Ewing sarcoma is an aggressive pediatric cancer of the bone and soft tissue, in 
which patients whose tumors have a poor histologic response to initial 
chemotherapy have a poor overall prognosis. Therefore, it is important to 
identify molecules involved in resistance to chemotherapy. Herein, we show that 
the DNA repair protein and transcriptional cofactor, EYA3, is highly expressed 
in Ewing sarcoma tumor samples and cell lines compared with mesenchymal stem 
cells, the presumed cell-of-origin of Ewing sarcoma, and that it is regulated by 
the EWS/FLI1 fusion protein transcription factor. We further show that EWS/FLI1 
mediates upregulation of EYA3 via repression of miR-708, a miRNA that targets 
the EYA3 3'-untranslated region, rather than by binding the EYA3 promoter 
directly. Importantly, we show that high levels of EYA3 significantly correlate 
with low levels of miR-708 in Ewing sarcoma samples, suggesting that this 
miR-mediated mechanism of EYA3 regulation holds true in human cancers. Because 
EYA proteins are important for cell survival during development, we examine, and 
show, that loss of EYA3 decreases survival of Ewing sarcoma cells. Most 
importantly, knockdown of EYA3 in Ewing sarcoma cells leads to sensitization to 
DNA-damaging chemotherapeutics used in the treatment of Ewing sarcoma, and as 
expected, after chemotherapeutic treatment, EYA3 knockdown cells repair DNA 
damage less effectively than their control counterparts. These studies identify 
EYA3 as a novel mediator of chemoresistance in Ewing sarcoma and define the 
molecular mechanisms of both EYA3 overexpression and of EYA3-mediated 
chemoresistance.