4 research outputs found
investigating integrin ανβ6 activation status in breast cancer
PhDbackground
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.Cancer Research UK
Integrin αVβ6-EGFR crosstalk regulates bidirectional force transmission and controls breast cancer invasion
The mechanical properties of the extracellular matrix within tumours control multiple cellular functions that drive cancer invasion and metastasis. However, the mechanisms controlling microenvironmental force sensation and transmission, and how these regulate transcriptional reprogramming and invasion, are unclear. Our aim was to understand how mechanical inputs are transmitted bidirectionally and translated into biochemical and transcriptional outputs to drive breast cancer progression. We reveal that adhesion receptor and growth factor receptor crosstalk regulates a bidirectional feedback mechanism co-ordinating force-dependent transcriptional regulation and invasion. Integrin αVβ6 drives invasion in a range of carcinomas and is a potential therapeutic target. αVβ6 exhibits unique biophysical properties that promote force-generation and increase matrix rigidity. We employed an inter-disciplinary approach incorporating proteomics, biophysical techniques and multi-modal live-cell imaging to dissect the role of αVβ6-EGFR crosstalk on transmission of mechanical signals bidirectionally between the extracellular matrix and nucleus. We show that αVβ6 expression correlates with poor prognosis in triple-negative breast cancer (TNBC) and drives invasion of TNBC cells. Moreover, our data show that a complex regulatory mechanism exists involving crosstalk between αVβ6 integrin and EGFR that impacts matrix stiffness, force transmission to the nucleus, transcriptional reprogramming and microenvironment rigidity. αVβ6 engagement triggers EGFR & MAPK signalling and αVβ6-EGFR crosstalk regulates mutual receptor trafficking mechanisms. Consequently, EGF stimulation suppresses αVβ6-mediated force-application on the matrix and nuclear shuttling of force-dependent transcriptional co-activators YAP/TAZ. Finally, we show that crosstalk between αVβ6 & EGFR regulates TNBC invasion. We propose a model whereby αVβ6-EGFR crosstalk regulates matrix stiffening, but also the transmission of extracellular forces into the cell in order to co-ordinate transcriptional reprogramming and invasion. To exploit adhesion receptors and receptor tyrosine kinases therapeutically, it will be essential to understand the integration of their signalling functions and how crosstalk mechanisms influence invasion and the response of tumours to molecular therapeutics