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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

Variability of EWS-ERG expression in mouse MPCs.A) Western blot analysis: three independent MPCs populations were infected with human ERG-1 (lanes 2, 5 and 8), EWS-ERG (lanes 3, 6 and 9) or an empty pMSCV puro vector (lanes 1,4 and 7). After confirming ERG-1 and EWS-ERG expression at the protein level 36 hours following infection (data not shown), cells were selected with 1.5 µg/ml puromycin for 14 days, and protein expression was re-assessed using a mouse anti v5 epitope mAb and a goat anti mouse-HRP conjugated antibody. Monoclonal mouse anti-actin was used as a loading control. (B) RT-PCR analysis of EWS-ERG infected MPC populations. RNA was extracted from puromycin-selected MPCs populations expressing (lanes 1 and 6) or not (lanes 2 and 4) EWS-ERG protein and from puromycin-selected MPC populations infected with an empty pMSCV puro vector (lanes 3 and 7). RT-PCR was performed using primers complementary to the 5′end of EWS and at the 3′end of ERG. A control lacking RT was included (lane 5) as well as a mouse β-actin amplification RNA control. (C) Genomic DNA analysis of EWS-ERG infected MPC populations. Lanes1 and 3: puromycin-selected MPC populations lacking EWS/ ERG protein (A lane 3) and showing degraded EWS-ERG RNA (B lane 4). Lanes 2 and 4: puromycin-selected MPC populations infected with an empty pMSCV puro vector. PCR was performed using primers either at the 5′end of EWS and at the 3′end of ERG-1 (lanes 3 and 4) or primers located on the pMSCV vector (lanes 1 and 2). D) Genomic DNA analysis of FUS-ERG infected MPCs populations. Genomic DNA was extracted from puromycin-selected MPCs populations infected with ESFT-associated FUS-ERG (lane 3), AML-associated FUS-ERG (lane 2) or an empty pMSCV puro vector (lane 1). PCR was performed using primers complementary to the 5′end of EWS and to the 3′end of FUS.
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pone-0002634-g004: Variability of EWS-ERG expression in mouse MPCs.A) Western blot analysis: three independent MPCs populations were infected with human ERG-1 (lanes 2, 5 and 8), EWS-ERG (lanes 3, 6 and 9) or an empty pMSCV puro vector (lanes 1,4 and 7). After confirming ERG-1 and EWS-ERG expression at the protein level 36 hours following infection (data not shown), cells were selected with 1.5 µg/ml puromycin for 14 days, and protein expression was re-assessed using a mouse anti v5 epitope mAb and a goat anti mouse-HRP conjugated antibody. Monoclonal mouse anti-actin was used as a loading control. (B) RT-PCR analysis of EWS-ERG infected MPC populations. RNA was extracted from puromycin-selected MPCs populations expressing (lanes 1 and 6) or not (lanes 2 and 4) EWS-ERG protein and from puromycin-selected MPC populations infected with an empty pMSCV puro vector (lanes 3 and 7). RT-PCR was performed using primers complementary to the 5′end of EWS and at the 3′end of ERG. A control lacking RT was included (lane 5) as well as a mouse β-actin amplification RNA control. (C) Genomic DNA analysis of EWS-ERG infected MPC populations. Lanes1 and 3: puromycin-selected MPC populations lacking EWS/ ERG protein (A lane 3) and showing degraded EWS-ERG RNA (B lane 4). Lanes 2 and 4: puromycin-selected MPC populations infected with an empty pMSCV puro vector. PCR was performed using primers either at the 5′end of EWS and at the 3′end of ERG-1 (lanes 3 and 4) or primers located on the pMSCV vector (lanes 1 and 2). D) Genomic DNA analysis of FUS-ERG infected MPCs populations. Genomic DNA was extracted from puromycin-selected MPCs populations infected with ESFT-associated FUS-ERG (lane 3), AML-associated FUS-ERG (lane 2) or an empty pMSCV puro vector (lane 1). PCR was performed using primers complementary to the 5′end of EWS and to the 3′end of FUS.

