First name
Alexia
Last name
Conte
Year of Study
Research Center
Thesis Title
Integration of modeling and experiments to define principles of EGFR activation and ubiquitination under physiological and pathological conditions
Thesis Abstract
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 clathrinmediated
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.
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 clathrinmediated
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.
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