ic-87114 and Cell-Transformation--Neoplastic

PI3-kinase has a crucial role in transformation mediated by the oncogenic c-Kit mutant D816V. In this study, we demonstrate that the c-Kit/D816V-mediated cell survival is dependent on an intact direct binding of PI3-kinase to c-Kit. However, mutation of this binding site had little effect on the PI3-kinase activity in the cells, suggesting that c-Kit/D816V-mediated cell survival is dependent on PI3-kinase but not its kinase activity. Furthermore, inhibition of the lipid kinase activity of PI3-kinase led only to a slight inhibition of cell survival. Knockdown of the predominant PI3-kinase isoform p110δ in c-Kit/D816V-expressing Ba/F3 cells led to reduced cell transformation both in vitro and in vivo without affecting the overall PI3-kinase activity. This suggests that p110δ has a lipid-kinase-independent role in c-Kit/D816V-mediated cell transformation. We furthermore demonstrate that p110δ is phosphorylated at residues Y524 and S1039 and that phosphorylation requires an intact binding site for PI3-kinase in c-Kit/D816V. Overexpression of p110δ carrying the Y523F and S1038A mutations significantly reduced c-Kit/D816V-mediated cell survival and proliferation. Taken together, our results demonstrate an important lipid-kinase-independent role of p110δ in c-Kit/D816V-mediated cell transformation. This furthermore suggests that p110δ could be a potential diagnostic factor and selective therapeutic target for c-Kit/D816V-expressing malignancies.

Topics: Adenine; Animals; Binding Sites; Blotting, Western; Cell Line; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Chlorocebus aethiops; Chromones; Class Ia Phosphatidylinositol 3-Kinase; COS Cells; Female; Mice, Inbred Strains; Mice, Nude; Morpholines; Mutation, Missense; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Binding; Proto-Oncogene Proteins c-kit; Quinazolines; RNA Interference; Serine; Tyrosine

Cancer-specific mutations in the iSH2 (inter-SH2) and nSH2 (N-terminal SH2) domains of p85alpha, the regulatory subunit of phosphatidylinositide 3-kinase (PI3K), show gain of function. They induce oncogenic cellular transformation, stimulate cellular proliferation, and enhance PI3K signaling. Quantitative determinations of oncogenic activity reveal large differences between individual mutants of p85alpha. The mutant proteins are still able to bind to the catalytic subunits p110alpha and p110beta. Studies with isoform-specific inhibitors of p110 suggest that expression of p85 mutants in fibroblasts leads exclusively to an activation of p110alpha, and p110alpha is the sole mediator of p85 mutant-induced oncogenic transformation. The characteristics of the p85 mutants are in agreement with the hypothesis that the mutations weaken an inhibitory interaction between p85alpha and p110alpha while preserving the stabilizing interaction between p85alpha iSH2 and the adapter-binding domain of p110alpha.

Topics: Adenine; Amino Acid Sequence; Animals; Base Sequence; Blotting, Western; Catalytic Domain; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Class I Phosphatidylinositol 3-Kinases; Dioxoles; Fibroblasts; Humans; Immunoprecipitation; Morpholines; Mutation; Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Binding; Protein Subunits; Pyrimidinones; Quinazolines; Thiazolidinediones; Transfection