Eleanor Cawthorne
undergraduate degree in Biochemistry with a Research Placement at The University of Huddersfield in 2012
Title: : NON-CLASSICAL SH3-DOMAIN MEDIATED SIGNALLING THROUGH THE VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR IN BREAST CANCER
Biography
Biography: Eleanor Cawthorne
Abstract
Introduction: In the absence of ligand stimulation, we have shown that a second tier of signalling is initiated by interactions between the SH3 domains of intracellular kinases and the proline rich motifs of receptor tyrosine kinases (RTKs). When this signalling is perturbed it can lead to pathological outcomes. This secondary tier of signalling has been shown to contribute to poorer outcomes with FGFR2 in ovarian cancer but it has not been widely explored for other RTKs or other types of cancers. One RTK of interest is VEGFR2, which is overexpressed in many cancer types including breast cancer (BC). VEGFR2 is a RTK that regulates angiogenesis, proliferation, survival and migration in response to the ligand VEGF-A. Inside a tumour mass, RTKs on the surface of cancer cells will have limited access to extracellular stimuli, including VEGF-A. Here, someof the response of VEGFR2 may be through these nonclassical interactions with non-activated RTKs and could possibly be used to aid in cancer progression and survival. Objective: To determine whether VEGFR2:SH3- domain-containing protein interactions occur under serum starved conditions in breast cancer, and to assess their impact on cancer cell behaviour. Methods: VEGFR2 expression levels were investigated in BC cell lines (SKBR3 and MCF7 cells) and HEK 293T cells as a control non-cancerous cell line under serum starved conditions for 24 hours. Protein-protein interactions (PPIs) were determined by endogenous Co-IP and via pull down of VEGFR2 constructs containing mutated SH3-binding sites in HEK 293T cells (low expression of endogenous VEGFR2), SKBR3 and MCF7 (high expression of endogenous VEGFR2) cells grown under serum starved conditions and visualised by immunoblotting. Protein expression and purification of the C-terminal tail of VEGFR2, containing only the c-terminal proline rich motif, was used in a pull down in cancer cell lysates and PPIs visualised by immunoblotting.