Mentions: Expression of the fusion and wild type proteins was tested by Western blot analysis. All of the fusion proteins as well as FLI-1 and ERG-1 were readily detected 36 hours following infection (Figure 3A). Densitometric analysis revealed that EWS-FLI-1, EWS-ERG and the ESFT-associated FUS-ERG isoform were expressed at comparable levels, whereas the DBDM, FLI-1, ERG-1 and the AML-associated FUS-ERG isoform displayed a 1.5 to 2.5 fold higher expression level (Figure S1). Expression of wild type FLI-1 and ERG-1, as well as that of the DBDM was stably maintained by MPCs infected with the corresponding viruses (Figure 3 B); EWS-FLI-1 protein expression was also stably maintained by MPCs, but at a 4-, 2- and 7 fold lower level than the DBDM and the FLI-1 and ERG-1 proteins, respectively, as assessed by densitometric analysis (Figure 3 B and Figure S1). Comparable expression levels of each of these proteins were observed in independently infected cells derived from the same primary population and in different MPC populations (data not shown). However, the same MPC populations displayed variable ability to maintain EWS-ERG expression and were unable to stably express either of the two FUS-ERG fusion proteins (Figure 4 and data not shown). The EWS-ERG fusion protein was maintained beyond 14 days of culture and selection in only one out of three MPC populations tested (figure 4A, lanes 3, 6, 9). Both genomic DNA and total RNA from cells that had lost EWS-ERG protein expression tested positive for the presence of EWS-ERG by PCR or RT-PCR using EWS-ERG specific primers. In some samples, RT-PCR analysis showed that loss of protein expression was accompanied by non-random EWS-ERG RNA processing (figure 4B, lane 4), reflected by the presence of two distinct transcripts of about 250 and 500 bp. PCR-amplification using primers complementary to the 5′end of EWS and the 3′end of ERG and subsequent sequence analysis of the amplicons revealed that the lower band was composed of the initial 5′ 175 bases of EWS fused to the 3′ 57 bases of ERG and the V5 tag. The higher band consisted of the 5′ 333 coding bases of EWS fused to the 3′ 139 coding bases of ERG and the V5 tag. In both cases the Ewing's sarcoma breakpoint was lost and translation showed no open reading frame (data not shown). No deamination was observed within the sequence suggesting the absence of RNA editing, and no point mutations were found in the EWS/ERG sequence.


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)

Variability of EWS-ERG expression in mouse MPCs.A) Western blot analysis: three independent MPCs populations were infected with human ERG-1 (lanes 2, 5 and 8), EWS-ERG (lanes 3, 6 and 9) or an empty pMSCV puro vector (lanes 1,4 and 7). After confirming ERG-1 and EWS-ERG expression at the protein level 36 hours following infection (data not shown), cells were selected with 1.5 µg/ml puromycin for 14 days, and protein expression was re-assessed using a mouse anti v5 epitope mAb and a goat anti mouse-HRP conjugated antibody. Monoclonal mouse anti-actin was used as a loading control. (B) RT-PCR analysis of EWS-ERG infected MPC populations. RNA was extracted from puromycin-selected MPCs populations expressing (lanes 1 and 6) or not (lanes 2 and 4) EWS-ERG protein and from puromycin-selected MPC populations infected with an empty pMSCV puro vector (lanes 3 and 7). RT-PCR was performed using primers complementary to the 5′end of EWS and at the 3′end of ERG. A control lacking RT was included (lane 5) as well as a mouse β-actin amplification RNA control. (C) Genomic DNA analysis of EWS-ERG infected MPC populations. Lanes1 and 3: puromycin-selected MPC populations lacking EWS/ ERG protein (A lane 3) and showing degraded EWS-ERG RNA (B lane 4). Lanes 2 and 4: puromycin-selected MPC populations infected with an empty pMSCV puro vector. PCR was performed using primers either at the 5′end of EWS and at the 3′end of ERG-1 (lanes 3 and 4) or primers located on the pMSCV vector (lanes 1 and 2). D) Genomic DNA analysis of FUS-ERG infected MPCs populations. Genomic DNA was extracted from puromycin-selected MPCs populations infected with ESFT-associated FUS-ERG (lane 3), AML-associated FUS-ERG (lane 2) or an empty pMSCV puro vector (lane 1). PCR was performed using primers complementary to the 5′end of EWS and to the 3′end of FUS.
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Related In: Results  -  Collection

