Flourescent Biosensor-Based, Cdc42 Activity Imaging For Understanding the Regulation of Epidermal Growth Receptor (EGFR) Signalling in Head and Neck Cancer
The Epidermal Growth Factor Receptor (EGFR) is overexpressed in several solid tumours including squamous cell carcinoma of the head & neck (SCCHN). Drugs that directly block the action of EGFR are currently available. However, a major unanswered question is; how best to select patients most likely to respond to these new treatments, as the response rate to EGFR-targeted mono-therapy in SCCHN, as well as other solid tumours, such as lung and colorectal cancer, is very low.Resistance to EGFR therapy may stem from aberrant receptor trafficking. Cdc42 is known to affect EGFR downregulation by sequestering c-Cbl, preventing it from catalyzing receptor ubiquitination. The aim of this project is to determine the role of Cdc42 in mediating cancer cell response to EGFR and ErbB-family targeted therapy.Using optical imaging techniques such as, Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FUM) I have analysed Cdc42 activity in Squamous Carcinoma Cell Lines after EGFR tyrosine kinase inhibitor (TKI) treatment, using the FRET biosensor Raichu-Cdc42. I have demonstrated that conversely, Raichu-Cdc42, and consequently endogenous Cdc42 activity increases significantly following EGFR TKI treatment. Further investigation revealed that the serine/threonine kinase, c-Jun NH2 Terminal Kinase 1 (JNK1) may modulate Cdc42 activity via a negative feedback mechanism, and JNK1 in turn regulates EGFR ubiquitination and downregulation. I have also demonstrated for the first time protein-protein interactions in pathological SCCHN tissue between members of the ErbB receptor family (EGFR and HER2) and between PKCa and Ezrin, using FRET and FUM. The novel results from this thesis provides further knowledge on factors influencing EGFR downregulation in SCCHN, that could account for the resistance to EGFR targeted therapies observed in a clinical setting. In addition the optical proteomic assays could be translated into new diagnostic/predictive tests, potentially allowing us to improve the outcome for head and neck cancer patients.
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