EWS/FLI mediates transcriptional repression via NKX2.2 during oncogenic transformation in Ewing's sarcoma.
EWS/FLI is a master regulator of Ewing's sarcoma formation. Gene expression studies in A673 Ewing's sarcoma cells have demonstrated that EWS/FLI downregulates more genes than it upregulates, suggesting that EWS/FLI, and/or its targets, function as transcriptional repressors. One critical EWS/FLI target, NKX2.2, is a transcription factor that contains both transcriptional activation and transcriptional repression domains, raising the possibility that it mediates portions of the EWS/FLI transcriptional signature. We now report that microarray analysis demonstrated that the transcriptional profile of NKX2.2 consists solely of downregulated genes, and overlaps with the EWS/FLI downregulated signature, suggesting that NKX2.2 mediates oncogenic transformation via transcriptional repression. Structure-function analysis revealed that the DNA binding and repressor domains in NKX2.2 are required for oncogenesis in Ewing's sarcoma cells, while the transcriptional activation domain is completely dispensable. Furthermore, blockade of TLE or HDAC function, two protein families thought to mediate the repressive function of NKX2.2, inhibited the transformed phenotype and reversed the NKX2.2 transcriptional profile in Ewing's sarcoma cells. Whole genome localization studies (ChIP-chip) revealed that a significant portion of the NKX2.2-repressed gene expression signature was directly mediated by NKX2.2 binding. These data demonstrate that the transcriptional repressive function of NKX2.2 is necessary, and sufficient, for the oncogenic phenotype of Ewing's sarcoma, and suggest a therapeutic approach to this disease.
Owen
Leah A
LA
Department of Oncological Sciences, University of Utah, Salt Lake City, Utah, United States of America.
Kowalewski
Ashley A
AA
Lessnick
Stephen L
SL
D000818:Animals; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008954:Models, Biological; D009687:Nuclear Proteins; D020411:Oligonucleotide Array Sequence Analysis; D015514:Oncogene Proteins, Fusion / Q000378:metabolism / Q000502:physiology*; D010641:Phenotype; D017434:Protein Structure, Tertiary; D051789:Proto-Oncogene Protein c-fli-1; D034802:RNA-Binding Protein EWS; D012512:Sarcoma, Ewing / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism* / Q000502:physiology*; D014158:Transcription, Genetic; D029961:Zebrafish Proteins
C106031:EWS-FLI fusion protein; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; C000715631:NKX2-2 protein, human; C000715627:Nkx2-2 protein, mouse; D009687:Nuclear Proteins; D015514:Oncogene Proteins, Fusion; D051789:Proto-Oncogene Protein c-fli-1; D034802:RNA-Binding Protein EWS; D014157:Transcription Factors; D029961:Zebrafish Proteins; C000715630:nkx2.2b protein, zebrafish