Discovery of microRNAs associated with breast cancer EMT using bioinformatics and next-generation sequencing

Abstract B093 : Breast cancer is the most common malignancy among women worldwide, with mortality often associated with distant metastasis. In recent years, microRNAs (miRs) have emerged as a new class of regulatory molecules that act as tumor suppressors or oncogenes and are capable of affecting metastatic processes such as epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET). Our study aimed to identify miRs associated with breast cancer EMT through miR profiling of EMT in PMC42 (ET vs LA, -/+ EGF) and MDA-MB-468 (control vs EGF, hypoxia) human breast cancer cells using microarray and Next Generation Sequencing (miR-Seq). Several miRs were reproducibly different between the untreated cells, as well as up- or down-regulated in response to epidermal growth factor (EGF). Expression levels of 36 miRs were validated in six breast cancer cell lines comprising a range of epithelial to mesenchymal cell lines. Nine miRs were then selected and the effects of manipulating these miRs were investigated in vitro and in vivo. MDA-MB-468 cells stably overexpressing (OE) the miRs of interest were produced, validated and tested for changes in in vitro migratory potential using a monolayer wound healing assay on the Cellomics platform. We found that the rate of wound closure was significantly faster (p<0.05) in MDA-MB-468 breast cancer cells OE miR-34b/c upon EGF induction even though there were no significant differences in migrative potential using Transwell migration assay. Although these cells showed no proliferative differences, MDA-MB-468 miR-34b/c OE cells showed decreased clonogenicity (p<0.01) compared to control cells. We extended this by looking at these manipulated cells in the MDA-MB-468 xenograft model of in vivo EMT. Our data suggests that there might be a slightly slower rate of primary tumor growth and an inhibition of lymph node metastasis in vivo. Other groups have also identified miR-34 family as a p53 target gene regulating Snail1-driven EMT (Kim et al., 2011) and have shown miR-34 to suppress breast cancer metastasis by targeting Fra-1 (Yang et al., 2012). Fra-1 and several other MEK pathway components were upregulated in the PMC42 and MDA-MB-468 EMT as determined by corresponding RNA-Seq analysis, and integrative analysis of the miR-Seq and RNA-Seq data is ongoing. Our work on EMT-associated miRs is targeted to provide a better understanding of miRs for their potential uses in diagnostic and therapeutic approaches to breast cancer.