investigating integrin ανβ6 activation status in breast cancer
Abstract
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.
Authors
Sproat, CarolineCollections
- Theses [4143]