Figure 5

SU11274 inhibition of MET in combination with EGFR inhibitor in erlotinib-resistant NSCLC cell signalling. (A) Potentiated inhibition of cellular cytoskeletal functions by combined MET–EGFR inhibition (SU11274 plus erlotinib) in H1975 cells under video microscopy. H1975 cells had constitutively activated cytoskeletal functions with enhanced cell motility and migration under the serum-starved culture conditions. Comparing with the untreated control (left panel), drug treatment using SU11274 (right panel) substantially inhibited the constitutively activated cytoskeletal functions of H1975 cells (Supplementary Figure 3). H1975 cells were cultured in serum-stimulated conditions (10% FBS) and treated with the following for video microscopy digital video recording as described in the Materials and Methods: (a) DMSO diluent control, (b) Erlotinib alone (2 μ M), (c) SU11275 alone (5 μ M), and (d) combined concurrent SU11274 (5 μ M)+erlotinib (2 μ M). Complete abrogation of cytoskeletal functions with inhibition of cell motility and migration was only evident in the dual SU11274/erlotinib TKI-treated cells (d). (B) MET inhibition using SU11274, in combination with EGFR inhibition (erlotinib), induced cooperative downstream signalling inhibition in A549 (left panel) and H1975 (right panel) cells in vitro. EGFR-TKI-resistant A549 and H1975 cells were cultured in serum-starved conditions with EGF and HGF dual ligands stimulation as described in the Materials and Methods section. The cells were treated with SU11274 alone, erlotinib alone, or combination SU11274 plus erlotinib, then analysed in immunoblotting as indicated. (C) MET inhibition using specific siRNA-MET, in combination with EGFR inhibition (erlotinib) induced cooperative downstream signalling inhibition in A549 cells in vitro. Cells were transfected with control siRNA or siRNA-MET as described in Methods. Forty-eight hours after transfection, cells were cultured in starved media overnight, then treated with or without erlotinib and alone or in combination with siRNA-MET as indicated. After 4 h of inhibitor treatment, cells were then stimulated with both EGF (100 ng ml⁻¹) and HGF (50 ng ml⁻¹) ligands as indicated for 15 min. Whole cell lysates were then collected for immunoblotting analysis as in panel B above. (D) Rescue from alternative RTK ligand-stimulated signalling (MET–HGF vs EGFR–EGF) against TKI in A549 cells. A549 cells were cultured under serum-starved conditions, and then treated with either HGF or EGF, and in the presence or absence of the corresponding targeted inhibitor SU11274 or erlotinib as indicated (lanes 2, 4). Dual ligand stimulation (HGF and EGF) with single or dual TKIs treatment was included as indicated (lanes 1, 3, 5, 6). Although receptor-specific TKI was able to inhibit the downstream signalling driven by the corresponding ligand stimulation, alternative ligand stimulation in the form of dual ligand stimulation rescued the inhibited downstream signals. Dual TKI SU11274 plus erlotinib inhibition was required to fully knockdown the dual ligand-stimulated downstream signal activation of AKT, ERK1/2, and STAT3 (lane 6). (E) MET inhibition with SU11274, in combination with EGFR inhibition using CL-387,785 (irreversible EGFR-TKI), induced cooperative downstream signalling inhibition in H1975 cells in vitro. Cells were cultured in starved media overnight, then treated with or without CL-387,785 and alone or in combination with MET inhibitor SU11274 as indicated. After 4 h of inhibitor treatment, cells were then stimulated with both EGF (100 ng ml⁻¹) and HGF (50 ng ml⁻¹) ligands as indicated for 15 min. Whole cell lysates were then collected for immunoblotting analysis. Similar to erlotinib, CL-387,785 further sensitised H1975 cells to SU11274 inhibition with enhanced cooperative inhibition of signalling pathways downstream of the two RTKs.