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The role of the insulin-like growth factor signaling axis in ETV6-NTRK3- mediated anchorage-independent growth and transformation Martin, Matthew Joseph


The insulin-like growth factor signaling axis is an important regulator of normal cell growth and proliferation, and is frequently dysregulated in cancer. Most dominantly acting oncoproteins tested to date require the type-1 insulin-like growth factor receptor (IGF-IR) for cellular transformation. ETV6-NTRK3 (EN) - the product of a t(12;15)(p13;q25) translocation that occurs in human pediatric spindle cell sarcomas and secretory breast carcinoma - is one such protein, as it fails to transform IGF-IR-null fibroblasts. EN binds and tyrosine phosphorylates the insulin-receptor substrate (IRS)-1, a major substrate of IGF-IR, which links EN to activation of the PI3K/Akt survival pathway and the Ras/Erk proliferative cascade. Here we show that EN specifically interacts with the phosphotyrosine-binding (PTB) domain of IRS-1, and in the absence of IRS-1 can bind its closely related homolog IRS-2. Disruption of these EN«IRS interactions through overexpression of the IRS-1 PTB domain, or depleting the cell of IRS-1 and IRS-2 together, inhibit EN-induced anchorage-independent growth. Further, we find that IGF-IR, through its ability to bind IRS molecules, serves to localize EN to the plasma membrane, which leads to acivation of the PI3K/Akt pathway. Nontransformed IGF-IR-null fibroblasts fail to activate this pathway in response to EN expression when plated under anchorage-indpendent conditions, and undergo detachment-induced cell death. Chemical inhibition of PI3K, or its downstream effector mTOR, significantly impairs EN-mediated transformation. Finally, I demonstrate that EN expression induces the ligand-independent tyrosine phosphorylation of IGF-IR. Both IGF-IR IRS-binding and kinase activity are required for this phenomenon, and IGF-IR mutants lacking either function do not display EN-mediated PI3K/Akt activation or subsequent oncogenesis. These observations point to EN as a regulator of a novel multicomponent membrane-localized signaling complex which potently stimulates the PI3K/Akt survival cascade, and they suggest that blocking the formation or activity of this complex would be a promising way to target EN-expressing tumors in vivo.

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