Worthington, J; (2013) Functional characterisation of targets of ErbB2-dependent signalling in breast cancer. Doctoral thesis , UCL (University College London).

Abstract

Amplification of the tyrosine kinase receptor ErbB2 in breast cancer correlates with disease progression, poor prognosis and recurrence. The mechanisms of downstream ErbB2 signalling and their effects on tumour progression remain ambiguous and thus the elucidation of pathways involved in ErbB2-dependent transformation is essential to realise novel diagnostic/prognostic markers and therapeutic targets. Previous profiling studies of an ErbB2 over-expressing cell system and tumour tissues identified gene products potentially implicated in ErbB2-dependent breast cancer. Candidate proteins were selected from the previous profiling experiments with respect to their relationship with ErbB2 over-expression and/or growth factor-dependent modulation. Particular interest was placed on poorly characterised proteins that were previously unknown to function in ErbB2-dependent malignancy. The protein expression profiles of selected candidates were established in a panel of HMLEC and breast cancer cell lines. Previous findings from the gene expression profiling studies, including the role of the candidate IGFBP3 in growth factor receptor interplay were validated at the protein level in a breast cancer cell line and/or an ErbB2 over-expressing cell system. Validated targets were functionally characterised by evaluating the effect of siRNA-mediated silencing of candidate expression on cellular invasion, proliferation, anchorage-independent growth and adhesion in ErbB2 over-expressing breast cancer cell lines. Prospective candidate interaction partners were identified by determining the effect of siRNA-dependent gene silencing on global protein expression using an in vivo quantitative mass labelling approach (SILAC) combined with LC-MS/MS technology. Bioinformatics analysis of data was used to define the functional consequences of siRNA-mediated knockdown and to link changes with cellular phenotype. The effects of ErbB2 knockdown were also evaluated to validate its role in cellular transformation and malignancy. Finally, novel downstream ErbB2 signalling targets and putative sites of phosphorylation were determined using a combination of SILAC labelling, phosphopeptide enrichment, LC-MS/MS and bioinformatics analysis in an ErbB2 over-expressing model cell system.

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