Src triggers ligand-independent EGFR signalling and trafficking

Src and EGFR are co-overexρressed or co-overactivated in ~ 70 % of breast cancers. This suggests that the two families of tyrosine kinases (TKs) may physically and functionally interact to promote breast cancer development. Thus, understanding the structure and functions of these TKs, and elucidation of their signalling and potential synergy, are important for designing new strategies to inhibit their action in pathological conditions. It is well known that multiple extracellular signals, including EGFR ligands, can activate Src which integrates these informations into cell survival, cell motility and cell proliferation. However, whether the opposite is also true, i.e. whether Src can activate EGFR and mimic its natural ligands, remains largely unknown. The general aim of this thesis is to better understand the role of Src and EGFR activation and signalling in epithelial cancer progression. The experimental strategy exploits MDCK epithelial cells bearing a thermosensitive v-Src mutant and expressing endogenous EGFR. We first analysed the mechanism of v-Src membrane association upon thermoactivation and especially its recruitment in raft domains. We found that thermo-activated v- Src showed dual membrane recruitment at non-raft domains (on endosomes?) and at plasma membrane lipid rafts. We further addressed whether this differential Src recruitment might specify its downstream signalling by analysing the two main arms of Src signalling, the MAPK and Akt pathways. We demonstrated that activation of v-Src led to sequential signalling, first via MAPK presumably at non-raft domains, then via Akt on a distinct set of rafts. Having shown that v-Src was able to be recruited to, and to signal at, plasma membrane rafts, we next investigated whether v-Src thermo-activation was sufficient to interact with EGFR and to trigger its signalling and endocytosis. We found that the effects of Src activation on EGFR mimic those of its natural ligand, EGF, including (i) EGFR phosphorylation of signalling tyrosine residues at the cell surface and subsequent Akt and Erk pathways; (ii) EGFR endocytosis via clathrin-coated pits; and (iii) sequestration in perinuclear/recycling endosomes with avoidance of Hiultivesicular bodies (MVB) and lysosomal degradation. Moreover, Src and EGFR were synergistic at signalling endosomes. In conclusion, we have established that ligand-independent Src activation can mimic and synergize EGF effects, including signalling to the Akt and ERK pathways. In many cases of human cancer, not only EGFR but also Src is overexpressed and activated without any mutation. Thus, SrciEGFR interactions at the cell surface could be sufficient to cause sustained mitogenic EGFR activation, even without ligand exposure and without requiring EGFR overexpression.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL