High content multiparametric functional screen for regulators of epithelial-mesenchymal transition identifies genes associated with chemoresistance

Metastasis accounts for the majority of treatment-refractory cancers and poor prognosis of the disease. The phenotypic transition of non-motile epithelial tumor cells to migratory and invasive ‘mesenchymal’ cells enables the escape of cancer cells from the primary tumor into the circulation. Morphological changes from cobblestone epithelial cells to elongated and spindle-shaped cells is a key feature of this process. This transition is also marked by upregulation of vimentin, an intermediate filament found in mesenchymal cells. We have previously developed a bladder cancer model with a step-wise enhancement in the metastatic cascade that we have used to interrogate the role of tumor cell plasticity in metastasis (1). Given the need to elucidate pathways and assign functions to particular genes involved in this process, we conducted a high content screening assay across genome wide siRNA, miRNA and kinase inhibitor (131 compounds) libraries to systematically identify modulators of EMT/MET. We identified candidate gene regulators of EMT in TSU-Pr1-B1 bladder cancer cells using the readouts of morphological change (cells stained with CMFDA) and a vimentin promoter activity (dsRed reporter construct). Following integration of the predicted target genes for miRNA hits and kinase inhibitors with siRNA hits, initial validation using a siRNA deconvolution strategy was performed for 400 targets. 221 siRNAs were classified as inducing statistically significant effects on cell shape and/or vimentin promoter reporter activity (defined as at least 2/4 duplexes inducing the effect for a given target). Gene ontology and pathway analyses feature cell cycle and various developmental pathways. Furthermore, the validated gene list was associated with bladder cancer histology and drug sensitivity in clinical specimens in multiple cancer types (Oncomine). Molecules identified in this assay will be further analysed using functional assays to determine their role in maintaining the epithelial phenotype and as regulators of chemoresistance.