Ewing sarcoma is characterized by the expression of the chimeric EWSR1-FLI1 transcription factor. Proteomic analyses indicate that the decrease of EWSR1-FLI1 expression leads to major changes in effectors of the dynamics of the actin cytoskeleton and the adhesion processes with a shift from cell-to-cell to cell-matrix adhesion. These changes are associated with a dramatic increase of in vivo cell migration and invasion potential. Importantly, EWSR1-FLI1 expression, evaluated by single-cell RT-ddPCR/immunofluorescence analyses, and activity, assessed by expression of EWSR1-FLI1 downstream targets, are heterogeneous in cell lines and in tumours and can fluctuate along time in a fully reversible process between EWSR1-FLI1high states, characterized by highly active cell proliferation, and EWSR1-FLI1low states where cells have a strong propensity to migrate, invade and metastasize. This new model of phenotypic plasticity proposes that the dynamic fluctuation of the expression level of a dominant oncogene is an intrinsic characteristic of its oncogenic potential.

Ewing sarcoma is a pediatric bone tumor characterized in 85% of cases by the fusion between EWS and FLI1 genes that results in the expression of the EWS-FLI1 aberrant transcription factor. Histologically, the Ewing tumor expresses high levels of the CD99 membrane glycoprotein. It has been recently described that CD99 expression contributes to the Ewing tumor oncogenesis by modulating growth and differentiation of tumor cells. Different studies have also shown that overexpression of EWS-FLI1 induces CD99 expression in non-Ewing cells. At the opposite, the knockdown of EWS-FLI1 expression by siRNA approaches has no significant effect on CD99 mRNA level in Ewing cells. Here, by in vivo and in vitro studies, we show that while EWS-FLI1 inhibition has only slight effects on the amount of CD99 transcript, it induces a dramatic decrease of the CD99 protein expression level, hence suggesting post-transcriptional regulations, possibly mediated by microRNAs. To further investigate this issue, we identified a set of 91 miRNAs that demonstrate EWS-FLI1 modulation, three of them being predicted to bind CD99 3 0 untranslated region (3 0 UTR). Among these, we show that miR-30a-5p has the ability to interact with the 3 0 UTR region of CD99 and to regulate its expression. Moreover, the re-expression of miRNA-30a-5p in Ewing cell line induces decreased cell proliferation and invasion. In this study, we therefore show that miR-30a-5p constitutes a major functional link between EWS-FLI1 and CD99, two critical biomarkers and therapeutic targets in Ewing sarcoma.