Abstract
Background:
Ewing's sarcoma is a malignancy characterized by a specific 11:22 chromosomal translocation which generates a novel EWS-FLI1 fusion protein functioning as an aberrant transcription factor. In the present study, we have further characterized the junction region of the EWS-FLI1 fusion protein.
Methods:
In-silico model of EWS-FLI1 fusion protein was analysed for ligand binding sites, and a putative region (amino acid (aa) 251-343 of the type 1 fusion protein) in the vicinity of the fusion junction was cloned and expressed using bacterial expression. The recombinant protein was characterized by Circular Dichroism (CD). We then expressed aa 251-280 ectopically in Ewing's sarcoma cell-line and its effect on cell proliferation, tumorigenicity and expression of EWS-FLI1 target genes were analysed.
Results:
Our modelling analysis indicated that Junction region (aa 251-343) encompasses potential ligand biding sites in the EWS-FLI1 protein and when expressed in bacteria was present as soluble form. Ectopically expressing this region in Ewing's sarcoma cells inhibited tumorigenicity, and EWS-FLI1 target genes indicating a dominant negative biological effect.
Conclusions:
Junction region can be exploited further as target for drug development in future to specifically target EWS-FLI1 in Ewing's Sarcoma.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Cell Line, Tumor
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Cell Proliferation*
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Circular Dichroism
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Cyclin D1 / genetics
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Cyclin D1 / metabolism
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Enhancer of Zeste Homolog 2 Protein
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Epithelial-Mesenchymal Transition / genetics
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Gene Expression Regulation, Neoplastic*
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Homeobox Protein Nkx-2.2
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Homeodomain Proteins / genetics
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Homeodomain Proteins / metabolism
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Humans
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Immunoblotting
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MCF-7 Cells
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Molecular Sequence Data
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Oncogene Proteins, Fusion / chemistry
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Oncogene Proteins, Fusion / genetics*
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Oncogene Proteins, Fusion / metabolism
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Peptides / genetics
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Peptides / metabolism
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Polycomb Repressive Complex 2 / genetics
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Polycomb Repressive Complex 2 / metabolism
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Proto-Oncogene Protein c-fli-1 / chemistry
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Proto-Oncogene Protein c-fli-1 / genetics*
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Proto-Oncogene Protein c-fli-1 / metabolism
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Proto-Oncogene Proteins c-myc / genetics
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Proto-Oncogene Proteins c-myc / metabolism
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RNA-Binding Protein EWS / chemistry
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RNA-Binding Protein EWS / genetics*
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RNA-Binding Protein EWS / metabolism
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Reverse Transcriptase Polymerase Chain Reaction
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Sarcoma, Ewing / genetics*
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Sarcoma, Ewing / metabolism
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Sarcoma, Ewing / pathology
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Transcription Factors / genetics
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Transcription Factors / metabolism
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Transfection
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Zebrafish Proteins
Substances
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EWS-FLI fusion protein
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Homeobox Protein Nkx-2.2
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Homeodomain Proteins
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MYC protein, human
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Oncogene Proteins, Fusion
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Peptides
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Proto-Oncogene Protein c-fli-1
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Proto-Oncogene Proteins c-myc
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RNA-Binding Protein EWS
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Recombinant Proteins
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Transcription Factors
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Zebrafish Proteins
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Cyclin D1
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EZH2 protein, human
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Enhancer of Zeste Homolog 2 Protein
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Polycomb Repressive Complex 2