Gene expression profile upon EWSR1::FLI1 knockdown in Ewing sarcoma cells A673 in vitro

Ewing sarcoma, a rare and aggressive pediatric cancer, is characterized by chromosomal translocations that give rise to chimeric transcription factors. The most frequent of these chromosomal translocations is the t(11;22) that produces the fusion of the EWSR1 and FLI1 genes to generate the chimeric transcription factor EWSR1::FLI1. EWSR1::FLI1 is the main oncogenic event in Ewing's sarcoma. Recently, it has been proposed that EWSR1::FLI1 levels may fluctuate in Ewing sarcoma cells, giving rise to two cell populations: cells expressing low levels of EWSR1::FLI1 are characterized by a more migratory and invasive phenotype, while cells expressing high levels of EWSR1::FLI1 are more proliferative. The identification and functional characterization of EWSR1::FLI1 gene targets is therefore relevant to understanding the pathobiology of Ewing sarcoma, which in turn could contribute to the identification of new therapeutic targets. Using this approach, we have observed that CD44, a transmembrane protein involved in cell adhesion and migration and associated with metastasis in various cancer types, is overexpressed in the EWSR1::FLI1-low phenotype. Our results suggest that CD44 may play a role in regulating cell migration in Ewing sarcoma cells and thus contribute to the spread of tumor cells. Overall design: A673 cells derived from Ewing sarcoma were genetically enginereed to express specific shRNAs against EWS::FLI1 upon doxycycline stimulation (A673/TR/shEF). Three independent clones from each enginereed cell were analyzed. Cells were stimulated with doxycycline for 72 hours to induce the expression of EWSR1:FLI1 shRNA. Afterwards, total RNA was isolated and gene expression profile analyzed by RNAseq. Gene expression profile in A673 cells in which EWSR1::FLI1 was downregulated (A673 EWSR1::FLI1 low expression) were compared to control cells (A673 EWSR1::FLI1 high expression).