Genome-wide DNA methylation analysis reveals estrogen-mediated epigenetic repression of metallothionein-1 gene cluster in breast cancer

Background: Recent genome-wide analysis has shown that DNA methylation spans long stretches of chromosome regions consisting of clusters of contiguous CpG islands or gene families. Hypermethylation of various gene clusters has been reported in many types of cancer. In this study, we conducted methyl-binding domain capture (MBDCap) sequencing (MBD-seq) analysis on a breast cancer cohort consisting of 77 patients and 10 normal controls, as well as a panel of 38 breast cancer cell lines. Results: Bioinformatics analysis determined seven gene clusters with a significant difference in overall survival (OS) and further revealed a distinct feature that the conservation of a large gene cluster (approximately 70 kb) metallothionein-1 (MT1) among 45 species is much lower than the average of all RefSeq genes. Furthermore, we found that DNA methylation is an important epigenetic regulator contributing to gene repression of MT1 gene cluster in both ER alpha positive (ER alpha+) and ER alpha negative (ER alpha-) breast tumors. In silico analysis revealed much lower gene expression of this cluster in The Cancer Genome Atlas (TCGA) cohort for ER alpha+ tumors. To further investigate the role of estrogen, we conducted 17 beta-estradiol (E2) and demethylating agent 5-aza-2'-deoxycytidine (DAC) treatment in various breast cancer cell types. Cell proliferation and invasion assays suggested MT1F and MT1M may play an anti-oncogenic role in breast cancer. Conclusions: Our data suggests that DNA methylation in large contiguous gene clusters can be potential prognostic markers of breast cancer. Further investigation of these clusters revealed that estrogen mediates epigenetic repression of MT1 cluster in ER alpha+ breast cancer cell lines. In all, our studies identify thousands of breast tumor hypermethylated regions for the first time, in particular, discovering seven large contiguous hypermethylated gene clusters.