ABSTRACT
Ewing's family tumors (EFTs) are characterized by recurrent chromosomal translocations that produce chimeric fusions between the EWS gene and one of five ETS transcription factors. The expression of EWS/FLI1, the predominant fusion product in EFTs, is believed to deregulate downstream target genes in an undefined tissue type and leads to development of EFTs. Attempts to generate model systems that represent EFTs have been hampered by an unexpected toxicity of the fusion gene. In the present study, we used gene expression analysis to identify tissue types based on the similarity of their expression profiles to those of EWS/FLI1-modulated genes. The data obtained from this screen helped to identify IMR-90 cells, a human fetal fibroblast, that upon further manipulation can maintain stable EWS/FLI1 expression without the reported toxicity. In addition, gene expression profiling of these cells revealed a significant overlap of genes that have been previously reported to be targets of EWS/FLI1. Furthermore, we show, for the first time, a partial transformation of these human primary fibroblasts with EWS/FLI1 expression. The experiments presented here provide a solid foundation for generation of a new model system for studying Ewing's sarcoma biology.
Subject(s)
Mesoderm/pathology , Models, Biological , Oncogene Proteins, Fusion/genetics , Sarcoma, Ewing/genetics , Transcription Factors/genetics , Apoptosis , Blotting, Western , Cell Line , Gene Expression Profiling , Gene Silencing , Humans , Mesenchymal Stem Cells/pathology , Polymerase Chain Reaction , Proto-Oncogene Protein c-fli-1 , RNA-Binding Protein EWSABSTRACT
Suppression of the expression of antiangiogenic factors has been closely associated with multiple malignancies. Thrombospondins 1 and 2 are members of a family of angiogenic inhibitors that are regulated by several oncogenes. In this study, we investigate the role of thrombospondins in Ewing's sarcoma and their regulation by EWS/ETS fusion oncoproteins. We show that the EWS/FLI1 fusion suppresses the expression of thrombospondins in both NIH3T3 fibroblasts and Ewing's sarcoma tumor-derived cell lines. This regulation depends on an intact EWS/FLI1 DNA-binding domain and may involve direct interactions between EWS/FLI1 and thrombospondin promoter regions. Forced expression of thrombospondins in Ewing's sarcoma cell lines inhibited the rate of tumor formation in vivo and markedly decreased the number of microvessels present in the tumors. These findings suggest that thrombospondins play a biologically significant role in tumor vascularization in Ewing's sarcoma and suggest potential therapeutic strategies for future therapeutic intervention.
Subject(s)
Gene Expression Regulation, Neoplastic , Neovascularization, Pathologic , Oncogene Proteins, Fusion/physiology , Sarcoma, Ewing/metabolism , Thrombospondins/biosynthesis , Transcription Factors/physiology , Animals , Cell Line, Tumor , Humans , Mice , Mice, Nude , Models, Genetic , NIH 3T3 Cells , Neoplasm Transplantation , Oligonucleotide Array Sequence Analysis , Proto-Oncogene Protein c-fli-1 , RNA-Binding Protein EWS , Sarcoma, Ewing/pathology , Time FactorsABSTRACT
Chromosomal translocations involving the N-terminal approximately 250 residues of the Ewings sarcoma (EWS) oncogene produce a group of EWS fusion proteins (EFPs) that cause several distinct human cancers. EFPs are potent transcriptional activators and interact with other proteins required for mRNA biogenesis, indicating that EFPs induce tumorigenesis by perturbing gene expression. Although EFPs were discovered more than a decade ago, molecular analysis has been greatly hindered by the repetitive EWS activation domain (EAD) structure, containing multiple degenerate hexapeptide repeats (consensus SYGQQS) with a conserved tyrosine residue. By exploiting total gene synthesis, we have been able to systematically mutagenize the EAD and determine the effect on transcriptional activation by EWS/ATF1 and cellular transformation by EWS/Fli1. In both assays, we find the following requirements for EAD function. First, multiple tyrosine residues are essential. Second, phenylalanine can effectively substitute for tyrosine, showing that an aromatic ring can confer EAD function in the absence of tyrosine phosphorylation. Third, there is little requirement for specific peptide sequences and, thus, overall sequence composition (and not the degenerate hexapeptide repeat) confers EAD activity. Consistent with the above findings, we also report that the EAD is intrinsically disordered. However, a sensitive computational predictor of natural protein disorder (PONDR VL3) identifies potential molecular recognition features that are tyrosine-dependent and that correlate well with EAD function. In summary we have uncovered several molecular features of the EAD that will impact future studies of the broader EFP family and molecular recognition by complex intrinsically disordered proteins.
