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

Owen LA, Kowalewski AA, Lessnick SL - PLoS ONE (2008)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncological Sciences, University of Utah, Salt Lake City, Utah, United States of America.

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.

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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.
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pone-0001965-g005: 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.

Mentions: TLE family members mediate transcriptional repression, in part, through recruitment of histone deacetylases (HDACs; ref. 23). Indeed, HDACs are recruited to NKX2.2-dependent promoters involved in oligodendrocyte development [24]. To determine if NKX2.2-mediated oncogenic transformation requires HDAC function, we used a clinically-approved HDAC inhibitor, vorinostat (Zolinza™, formerly suberoylanilide hydroxamic acid, SAHA), in transformation assays. Inclusion of vorinostat effectively prevented Ewing's sarcoma cell growth in tissue culture, and colony formation in soft-agar, in a dose-dependent manner (Figure 5A–5B). These data complement recently published work demonstrating that multiple HDAC inhibitors are effective against Ewing's sarcoma in preclinical studies including murine xenografts [25], [26], [27].


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

Owen LA, Kowalewski AA, Lessnick SL - PLoS ONE (2008)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2291578&req=5

pone-0001965-g005: 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.
Mentions: TLE family members mediate transcriptional repression, in part, through recruitment of histone deacetylases (HDACs; ref. 23). Indeed, HDACs are recruited to NKX2.2-dependent promoters involved in oligodendrocyte development [24]. To determine if NKX2.2-mediated oncogenic transformation requires HDAC function, we used a clinically-approved HDAC inhibitor, vorinostat (Zolinza™, formerly suberoylanilide hydroxamic acid, SAHA), in transformation assays. Inclusion of vorinostat effectively prevented Ewing's sarcoma cell growth in tissue culture, and colony formation in soft-agar, in a dose-dependent manner (Figure 5A–5B). These data complement recently published work demonstrating that multiple HDAC inhibitors are effective against Ewing's sarcoma in preclinical studies including murine xenografts [25], [26], [27].

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncological Sciences, University of Utah, Salt Lake City, Utah, United States of America.

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.

Show MeSH
Related in: MedlinePlus