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Title: investigating integrin ανβ6 activation status in breast cancer
Author: Sproat, Caroline
ISNI:       0000 0004 7653 4658
Awarding Body: Queen Mary University of London
Current Institution: Queen Mary, University of London
Date of Award: 2017
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background The extracellular matrix receptor integrin ανβ6 is known to potentiate breast cancer (BrCa) cell invasion, metastasis and tumour-trophic growth factor receptor crosstalk during tumourigenesis. Monoclonal antibody blockade of ανβ6 diminishes invasion in vitro and arrests BrCa tumour growth and metastasis in vivo. Aberrant integrin activation status has been implicated in progression to metastatic disease in BrCa; with differential internalisation and endocytic trafficking kinetics reported for active versus inactive integrin species in malignant disease. Despite its emerging potential for targeted therapy, little is known regarding regulation of integrin ανβ6-mediated activation and signalling during progression to an invasive, metastatic state. It is hypothesised that the aetiopathological significance of integrin ανβ6 during neoplastic transformation and malignant progression in BrCa is dependent specifically upon its activation status and associated conformation, since this active state will permit establishment of known integrin-mediated oncogenic signalling underpinning acquisition of a malignant phenotype, including activation of invasion and metastasis. results Canonical integrin activation studies using divalent cations and cognate ligand stimulation indicated antibodies 6.2E5 and 6.2G2 recognise activation-associated epitopes, which are also ligand-induced binding sites (LIBS) in live-labelled cells by FCM and IMF. However, their utility to discriminate the active fraction distinct from the total or inactive fractions of ανβ6 by IHC in primary BrCa samples could not be robustly established. Evaluation of the 6.2E5 and 6.2G2 epitopes in the MCF10 isogenic model revealed that relative surface abundance of these active epitopes determined by FCM was not significantly altered; but their subcellular redistribution upon neoplastic transformation and malignant progression was observed by IMF, implicating derailed internalisation and trafficking of active ανβ6 during breast tumourigenesis and metastatic disease progression. Proteomic interrogation and network analysis of the 2D-enriched adhesion assays identified 7 novel putative molecular regulators of a ligand-engaged, activated ανβ6-mediated adhesion environment (DMBT-1, MARCKS, MXRA5, SEPT6, SEPT9, MYH9, MYH10) in the BT-20 TNBC cell line. Functional validation of these candidate mediators of the "β6 adhesome" by siRNA strategies was not achieved due to inconsistent stable knockdown. Phosphoproteomic definition of LAP ligand-engaged, active ανβ6-mediated signalling ("β6 kinome") during receptor-ligand internalisation revealed EGFR-dependency for downstream ERK1/2 signal activation in BT-20 and SUM159, but not MDA-MB-468 TNBC cells. Kinase substrate enrichment analysis (KSEA) identified 5 novel putative mediators of downstream ανβ6 signalling (COT, MAPKAPK2, PDPK1, Nuak1, TBK1) and implicated Akt1 isoform-specific activation downstream of ανβ6-LAP internalisation. Following LAP-induced ανβ6 activation and internalisation, EGFR underwent phosphorylation at multiple known activation sites, including a residue (Thr693) critical for EGFR receptor internalisation; suggesting integrin ανβ6-EGFR reciprocity during respective receptor activation and internalisation. conclusion The active conformer of integrin ανβ6 may be studied using antibodies 6.2E5 and 6.2G2 in live-labelled cells by FCM and IMF. Subcellular redistribution of activation-associated epitopes during BrCa progression implicates derailed internalisation and intracellular trafficking kinetics of active ανβ6 during tumourigenesis, while protein expression studies identified 7 putative molecular regulators of ligand-engaged, active ανβ6-mediated adhesion. Integrin ανβ6-mediated signalling during internalisation revealed an ανβ6-EGFRAkt1 signalling axis during breast tumourigenesis and disease progression, while further understanding of integrin biology and growth factor receptor crosstalk may provide additional rationale for potential combination therapies in breast cancer.
Supervisor: Not available Sponsor: Cancer Research UK
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Breast cancer ; Tumour Biology