EWS/FLI1 regulates EYA3 in Ewing sarcoma via modulation of miRNA-708, resulting in increased cell survival and chemoresistance

Mol Cancer Res. 2012 Aug;10(8):1098-108. doi: 10.1158/1541-7786.MCR-12-0086. Epub 2012 Jun 20.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bone Neoplasms* / drug therapy
  • Bone Neoplasms* / metabolism
  • Bone Neoplasms* / pathology
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Cell Survival / genetics
  • DNA-Binding Proteins* / genetics
  • DNA-Binding Proteins* / metabolism
  • Doxorubicin / pharmacology
  • Drug Resistance, Neoplasm / genetics
  • Etoposide / pharmacology
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Mesenchymal Stem Cells / metabolism
  • MicroRNAs* / genetics
  • MicroRNAs* / metabolism
  • Oncogene Proteins, Fusion* / genetics
  • Oncogene Proteins, Fusion* / metabolism
  • Protein Tyrosine Phosphatases* / genetics
  • Protein Tyrosine Phosphatases* / metabolism
  • Proto-Oncogene Protein c-fli-1* / genetics
  • Proto-Oncogene Protein c-fli-1* / metabolism
  • RNA-Binding Protein EWS* / genetics
  • RNA-Binding Protein EWS* / metabolism
  • Sarcoma, Ewing* / drug therapy
  • Sarcoma, Ewing* / metabolism
  • Sarcoma, Ewing* / pathology

Substances

  • DNA-Binding Proteins
  • EWS-FLI fusion protein
  • MIRN708 microRNA, human
  • MicroRNAs
  • Oncogene Proteins, Fusion
  • Proto-Oncogene Protein c-fli-1
  • RNA-Binding Protein EWS
  • Etoposide
  • Doxorubicin
  • EYA3 protein, human
  • Protein Tyrosine Phosphatases