miR-200b-mediated reversion of a spectrum of epithelial to mesenchymal transition states in recessive dystrophic epidermolysis bullosa squamous cell carcinomas

Abstract Background Cutaneous squamous cell carcinomas (SCC) are the leading cause of death in patients suffering from recessive dystrophic epidermolysis bullosa (RDEB). However, the survival time from first diagnosis differs among patients, and some tumours spread particularly fast, while others may remain localised over years. As treatment options are limited, there is an urgent need to provide further insights into the pathomechanisms elucidating the clinical heterogeneity of RDEB tumours to foster therapy development and support clinical decision-making. Objectives Investigate differences in RDEB tumours of diverging aggressiveness at the molecular and phenotypic level, with a particular focus on epithelial to mesenchymal (EMT) transition states and thus miR-200b as a regulator. Methods Primary RDEB-SCC keratinocyte lines were characterised with respect to their EMT state. For this purpose, cell morphology was classified, and the expression of EMT markers was analysed using immunofluorescence, flow cytometry, sqRT-PCR, and western blotting. The motility of RDEB-SCC cells was determined and conditioned medium of RDEB-SCC cells was used to treat endothelial cells in an angiogenesis assay. In addition, we mined previously generated microRNA (miRNA) profiling data to identify a candidate with potential therapeutic relevance and performed transient miRNA transfection studies to investigate the candidate’s ability to reverse EMT characteristics. Results We observed a high variability in the EMT state among the RDEB-SCC cell lines, which correlated with in situ analysis of two available patient biopsies and respective clinical disease course. Furthermore, we identified miR-200b-3p to be downregulated in RDEB-SCCs, with the extent of deregulation significantly correlating with EMT features of the various tumour lines. miR-200b-3p was reintroduced into RDEB-SCC cell lines with pronounced EMT features, resulting in a significant increase in epithelial characteristics, including cell morphology, EMT marker expression, migration and angiogenic potential. Conclusions In summary, RDEB-SCCs exist in different EMT states and the level of miR-200b is indicative of how far an RDEB-SCC has gone down the EMT path. Moreover, reintroduction of miR-200b significantly reduced mesenchymal features.