EWSR1‐FLI1‐dependent Ezrin expression and FAK Y397 phosphorylation in an EwS in vivo model and primary human tumor samples. (A) DOX‐inducible knockdown of EWSR1‐FLI1 in A673 cells showed downregulation of FLI‐1 and Ezrin mRNA levels in A673 EwS cells, while FAK levels remained unaffected (each n = 3, Dunnett’s multiple comparison test). (B) Immunohistochemistry confirmed the downregulation of Ezrin on protein level along with a decrease in Y397 phosphorylation of FAK in A673‐derived xenografts, while FAK protein levels remained unaltered (each n = 3, Dunnett’s multiple comparison test). (C) siRNA‐based knockdown of Ezrin led to a significant decrease in Y397 phosphorylation of FAK in EwS cells. (D) IHC analyses of FAK/Ezrin expression and Y397 phosphorylation of FAK in tissue samples derived from 97 EwS patients treated in the CESS trial. All EwS tumor samples were positive for FAK protein in immunohistochemistry, and the majority of tumor samples showed moderate‐to‐strong expression of Ezrin and Y397 phosphorylation of FAK (85% and 84% of cases, respectively). All pFAK+ cases were Ezrin+. Survival analyses showed no significant association between Ezrin expression/FAK phosphorylation and overall survival (P = 0.3203 and 0.9438, respectively; n = 97; Kaplan–Meier method). *p < 0.05; **p < 0.01; ***p < 0.001.