Mutation and downregulation of Mig6 are often observed in human lung cancer cell lines and also correlate with a reduced survival rate in breast cancer patients. Secondly, ubiquitin dependent EGFR degradation mediated by Cbl is enhanced in the L858R cells. Both of besides these two characteristics seem to contribute to the negative regulation of the EGFR signaling pathway. However, no mechanistic explanation has been found for the contributions of these molecules to the gefitinib sen sitivity of the L858R mutation. Recent studies showed that dynamics and regulation of the intracellular signaling cascades are efficiently elu cidated with an assistance of computational simulations. To obtain a logical understanding of the gefiti nib sensitivity associated with L858R mutation, the mathematical analysis Inhibitors,Modulators,Libraries of the EGFR signaling pathway should be more preferable rather than sole experimental representations.

In this study, we used experimental and computational approaches to investigate regulatory mechanisms that dis tinguish cell specific Inhibitors,Modulators,Libraries gefitinib sensitivity in H1299 human NSCLC cell lines. We have modified the existing kinetic model of Inhibitors,Modulators,Libraries the EGFR signaling pathway and built new mod els for H1299 wild type, H1299 with overexpressed wild type EGFR, and H1299 overexpressing the EGFR with L858R mutation. The three types of cells showed different signaling dynamics in response to EGF stimulation. Over expression of wild type EGFR induced high and sustained phosphorylation of EGFR, Shc, MEK and ERK, while the L858R mutation reduced these response levels.

In addition, H1299L858R, which is sup posed to be more sensitive to gefitinib than H1299EGFR WT, was effectively inhibited by Inhibitors,Modulators,Libraries gefitinib administration at the downstream part of the signaling pathway compared with H1299EGFR WT, but, surprisingly, not at the upstream part of the pathway. The model incorporated Mig6, Inhibitors,Modulators,Libraries but not Cbl overexpres sion, successfully captured the signaling behavior observed in our experimental data, implying that Mig6 is responsi ble for enhancing gefitinib sensitivity. Detailed computa tional analyses revealed that Mig6 amplifies gefitinib sensitivity at the steps of MEK phosphorylation depho sphorylation and ERK phosphorylation dephosphorylation. We experimentally verified that overexpression of Mig6 contributed to suppressing cellular growth in the presence of gefitinib.

Our analysis further suggested that the combi nation of Mig6 and gefitinib exhibits a synergistic effect in inhibiting EGFR signaling pathway. Methods Cell culture H1299 human lung cancer derivatives, H1299WT, H1299EGFR WT and H1299L858R, were sellckchem established as described elsewhere. Cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum and 1 mM sodium pyruvate. Prior to growth hormone treatment, the cells were serum starved for 16 24 hours.