EWS/FLI mediates transcriptional repression via NKX2.2 during oncogenic transformation in Ewing's sarcoma

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.

Figure 1. Transcriptional repression and DNA binding domains are required for NKX2.2-mediated Ewing's sarcoma cell oncogenic transformation.

(A) Schematic of wild type and 3x-FLAG tagged NKX2.2 constructs. The positions of the transcriptional repressor domain (TN), the homeodomain (HD), the NK2-specific domain (SD), and the transcriptional activation domain (TAD) are shown. (B) Soft agar colony formation of A673 cells infected with the indicated RNAi and cDNA constructs. Error bars indicate standard deviations of duplicate assays. (C) Western blot analysis of A673 cells infected with the indicated RNAi and cDNA constructs, using anti-NKX2.2 antibody (to determine the expression of endogenous NKX2.2 following knockdown using the NKX-RNAi construct), anti-FLAG (to assess the expression of cDNA constructs), or anti-tubulin (as a loading control). The positions of endogenous NKX2.2 (end. NKX2.2) and of the exogenous NKX2.2 constructs (exog. NKX2.2) are indicated.

Figure 2. The TN and HD domains are sufficient to rescue NKX2.2-mediated Ewing's sarcoma oncogenic transformation.

(A) Schematic diagram of the 3xFLAG-tagged TN-HD fusion protein. (B) Soft agar colony formation of A673 cells infected with the indicated RNAi and cDNA constructs demonstrate that the TN-HD fusion rescues oncogenic transformation as efficiently as wild-type (wt) NKX2.2. Error bars indicate standard deviations of duplicate assays. (C) Western blot analysis of A673 cells infected with either control luc-RNAi, or NKX-RNAi, constructs, and the TN-HD fusion, or wild-type NKX2.2, using anti-NKX2.2 antibody (to determine the expression of endogenous NKX2.2 following knockdown using the NKX-RNAi construct), anti-FLAG (to assess the expression of cDNA constructs), or anti-tubulin (as a loading control).

Figure 3. NKX2.2 downregulated target genes are enriched in the EWS/FLI downregulated dataset.

(A) Venn diagram representation of the overlap between EWS/FLI downregulated genes and NKX2.2 downregulated genes. The Chi square-determined p-value is indicated. (B) Gene set enrichment analysis (GSEA) using EWS/FLI-regulated genes as the rank-ordered dataset and NKX2.2 downregulated genes as the geneset. EWS/FLI-regulated genes are shown on the x-axis, with upregulated genes toward the left, and downregulated genes toward the right. The positions of genes that were downregulated by NKX2.2 are indicated by the black vertical lines in the center portion of the panel. Ranking metric scores are shown in the bottom portion of the panel. The normalized enrichment score (NES) and p-value are shown.

Figure 4. Role of TLE corepressors in NKX2.2-mediated gene repression.

(A) Western blot showing retroviral-mediated expression of dominant-negative TLE (AES) in A673 Ewing's sarcoma cells as compared to an empty vector control (“none”). Tubulin is shown as a loading control. (B) Tissue culture growth of A673 Ewing's sarcoma cells expressing AES was unchanged as compared to empty vector control bearing cells (“none”). These data are a representative example from multiple experimental replicates. (C) Colony formation of A673 Ewing's sarcoma cells in soft agar was inhibited by AES expression, as compared to empty vector control infected cells (“none”). Error bars indicate standard deviations of duplicate assays.

Figure 5. HDACs mediate repression of NKX2.2 targets.

(A) Inclusion of the indicated concentrations of vorinostat inhibited the short-term tissue culture growth of A673 Ewing's sarcoma cells in a dose-dependent manner. The OD595 of crystal violet staining is shown on the y-axis, and is proportional to cell number. (B) Addition of the indicated concentrations of the HDAC inhibitor vorinostat caused a dose-dependent decrease in soft agar colony formation of A673 Ewing's sarcoma cells. Error bars indicate standard deviations of duplicate assays. (C) 0.6 µM of vorinostat is sufficient to cause an increase of acetylated histone H4 in A673 Ewing's sarcoma cells, as observed by Western blot, as compared to vehicle control. Total histone H4 levels are shown as a loading control. (D) Gene set enrichment analysis (GSEA) using vorinostat-regulated genes in A673 Ewing's sarcoma cells as the rank-ordered dataset and NKX2.2 downregulated genes as the geneset. Vorinostat-regulated genes are shown on the x-axis, with upregulated genes toward the left, and downregulated genes toward the right. The positions of genes that were downregulated by NKX2.2 are indicated by the black vertical lines in the center portion of the panel. Ranking metric scores are shown in the bottom portion of the panel. The normalized enrichment score (NES) and p-value are shown. (E) Gene set enrichment analysis (GSEA) using vorinostat-regulated genes in A673 Ewing's sarcoma cells as the rank-ordered dataset and the 72 genes downregulated by both EWS/FLI and NKX2.2 as the geneset. The normalized enrichment score (NES) and p-value are shown. (F) Gene set enrichment analysis (GSEA) using NKX2.2-regulated genes as the rank-ordered dataset and NKX2.2 ChIP-chip targets as the geneset. The normalized enrichment score (NES) and p-value are shown.