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The role of the insulin-like growth factor pathway in ETV6-NTRK3-mediated transformation Morrison, Kevin B.


Congenital fibrosarcoma (CFS) and cellular congenital mesoblastic nephroma (CMN) are pediatric spindle cell malignancies that are characterized by two specific cytogenetic abnormalities: trisomy of chromosome 11 and a t(12;15)(pl3;q25) rearrangement. The t(12;15) translocation creates a transcriptionally active fusion gene that encodes a chimeric protein, ETV6-NTRK3 (EN). This fusion oncogene transforms NIH3T3 fibroblasts through constitutive activation of the Ras-MAP kinase (MAPK) pathway. In this study we demonstrate that trisomy 11 correlates with high expression of insulin-like growth factor-II (IGF-II) in CFS and CMN tumor samples using Northern blotting. We provide evidence that an intact IGF signaling axis is essential for EN-mediated transformation by expression of EN in IGF-I receptor (IGF-IR) knockout murine fibroblasts. In the absence of IGF-IR, EN is only partially transforming as measured by colony formation in soft agar. We have investigated the signaling pathways activated by IGF-IR and the EN fusion protein in NIH3T3 and IGF-IR knockout murine fibroblasts (R-cells) and we have found significant overlap and interdependence between these two pathways. Along with constitutive activation of the Ras-MAPK pathway, EN transformation is also dependent on constitutive activation of the phosphatidylinositol-3' kinase (PI-3K)-Akt survival pathway as demonstrated through the use of the specific PI-3K blocking agents, LY294002 and wortmannin, that revert transformation. The levels of activation of these key transformation pathways are significantly reduced in the absence of IGF-IR. In the presence and absence of IGF-IR, however, EN appears to interact with the major downstream substrate of IGF-IR, the insulin-receptor substrate-1 (IRS-1), as demonstrated by co-immunoprecipitation experiments. IRS-1 appears to be a substrate for the kinase domain of EN as it is constitutively tyrosine-phosphorylated in the presence and absence of IGF-IR in EN expressing cells. Also, IRS-1 serves as a docking site for recruitment of Grb2 and p85 which are key elements involved in the activation of the PI-3K-Akt and Ras-MAPK pathways. Despite its ability to activate IRS-1, EN fails to confer a fully transformed phenotype to IGF-IR knockout cells. We speculate that IGF-IR may allow maximal activation of the PI-3K-Akt and MAPK pathways through direct binding and activation of upstream elements of these pathways independent of IRS-1 or that IGF-IR plays a role in translocating activated IRS-1 and EN to the inner cell membrane where they can act efficiently in a subcellular compartment enriched for upstream regulatory elements of the PI-3K-Akt and MAPK pathways.

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