease, and various subtypes have been defined at the level of gene expression and epigenetic modifications, such as DNA methylation. Epigenetic alterations are attractive candidates for the development of novel biomarkers or as targets for new therapeutic approaches. Mouse models allow monitoring of tumor development from early time points of initiation to final stages of tumorigenesis, but are often poorly characterized with respect to alterations of the epigenetic landscape. Therefore, the aim of this thesis was to generated genome-wide profiles of DNA methylation and histone modifications for the C3(1)SV40TAg (C3(1)) mouse model of basal-like breast cancer and to investigate the epigenetic regulation of long noncoding RNAs. Using a genome-wide screen with Methyl CpG Immunoprecipitation followed by next generation sequencing, we identified several thousand regions with recurrent methylation alterations at different stages of C3(1) tumorigenesis. Differentially methylated genes pointed towards a luminal progenitor as tumor cell of origin in the C3(1) model, and we confirmed a link between DNA methylation and gene expression for five of these genes. Comparisons at the level of promoter methylation revealed general similarity of the C3(1) methylome to human breast cancer. Generation of a chromatin map of the C3(1) model from four histone modifications (H3K4me3, H3K4me1, H3K27ac, H3K27me3) allowed an integrative investigation of methylation changes at breast tissue-specific enhancer regions. We linked tissue-specific alterations of chromatin states in combination with DNA methylation to changes in the expression of genes with importance for mammary gland development and breast cancer. These results unveiled a potential involvement of transcription factors Etv4 (ETS variant 4) and Runx1 (runt related transcription factor 1) in C3(1) tumorigenesis that might help to understand tumor development in basal-like breast cancer. An emerging theme in epigenetic research is the capacity of long noncoding RNAs (lncRNAs) to modulate gene expression by recruitment of gene-silencing or activating complexes. Since regulation of lncRNAs expression is poorly characterized, we investigated DNA methylation changes during carcinogenesis at lncRNA promoters and their influence on neighboring proteincoding genes. Exemplarily, we demonstrated coordinated overexpression of Esrp2 (Epithelial splicing regulatory protein 2) and the lncRNA Esrp2-as (Esrp2-antisense) in C3(1) tumors that was inversely correlated with DNA methylation levels. Knockdown and overexpression of the transcripts did not provide evidence for reciprocal regulation of transcript expression. In contrast, luciferase reporter assays suggested that co-expression of both transcripts is controlled by differential methylation at a common enhancer region. These results are of clinical relevance as high levels of ESRP2 expression in human breast cancer are linked to unfavorable prognosis.