Metastasis accounts for the majority of treatment-refractory cancers and poor prognosis of the disease. The phenotypic transition of non-motile epithelial tumour cells to migratory and invasive mensenchymal cells (epithelial-mesenchymal transition (EMT)) enables the escape of cells from primary tumor site into the circulation. Morphological change from cobblestone to elongated and spindle-shaped like cells is a primary feature of this process. This transition is also marked by upregulation of biomarker vimentin, an intermediate filament found in mesenchymal cells. The reverse process, mesenchymal to epithelial transition (MET) is postulated to allow these disseminated mesenchymal-like cells to establish macrometastasis at secondary sites. Our lab has previously developed the TSU-Pr1 bladder cancer model, which we have used to demonstrate the relationship between epithelial/mesenchymal state and metastasis (1). Given the need to elucidate the pathways and assign functions to particular genes involved in this process, we conducted a high content screening assay using TSU-Pr1-B1 cells together with genome wide siRNA, miRNA and kinase inhibitor libraries to systematically identify modulators of EMT/MET. Using morphological change and a vimentin promoter-reporter system (Vimentin/dsRed) that is responsive to alterations in cellular epithelial and mesenchymal state as primary readouts, screening in this model has identified candidate genes and miRNAs involved in the regulation of EMT/MET. Interestingly, a number of miRNA hits (z-score ≥ 2) predicted target genes overlap with siRNA hits (morphological change z-score ≥ 4 and/or Vimentin/dsRed z-score ≥ 5). These genes (n=310), together with an additional ninety genes identified using the siRNA and kinase libraries, are currently being validated. This robust multiparameter assay has the power to generate high confidence hits that will enable the identification of novel molecules involved in this process, in addition to confirming the involvement of some established genes in regulating EMT/MET. This unbiased genome wide approach will contribute to our understanding of this essential process.
Project supported by the National Breast Cancer Foundation (Australia), a NHMRC Career Development Award (EDW) and a Malaysia Government scholarship (NABMS).
(1) Chaffer et al. (2005) Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in transitional cell carcinoma of the bladder. Clin Exp Metastasis 22: 115-25.