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pone-0002634-g004: Variability of EWS-ERG expression in mouse MPCs.A) Western blot analysis: three independent MPCs populations were infected with human ERG-1 (lanes 2, 5 and 8), EWS-ERG (lanes 3, 6 and 9) or an empty pMSCV puro vector (lanes 1,4 and 7). After confirming ERG-1 and EWS-ERG expression at the protein level 36 hours following infection (data not shown), cells were selected with 1.5 µg/ml puromycin for 14 days, and protein expression was re-assessed using a mouse anti v5 epitope mAb and a goat anti mouse-HRP conjugated antibody. Monoclonal mouse anti-actin was used as a loading control. (B) RT-PCR analysis of EWS-ERG infected MPC populations. RNA was extracted from puromycin-selected MPCs populations expressing (lanes 1 and 6) or not (lanes 2 and 4) EWS-ERG protein and from puromycin-selected MPC populations infected with an empty pMSCV puro vector (lanes 3 and 7). RT-PCR was performed using primers complementary to the 5′end of EWS and at the 3′end of ERG. A control lacking RT was included (lane 5) as well as a mouse β-actin amplification RNA control. (C) Genomic DNA analysis of EWS-ERG infected MPC populations. Lanes1 and 3: puromycin-selected MPC populations lacking EWS/ ERG protein (A lane 3) and showing degraded EWS-ERG RNA (B lane 4). Lanes 2 and 4: puromycin-selected MPC populations infected with an empty pMSCV puro vector. PCR was performed using primers either at the 5′end of EWS and at the 3′end of ERG-1 (lanes 3 and 4) or primers located on the pMSCV vector (lanes 1 and 2). D) Genomic DNA analysis of FUS-ERG infected MPCs populations. Genomic DNA was extracted from puromycin-selected MPCs populations infected with ESFT-associated FUS-ERG (lane 3), AML-associated FUS-ERG (lane 2) or an empty pMSCV puro vector (lane 1). PCR was performed using primers complementary to the 5′end of EWS and to the 3′end of FUS.
Mentions: Expression of the fusion and wild type proteins was tested by Western blot analysis. All of the fusion proteins as well as FLI-1 and ERG-1 were readily detected 36 hours following infection (Figure 3A). Densitometric analysis revealed that EWS-FLI-1, EWS-ERG and the ESFT-associated FUS-ERG isoform were expressed at comparable levels, whereas the DBDM, FLI-1, ERG-1 and the AML-associated FUS-ERG isoform displayed a 1.5 to 2.5 fold higher expression level (Figure S1). Expression of wild type FLI-1 and ERG-1, as well as that of the DBDM was stably maintained by MPCs infected with the corresponding viruses (Figure 3 B); EWS-FLI-1 protein expression was also stably maintained by MPCs, but at a 4-, 2- and 7 fold lower level than the DBDM and the FLI-1 and ERG-1 proteins, respectively, as assessed by densitometric analysis (Figure 3 B and Figure S1). Comparable expression levels of each of these proteins were observed in independently infected cells derived from the same primary population and in different MPC populations (data not shown). However, the same MPC populations displayed variable ability to maintain EWS-ERG expression and were unable to stably express either of the two FUS-ERG fusion proteins (Figure 4 and data not shown). The EWS-ERG fusion protein was maintained beyond 14 days of culture and selection in only one out of three MPC populations tested (figure 4A, lanes 3, 6, 9). Both genomic DNA and total RNA from cells that had lost EWS-ERG protein expression tested positive for the presence of EWS-ERG by PCR or RT-PCR using EWS-ERG specific primers. In some samples, RT-PCR analysis showed that loss of protein expression was accompanied by non-random EWS-ERG RNA processing (figure 4B, lane 4), reflected by the presence of two distinct transcripts of about 250 and 500 bp. PCR-amplification using primers complementary to the 5′end of EWS and the 3′end of ERG and subsequent sequence analysis of the amplicons revealed that the lower band was composed of the initial 5′ 175 bases of EWS fused to the 3′ 57 bases of ERG and the V5 tag. The higher band consisted of the 5′ 333 coding bases of EWS fused to the 3′ 139 coding bases of ERG and the V5 tag. In both cases the Ewing's sarcoma breakpoint was lost and translation showed no open reading frame (data not shown). No deamination was observed within the sequence suggesting the absence of RNA editing, and no point mutations were found in the EWS/ERG sequence.

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