Identification of a DNA methylation signature and distinct microRNA variants in breast cancer

Abstract

Summary Deregulation of factors involved in the epigenetic regulation of gene expression is a hallmark of cancer. In this dissertation, DNA methylation and microRNA (miRNA) expression, as two components implicated in epigenetic regulation, were studied in breast cancer. In the first study, the establishment of a DNA methylation signature for breast cancer was aimed at, based on the early occurrence and stability of abnormal DNA methylation patterns in tumors. Specialized oligonucleotide microarrays were developed and optimized to screen DNA methylation patterns of the candidate loci associated with breast cancer. The assay was applied to analyze DNA methylation patterns of breast cancer and control samples. The profiling data was subjected to multiple bioinformatic analyses in order to identify a DNA methylation signature with appropriately high potential for diagnosis. Methylation patterns of CpG sites associated with two genes, SFRP2 and GHSR, were identified as promising markers. Up-regulation and activation of estrogen receptor α (ERα) signaling is a common feature of almost 70% of breast cancers. However, our understanding of the molecular mechanisms underlying deregulation of this signaling pathway is scarce. In the second study, based on miRNA profiling data of human mammary cell lines, miR-375 was identified as an overexpressed miRNA in ERα-positive cells. Analysis of the miRNA locus revealed that miR-375 overexpression is mainly caused by the loss of epigenetic marks, such as H3K9me2 and local DNA hypomethylation, which, in turn, triggers the dissociation of the transcriptional repressor CCCTC-binding factor (CTCF) from the promoter and enables interactions of ERα with regulatory regions of miR-375. Inhibition of miR-375 in ERα-positive MCF7 cells resulted in reduced ERα activation and cell proliferation. In addition, RASD1, an estrogen inducible gene, was identified as a miR-375 target mediating miR-375 effect on ERα. These data indicate the existence of a positive feedback regulation between ERα and miR-375. The methylation signature identified shows a promising potential to be applicable for diagnosis/risk assessment of breast cancer. Furthermore, our findings from miRNA study provide significant insight into the deregulation of ERα pathway, which may open new therapeutic strategies for breast cancer.