Abstract
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.
Highlights
Ewing’s sarcoma is an aggressive bone and soft tissue tumor of adolescents and young adults [1]
We recently demonstrated that expression of the transcription factor NKX2.2 is upregulated by EWS/FLI in Ewing’s sarcoma and is required for the oncogenic phenotype of the disease [8,15,16,17]
To determine the mechanism by which NKX2.2 contributes to oncogenic transformation in Ewing’s sarcoma, we analyzed a series of NKX2.2 mutants (Figure 1A) using a ‘‘knockdown/ rescue’’ approach
Summary
Ewing’s sarcoma is an aggressive bone and soft tissue tumor of adolescents and young adults [1]. EWS/FLI expression is necessary for the oncogenic phenotype of Ewing’s sarcoma cells, and is sufficient to mediate oncogenic transformation of heterologous NIH3T3 cells [6,7,8]. These data suggest that EWS/ FLI functions as a transcriptional activator to mediate oncogenesis in Ewing’s sarcoma.
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