Proteomic and phosphoproteomic analysis of signalling by adhesion and growth factor receptors in mammary epithelial cells

Abstract

Cell adhesion and communication are essential for tissue morphogenesis and repair in healthy multicellular organisms. However, dysregulation of these processes can drive disease progression in conditions such as cancer. Selective cell adhesion to the extracellular matrix is mediated by integrins, a family of transmembrane receptors that compartmentalise signalling and organise the cytoskeleton. Adhesion receptors provide spatial cues to cells to allow them to respond to growth factor and cytokine signals from the tissue microenvironment. This is of key importance during epithelial morphogenesis of the mammary gland. Polarised mammary epithelial cells (MECs) receive adhesion signals from the basement membrane and growth factor signals from the surrounding stroma. This combination of signalling drives cell fate processes such as migration, proliferation, differentiation and apoptosis. The ErbB family of growth factor receptors (GFRs), which includes EGFR, have key roles during both mammary development and breast carcinoma progression. However, the specific signalling circuitry downstream of adhesion and GFRs has yet to be determined. Using a global, un-biased proteomics approach this study determined the composition of MEC adhesion complexes and elucidated how connections between signalling proteins were altered upon growth factor stimulation. A methodology was generated for the specific enrichment of adhesion complexes from MECs following EGF treatment. This was coupled to a quantitative mass spectrometry analysis workflow and bioinformatic protein-protein interaction network and Gene Ontology analyses to identify EGF-induced changes to isolated adhesion complexes. Analysis of adhesion complexes isolated from EpH4 MECs revealed a decrease in the abundance of adhesion proteins 5 minutes after EGF stimulation. In addition, phosphoproteomic analysis of isolated adhesion complexes revealed differential phosphorylation of adhesion proteins upon EGF treatment. Together with network and Gene Ontology analyses, these data suggested a mechanism of EGF induced receptor endocytosis and adhesion complex turnover, which may regulate cell migration. Network analysis of EGFR and β1-integrin co-interacting proteins identified Eps8 as a possible candidate regulator of receptor internalisation and cytoskeletal remodelling in MECs. Current knockdown studies are addressing whether Eps8 and its homologue Eps8l2 are functionally involved in these processes. This study presents the first proteomic analysis of isolated adhesion complexes in epithelial cells, and the first proteomic analysis of adhesion-GFR crosstalk in MECs. Further work should provide insights into the altered interaction between adhesion and GFR signalling in breast cancer progression.

Date of Award	1 Aug 2014
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Martin Humphries (Supervisor) & Charles Streuli (Supervisor)