Figure 4

Signalling cross-activation between MET and EGFR signalling pathways. (A) Cross-activation between MET and EGFR signalling in lung cancer cells, A549 and H1975. A549 or H1975 cells were cultured in serum-starved conditions with exogenous stimulation with RTK ligands: EGF alone, HGF alone, or both EGF and HGF. Cells without any ligand stimulation were included as control. Both MET and EGFR signalling pathways are functional and ligand-sensitive in A549 and H1975 cells. There was augmented downstream signalling with combined EGF-HGF co-stimulation, with also more durable signalling induction. In A549 cells, although HGF alone did not activate EGFR phosphorylation appreciably, under co-stimulation conditions with EGF together, HGF further enhanced the EGFR phosphorylation in A549 cells to a level higher than that with EGF alone. On the other hand, EGF stimulation of H1975 cells co-activated MET receptor to enhance the level of MET phosphorylation. (B) MET–EGFR cross-activation in lung cancer. Left panel (A549), HGF cross-activated p-EGFR in A549 cells in the presence of co-stimulation with EGF. A549 cells were cultured in serum-starved conditions overnight, then stimulated with EGF alone (100 ng ml⁻¹, 15 min), HGF alone (50 ng/ml, 15 min), or both. Whole cell lysates were collected for immunoprecipitation with EGFR antibody, followed by immunoblotting (WB) with antibodies against p-EGFR[Y1068] (upper panel) and total EGFR (lower panel). Right panel (H1975), EGF cross-activates phospho-MET in H1975 cells. H1975 cells were cultured in starved media overnight, then stimulated with EGF alone (100 ng ml⁻¹, 15 min), HGF alone (50 ng ml⁻¹, 15 min), or both. Whole cell lysates were collected for immunoprecipitation with MET antibody (C-12), followed by immunoblotting with antibodies against p-MET[Y1234/1235] (upper panel) and total MET (lower panel). The MET and EGFR genotypes of the A549 and H1975 cells, as well as their MET genomic copy numbers, are shown in the bottom.