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.
No description available.
Alterations to the tumour microenvironment is a common feature of many cancers, including breast cancer, and there is increasing evidence that alterations to the microenvironment, including; increased integrin expression, ECM deposition and protease activity, promote cancer progression. Most invasive breast cancers arise from a preinvasive stage, ductal carcinoma in situ (DCIS). Previous work in our laboratory has shown the microenvironment of DCIS is altered, such that myoepithelial cells (MECs) switch to a tumour-promoting phenotype, associated with upregulation of integrin αvβ6 and fibronectin (FN) expression. Mechanisms by which integrin αvβ6 and FN expression are regulated is unclear. We show DCIS progression into invasion is accompanied by an increase in MEC expression of integrin αvβ6 and periductal FN deposition, and their expression were associated in DCIS. These findings were modelled in isolated primary DCIS-MECs, primary normal MECs and MEC lines, with and without integrin αvβ6 expression, where integrin αvβ6-positive MECs upregulating FN expression. We identified integrin αvβ6-positive DCIS ducts were larger than integrin αvβ6-negative DCIS ducts, and mechanical stretching of primary normal MECs and a normal MEC line led to upregulation of integrin αvβ6 expression and FN deposition in a TGFβ-dependent manner. We further show upregulation of integrin αvβ6 and FN by MECs mediate TGFβ-dependent upregulation of MMP13 which promotes breast cancer cell invasion in vitro. These data show altered tissue mechanics in DCIS and MEC expression of integrin αvβ6 and FN deposition are linked, and implicate TGFβ in their activation. These findings suggest integrin αvβ6 and FN may be used as markers to stratify DCIS patients.
Lomas, Andrew Philip
Lactate Dehydrogenase-A (LDH-A) is up-regulated in a broad array of cancers and is associated with poor prognosis. Involved in the hypoxic response, LDH-A is a HIF-1 target and is responsible for the enzymatic reduction of pyruvate to lactate. This is important for several reasons, chiefly (1) the regeneration of NAD+ which feeds back into earlier glycolytic stages and (2) the depletion of intracellular pyruvate concentrations. High intracellular pyruvate is known to inhibit HDACs and is associated with increased apoptosis. LDH-A is also known to be controlled by oncogenes such as c-Myc suggesting an oncogenic role. Studies have shown that the knock-out of LDH-A reduces proliferation and tumourgenicity, and stimulates the mitochondria. This thesis therefore had three aims: firstly, to validate LDH-A inhibition and elucidate its full nature in terms of the implications for tumour survival; secondly, to ascertain the role of LDH-B in order to determine whether selectivity towards LDH-A would be a necessary feature of any small molecule; lastly, to recapitulate siRNA mediated LDH-A inhibition with small molecule inhibitors that had the potential for clinical application. The thesis examined both clinical data and a broad panel of cultured cancer cell types in order to select appropriate model in which to validate siRNA mediated inhibition of LDH-A and LDH-B. After it was demonstrated that LDH-A inhibition reduced the growth of cultured cells, a range of techniques were used to quantify this reduced growth in terms of cell death and changes in metabolism. Further to this, literature studies had proposed a role for LDH-B in maintaining lactate fuelled tumour growth; however, this thesis shows that in the cell lines studied, lactate-fuelled tumour growth was an LDH-A dependent phenomenon. Finally, a high throughput assay system was designed and validated and a library of small molecules was selected, synthesized, and screened in order to identify selective inhibitors of LDH-A.
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