Investigation of novel receptor tyrosine kinase mediated non-canonical signalling in breast cancer

Introduction: It has been discovered by our laboratory group that a novel cell signalling mechanism (Tier 2 signalling) can occur via receptor tyrosine kinases and SH3 domain-containing proteins when cellular environmental conditions are perturbed. Aberrant Tier 2 signalling in response to stress-induced changes to protein expression is implicated in tumorigenesis and is physiologically relevant to tumour cells under microenvironmental stress. The role of aberrant receptor tyrosine kinases expression in breast cancer pathogenesis and the high levels of anti-receptor tyrosine kinase resistance, suggest that aberrant Tier 2 signalling may play a role in breast cancer progression and drug-resistance. Objectives: To investigate whether overexpression or upregulated expression of receptor tyrosine kinases and SH3 domain-containing proteins in response to growth factor deprivation leads to the non-canonical activation of SH3 domain containing proteins in breast cancer cells. The possible aberrant downstream Tier 2 signalling mechanisms which could lead to an oncogenic outcome in breast cancer cells, was also examined. Methods: Relative cell protein levels were determined via immunoblotting. Various protein-protein interaction methods were utilised, including the use of Co-IP, pull-downs and FRET, to validate candidate non-canonical protein-protein interactions. Phenotypic cellular assays were performed to determine the phenotypic consequence of non-canonical activation of SH3 domain-containing proteins. Results: The work presented here demonstrates that breast cancer cells change their proteome in response to growth under serum-deprivation stress. A particular non-canonical protein-protein interaction was established to occur between the second proline-rich motif of the receptor tyrosine kinase VEGFR2 and the SH3-domain of Src-kinase in breast cancer cells, under serum-deprived conditions. The VEGFR2-mediated non-canonical activation of Src was observed to play a role in breast cancer cell migration. Conclusions: The data within this thesis supports a novel Tier 2 signalling pathway in breast cancer cells, which is upregulated in response to growth under serum-deprived stress and may be implicated in breast cancer cell migration. This aberrant Tier 2 mechanism may play an important role in breast cancer progression and resistance mechanisms, when under serum-deprived microenvironmental stress.