Ewing's sarcoma is a common pediatric tumor that arises in bone and soft tissues. Chromosomal translocations create fusion proteins between the N-terminus of EWS and the DNA binding domain from the closely related ets proteins Fli or Erg in at least 95% of Ewing's sarcomas. Related fusion proteins have been identified in five other types of human sarcomas and in myeloid leukemia. The EWS/Fli and EWS/Erg chimeric proteins can function as transcription factors and they can transform NIH-3T3 cells. Transcription activation and cellular transformation appear to be inter- related, but distinct functions of the fusion proteins. The exact process by which EWS/ets fusion genes contribute to tumorigenesis is unknown. The proposed studies will investigate the hypothesis that the EWS domain contributes unique properties to the chimeric proteins that play a critical role in the genesis of Ewing's sarcomas through interactions with other cellular proteins. In preliminary studies leading to this application, five proteins that interact with the tumor- associated domain of EWS were isolated multiple time in a yeast two- hybrid assay. Several lines of in vitro and in vivo experimentation are proposed to identify functional components of the EWS domain and determine the effects of protein-protein interactions on EWS/Fli activity. EWS-interacting proteins will be evaluated for their effect on transactivation and cellular transformation by EWS/Fli using cotransfection and cell culture assays. In addition, deletions and point mutations in the domain of EWS will be tested to define the residues required for protein-protein associations as detected by in vitro and cell culture assays. EWS/Fli proteins containing mutations in the interaction domains will be analyzed to determine the contribution of protein-protein interactions to transactivation and cellular transformation by EWS/Fli. Finally, we will identify the cell-types that are susceptible to transformation by EWS/Fli and study tumorigenesis in vivo using transgenic mice. Expression of functionally relevant EWS- interacting genes will be examined in Ewing's sarcoma cell lines and primary Ewing's sarcoma tissue. The results of these studies should provide a better understanding of the role of EWS/ets chimeric proteins in Ewing's sarcoma, and may identify common factors that contribute to tumorigenesis in human sarcomas and leukemias containing related fusionproteins.
Showing the most recent 10 out of 124 publications
