Abstract 3322: DNA hypermethylation plays an important role in the epithelial-mesenchymal transition in triple negative breast cancer

Abstract

Abstract We have developed an in vitro model of triple negative breast epithelial cell lines which represents the progression of the basal breast cancer subtype (Russo et al, FASEB 2006; Huang et al, Cancer Res 2007). This model consists of three cell lines, MCF10F, trMCF and bsMCF representing respectively the normal, transformed and tumorigenic phases of the cancer progression. There are genomic differences among these cells that are characteristics of epithelial-mesenchymal transition (EMT). In the present work, we evaluated the changes in methylation throughout the EMT by performing MBD-Cap sequencing of MCF10F, trMCF and bsMCF. Methylated DNA was separated using MethylMiner™ Methylated DNA Enrichment Kit (Life Technologies), and the samples were used for library preparation and sequencing using Illumina platform. To identify the changes in the methylation profile of these cell lines during the process of EMT we performed the comparisons MCF10F vs. trMCF; trMCF vs. bsMCF; and MCF10F vs. bsMCF. Genes were considered differentially methylated when they showed adjusted p-value (Benjamini and Hochberg) < 0.01 and at least twofold difference in methylation levels. We found that overall the methylation patterns increase as the EMT takes place. We observed 816 genes hypermethylated in trMCF compared to MCF10F, 229 genes in bsMCF compared to trMCF, and 1269 hypermethylated genes in bsMCF vs. MCF10F. Gene ontology and pathway enrichment analyses revealed that differentiation, morphogenesis, motility and adhesion were processes over-represented among the genes that were hypermethylated throughout the EMT process. One of the hypermethylated genes was NRG1 (neuregulin 1), which encodes a cell adhesion molecule and has been shown to be methylated in breast cancer tissue. Although in smaller numbers, we also observed some genes hypomethylated during the EMT. There were 29 hypomethylated genes in trMCF compared to MCF10F, 94 in bsMCF vs. trMCF, while 45 hypomethylated genes were observed in bsMCF compared to MCF10F. These data show that methylation is an important epigenetic molecular mechanism during the cancer progression in this in vitro model. Most of the DNA methylation changes occur during the first step of malignant transformation, from normal to transformed cells (845 genes with differentially methylated areas). Moreover, the additional epigenetic modifications during the EMT (323 genes in bsMCF vs. trMCF) contribute to the cellular development of tumorigenic properties. These results point us to the potential use of demethylating agents, such as DNMT inhibitors, as therapeutic drugs against triple negative breast cancer. (This work was supported by The Pennsylvania Cancer Cure Grant 6914101, the Barbara and Joseph Breitman donation, the Flyers Wives and by NIH core grant CA06927). Citation Format: Julia Santucci-Pereira, Yanrong Su, Jose Russo. DNA hypermethylation plays an important role in the epithelial-mesenchymal transition in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3322.