Poster Presentation 25th Lorne Cancer Conference 2013

Discovery of microRNAs associated with Breast Cancer Epithelial Mesenchymal Plasticity using Bioinformatics and Next Generation Sequencing (#362)

Eliza Soo 1 2 , Tony Blick 1 , Mark Waltham 1 , Gregory Goodall 3 , Angels Fabra-Fres 4 , Izhak Haviv 5 , Kaylene Simpson 6 , Erik Thompson 1 2 , Bryce van Denderen 1
  1. St Vincent's Institute, Fitzroy, VIC, Australia
  2. Surgery, University of Melbourne, Melbourne, Victoria, Australia
  3. Center for Cancer Biology, SA Pathology, Adelaide, South Australia, Victoria
  4. IDIBELL, Barcelona, Spain
  5. Bar Ilan University, Ramat Gan, Israel
  6. Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia

MicroRNAs (miRs) are a class of small RNA molecules that regulate gene transcript stability and processing by binding to discreet motifs in the 3’ and 5’ UTRs of mRNAs. They play important roles in development, including epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET), which have been associated with cancer metastasis. Epithelial mesenchymal plasticity (EMP) encompasses the dynamic interconversion between epithelial and mesenchymal states, and hybrid states thereof. miR profiling of control and EMT-induced PMC42-ET (ET), PMC42-LA (LA) and MDA-MB-468 cells using the mirVana probe set V1 and Next Generation Sequencing (miRSeq) was undertaken. Several miRs were reproducibly up- or down-regulated between the untreated cells, as well as in response to EGF. Variations in miR expression were also assessed bioinformatically using publically available data from >50 human breast cancer cell lines. Whilst a number of these have already been implicated in cancer, other novel miRs consistently associated with EMP were also identified. The expression levels of >20 miRs were validated using TaqMan® miR assays in 6 breast cancer cell lines. Stable over-expression of miRs in cell lines with low endogenous expression was achieved by lentiviral transduction. Breast cancer cell lines stably over-expressing the miRs of interest were produced and examined by qRT-PCR for EMP-associated gene expression, and were tested for changes in in vitro migratory potential using a monolayer wound healing assay on the Cellomics platform. The rate of wound closure was significantly faster in MDA-MB-468 breast cancer cells overexpressing miR-34b/c cluster (p=0.033) upon EGF induction. Several other miRs look promising and are currently being pursued. The migratory potential of these cells will be assessed further using the Transwell migration assay. MDA-MB-468 cells with manipulated miR expressions are also being examined in the MDA-MB-468 xenograft model of in vivo EMP 1.

  1. Gunasinghe et al., Cancer Metastasis Reviews, 2012.