INTEGRATION OF MODELING AND EXPERIMENTS TO DEFINE PRINCIPLES OF EGFR ACTIVATION AND UBIQUITINATION

Epidermal growth factor receptor (EGFR)-dependent signaling is involved in numerous physiological processes, and its deregulation leads to cellular dysfunctions and pathologies, first and forecast, cancer. Endocytosis has a crucial impact on the downstream EGFR signaling response and it is regulated by ligand concentration. Indeed, depending on the EGF dose, the EGFR can be internalized through clathrin-mediated endocytosis (CME) or non-clathrin endocytosis (NCE). The switch between these two internalization mechanisms occurs over a narrow range of EGF concentrations (1-10 ng/ml). Importantly, EGFR ubiquitination shows a threshold response over the same range of EGF doses and is responsible for the commitment of EGFR to NCE, and thus, for EGFR signal extinction through receptor degradation. In this project, we were interested in elucidating the cellular mechanisms that regulate and coordinate the choice between these two endocytic routes, in addition, we aim to clarify how the integration of the two pathways influences EGFR downstream signaling. In order to deal with the complexity of the system, we adopted an integrated research approach combining mathematical modeling with wet-lab experiments. To this purpose, in collaboration with the Systems Biology group at our Institute, we developed a mathematical model of early EGFR activation that quantitatively accounts for the ubiquitination threshold observed at 2 minutes of EGF stimulation. The ‘early model’ was able to generate important predictions; in particular, it predicts a weakness in the system that is unveiled in the presence of high EGF concentrations and EGFR overexpression, two conditions frequently observed in cancer. We tested these predictions using different cell-based model systems subjected to varying perturbations. A challenge in the biological validation of the model, was obtaining quantitative reproducible data. To this aim, we optimized a quantitative ELISA-based assay to measure EGFR ubiquitination/phosphorylation upon different perturbations. This assay revealed to be powerful and allowed us to validate the predictions generated by the model. Thanks to our integrative approach, we identified Cbl as the limiting and weak element of the system. We expect that our model of EGFR activation will provide novel insights into the role of EGFR endocytosis, controlling the balance between EGFR signaling and downmodulation, frequently altered in cancer.