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IGF1 is a common target gene of Ewing's sarcoma fusion proteins in mesenchymal progenitor cells.

Cironi L, Riggi N, Provero P, Wolf N, Suvà ML, Suvà D, Kindler V, Stamenkovic I - PLoS ONE (2008)

Bottom Line: Whereas all MPC isolates tested could stably express EWS-FLI-1, only some sustained stable EWS-ERG expression and none could express FUS-ERG for more than 3-5 days.However, all three fusion proteins, but neither FLI-1 nor ERG-1 alone, activated the IGF1 promoter and induced IGF1 expression.Whereas expression of different ESFT-associated fusion proteins may require distinct cellular microenvironments and induce transcriptome changes of limited similarity, IGF1 induction may provide one common mechanism for their implication in ESFT pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Pathology, Institute of Pathology CHUV, University of Lausanne, Lausanne, Switzerland.

ABSTRACT

Background: The EWS-FLI-1 fusion protein is associated with 85-90% of Ewing's sarcoma family tumors (ESFT), the remaining 10-15% of cases expressing chimeric genes encoding EWS or FUS fused to one of several ets transcription factor family members, including ERG-1, FEV, ETV1 and ETV6. ESFT are dependent on insulin-like growth factor-1 (IGF-1) for growth and survival and recent evidence suggests that mesenchymal progenitor/stem cells constitute a candidate ESFT origin.

Methodology/principal findings: To address the functional relatedness between ESFT-associated fusion proteins, we compared mouse progenitor cell (MPC) permissiveness for EWS-FLI-1, EWS-ERG and FUS-ERG expression and assessed the corresponding expression profile changes. Whereas all MPC isolates tested could stably express EWS-FLI-1, only some sustained stable EWS-ERG expression and none could express FUS-ERG for more than 3-5 days. Only 14% and 4% of the total number of genes that were respectively induced and repressed in MPCs by the three fusion proteins were shared. However, all three fusion proteins, but neither FLI-1 nor ERG-1 alone, activated the IGF1 promoter and induced IGF1 expression.

Conclusion/significance: Whereas expression of different ESFT-associated fusion proteins may require distinct cellular microenvironments and induce transcriptome changes of limited similarity, IGF1 induction may provide one common mechanism for their implication in ESFT pathogenesis.

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Related in: MedlinePlus

Schematic representation of IGF1 promoter fragments cloned into the pGL3 basic vector (A) and the corresponding luciferase activity (B) in human mesenchymal stem cells expressing EWS-FLI-1.Restriction sites used for cloning are indicated. The exon 1 start codon, putative promoter regions GXP79580 and GXP641910 according to GEM analysis (Genomatix) are shown. Levels of luciferase activity, calculated as in Figure 6, are normalized to values obtained with the –1682 construct which was arbitrarily set to 100%. Error bars represent the S.E.M. of three independent experiments.
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pone-0002634-g008: Schematic representation of IGF1 promoter fragments cloned into the pGL3 basic vector (A) and the corresponding luciferase activity (B) in human mesenchymal stem cells expressing EWS-FLI-1.Restriction sites used for cloning are indicated. The exon 1 start codon, putative promoter regions GXP79580 and GXP641910 according to GEM analysis (Genomatix) are shown. Levels of luciferase activity, calculated as in Figure 6, are normalized to values obtained with the –1682 construct which was arbitrarily set to 100%. Error bars represent the S.E.M. of three independent experiments.

Mentions: GEMS analysis [22], used to search for transcription factor binding sites (MatInspector) and sequence models (ModelInspector), of the –1682 to +1 region of the human IGF-1 promoter, revealed the presence of potential binding sites for several different transcription factors, including ets family members, supporting the possibility of direct interaction of Ewing's sarcoma fusion proteins with the IGF-1 promoter. Comparative Genomix analysis, on the other hand, showed no conserved putative ets binding sites in this region, whereas other transcription factor binding sites, such as RFX1, were found to be conserved. To define the relevance of the 2 putative promoter regions GXP79580 and GXP641910, identified by Genomatix analysis, 3 additional luciferase fusion plasmids were generated by restriction enzyme cleavage and ligation to the pGL3 vector (Figure 8 A). Constructs –1098pGL3 and –913pGL3, generated respectively by XhoI/HindIII, and KpnI/HindIII digestion, included the entire GXP79580 promoter, a short sequence of GXP641910 being included in the –1098pGL3 construct. The –673pGL3 plasmid, obtained by AvaI/HindIII digestion, contained only part of the GXP79580 promoter.


IGF1 is a common target gene of Ewing's sarcoma fusion proteins in mesenchymal progenitor cells.

Cironi L, Riggi N, Provero P, Wolf N, Suvà ML, Suvà D, Kindler V, Stamenkovic I - PLoS ONE (2008)

Schematic representation of IGF1 promoter fragments cloned into the pGL3 basic vector (A) and the corresponding luciferase activity (B) in human mesenchymal stem cells expressing EWS-FLI-1.Restriction sites used for cloning are indicated. The exon 1 start codon, putative promoter regions GXP79580 and GXP641910 according to GEM analysis (Genomatix) are shown. Levels of luciferase activity, calculated as in Figure 6, are normalized to values obtained with the –1682 construct which was arbitrarily set to 100%. Error bars represent the S.E.M. of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2481291&req=5

pone-0002634-g008: Schematic representation of IGF1 promoter fragments cloned into the pGL3 basic vector (A) and the corresponding luciferase activity (B) in human mesenchymal stem cells expressing EWS-FLI-1.Restriction sites used for cloning are indicated. The exon 1 start codon, putative promoter regions GXP79580 and GXP641910 according to GEM analysis (Genomatix) are shown. Levels of luciferase activity, calculated as in Figure 6, are normalized to values obtained with the –1682 construct which was arbitrarily set to 100%. Error bars represent the S.E.M. of three independent experiments.
Mentions: GEMS analysis [22], used to search for transcription factor binding sites (MatInspector) and sequence models (ModelInspector), of the –1682 to +1 region of the human IGF-1 promoter, revealed the presence of potential binding sites for several different transcription factors, including ets family members, supporting the possibility of direct interaction of Ewing's sarcoma fusion proteins with the IGF-1 promoter. Comparative Genomix analysis, on the other hand, showed no conserved putative ets binding sites in this region, whereas other transcription factor binding sites, such as RFX1, were found to be conserved. To define the relevance of the 2 putative promoter regions GXP79580 and GXP641910, identified by Genomatix analysis, 3 additional luciferase fusion plasmids were generated by restriction enzyme cleavage and ligation to the pGL3 vector (Figure 8 A). Constructs –1098pGL3 and –913pGL3, generated respectively by XhoI/HindIII, and KpnI/HindIII digestion, included the entire GXP79580 promoter, a short sequence of GXP641910 being included in the –1098pGL3 construct. The –673pGL3 plasmid, obtained by AvaI/HindIII digestion, contained only part of the GXP79580 promoter.

Bottom Line: Whereas all MPC isolates tested could stably express EWS-FLI-1, only some sustained stable EWS-ERG expression and none could express FUS-ERG for more than 3-5 days.However, all three fusion proteins, but neither FLI-1 nor ERG-1 alone, activated the IGF1 promoter and induced IGF1 expression.Whereas expression of different ESFT-associated fusion proteins may require distinct cellular microenvironments and induce transcriptome changes of limited similarity, IGF1 induction may provide one common mechanism for their implication in ESFT pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Pathology, Institute of Pathology CHUV, University of Lausanne, Lausanne, Switzerland.

ABSTRACT

Background: The EWS-FLI-1 fusion protein is associated with 85-90% of Ewing's sarcoma family tumors (ESFT), the remaining 10-15% of cases expressing chimeric genes encoding EWS or FUS fused to one of several ets transcription factor family members, including ERG-1, FEV, ETV1 and ETV6. ESFT are dependent on insulin-like growth factor-1 (IGF-1) for growth and survival and recent evidence suggests that mesenchymal progenitor/stem cells constitute a candidate ESFT origin.

Methodology/principal findings: To address the functional relatedness between ESFT-associated fusion proteins, we compared mouse progenitor cell (MPC) permissiveness for EWS-FLI-1, EWS-ERG and FUS-ERG expression and assessed the corresponding expression profile changes. Whereas all MPC isolates tested could stably express EWS-FLI-1, only some sustained stable EWS-ERG expression and none could express FUS-ERG for more than 3-5 days. Only 14% and 4% of the total number of genes that were respectively induced and repressed in MPCs by the three fusion proteins were shared. However, all three fusion proteins, but neither FLI-1 nor ERG-1 alone, activated the IGF1 promoter and induced IGF1 expression.

Conclusion/significance: Whereas expression of different ESFT-associated fusion proteins may require distinct cellular microenvironments and induce transcriptome changes of limited similarity, IGF1 induction may provide one common mechanism for their implication in ESFT pathogenesis.

Show MeSH
Related in: MedlinePlus