AbstractThe epidermal growth factor receptor (EGFR) plays a pivotal role in cellular proliferation, differentiation and migration and is found to be overexpressed in a variety of tumours including the basal-like subtype of breast cancer. Despite decades of basic research in the HER signalling field, and many EGFR-targeted anti-cancer drugs are used clinically, the success rate for these agents is low, particularly in terms of improvement of overall survival. In breast cancer, the clinical response rates vary between clinical studies, ranging from 6-49%, in part, depending on the type of breast cancer, the stage of the disease, and the treatment used. These marginal benefits are mainly due to insufficient patient selection, and potentially also due to an inadequate inhibition of HER signalling and tumour escape via alternate signalling routes (such as HER2/3/4/c-Met).
The first objective of this work was to use an in-house semi-automated protein activity sensing/imaging technique, to interrogate the EGFR-centric subnetwork of proteins, in order to obtain better understanding of molecular factors influencing the therapeutic sensitivity of the EGFR network.
To monitor EGFR activity in cells we use a CrkII-based biosensor (CrkII protein flanking with eGFP and mRFP1), which undergoes conformational changes upon phosphorylation of tyrosine-221 by EGFR and other receptor tyrosine kinase (RTK)s including c-Met and PDGFβR. This changes are detected by fluorescence lifetime imaging microscopy (FLIM) combined with fluorescence resonance energy transfer (FRET). Using the sensor, we have conducted a high-content FLIM screen utilising a library of short interfering (si)RNA consist of 533 genes interconnected with EGFR directly or indirectly, chosen from the Human Protein Reference Database. Genes that modulate the EGF-induced biosensor conformational changes were identified. Including EGFR, we identified 22 possible targets (4.1% from the siRNA library) and have selected a few for further validation to establish the biological importance of the genes identified as primary hits.
The same biosensor was also transfected using a liposome-based delivery system into murine models of basal-like breast cancer; and demonstrated a significant degree of intratumoural heterogeneity in EGFR activity, as well as the effect of EGFR inhibitors in situ.
In the long term, the knowledge obtained in this thesis can potentially be translated to biomarkers that are based on a better understanding of tumour cell sensitivity to EGFR inhibition and may thereby suggest new combination treatments.
|Date of Award||2013|
|Supervisor||Tony Ng (Supervisor) & Cheryl Gillett (Supervisor)|