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Integrin α9β1 promotes malignant tumor growth and metastasis by potentiating epithelial-mesenchymal transition.

Gupta SK, Oommen S, Aubry MC, Williams BP, Vlahakis NE - Oncogene (2012)

Bottom Line: In addition, we found that α9β1 forms a tri-partite protein complex with β-catenin and E-cadherin, which dissociates following integrin activation and subsequent src and β-catenin phosphorylation.These in vitro results are biologically significant as α9β1-expressing cancer cells induce greater tumor growth and metastases in mice as compared to the cells without α9β1 expression or when integrin expression is suppressed.Furthermore, integrin α9β1 is expressed in primary human small cell lung cancer and patients having a high expression of α9β1 demonstrated significantly worse long-term survival compared with patients with low α9β1 expression.

View Article: PubMed Central - PubMed

Affiliation: Thoracic Disease Research Unit, Division of Pulmonary & Critical Care Medicine, Mayo Clinic, Rochester 55905, MN, USA.

ABSTRACT
The integrin α9β1 binds a number of extracellular matrix components to mediate cell adhesion, migration and tissue invasion. Although expressed in a variety of normal human cells including endothelium, it is also expressed in cancer cells. We have previously shown that α9β1 binds VEGF-A to facilitate angiogenesis, an important component of the tumor microenvironment. As α9β1 induces accelerated cancer cell migration, we wished to determine what role it played in cancer growth and metastasis. In this study, we show that α9β1 expression induces molecular changes consistent with epithelial-mesenchymal transition. In addition, we found that α9β1 forms a tri-partite protein complex with β-catenin and E-cadherin, which dissociates following integrin activation and subsequent src and β-catenin phosphorylation. These findings were consistent in cells in which: α9β1 was exogenously over-expressed, or when its expression was suppressed in cancer cells endogenously expressing α9β1. These in vitro results are biologically significant as α9β1-expressing cancer cells induce greater tumor growth and metastases in mice as compared to the cells without α9β1 expression or when integrin expression is suppressed. Furthermore, integrin α9β1 is expressed in primary human small cell lung cancer and patients having a high expression of α9β1 demonstrated significantly worse long-term survival compared with patients with low α9β1 expression. These findings highlight a novel mechanism of integrin α9β1 function in human cancer.

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α9β1 co-associates with E-cadherin and β-catenin inducing EMT through src signalingA, Immunoblots for α9 or src from immunoprecipitates of SW480 cells cultured with or without the α9β1 specific ligand, TnfnRAA and/or the src inhibitor, PP1. B, Immunoblots for β1, α9 or Src from immunoprecipitates of matrix activated SW-480-mock (pFN) or SW480-α9 cells (TnfnRAA). C, Immunoblots for α9, Src or β-catenin in lysates from SW480-mock or α9 cells grown with pFN or TnfnRAA and/or PP1. D, Confocal images of SW480-α9 cells to determine co-localization of α9β1 (red) with β-catenin (green, top panel) or E-cadherin (green, bottom panel); nuclei stained blue with DAPI. E, Immunoblots, of immunoprecipitates from SW480-α9 grown on plastic (no matrix) or TnfnRAA (right panel), to determine co-association of α9 with β-catenin or E-cadherin. F, Left panel, Immunoblots for β-catenin, src, E-cadherin or vimentin in lysates from SW480-α9 cells transfected with non-targeted siRNA or siRNA targeted to src or β-catenin; Right panel: Migration (left), invasion (middle) or proliferation (right) assays in SW480-α9 cells following transfection with siRNA as indicated.
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Figure 2: α9β1 co-associates with E-cadherin and β-catenin inducing EMT through src signalingA, Immunoblots for α9 or src from immunoprecipitates of SW480 cells cultured with or without the α9β1 specific ligand, TnfnRAA and/or the src inhibitor, PP1. B, Immunoblots for β1, α9 or Src from immunoprecipitates of matrix activated SW-480-mock (pFN) or SW480-α9 cells (TnfnRAA). C, Immunoblots for α9, Src or β-catenin in lysates from SW480-mock or α9 cells grown with pFN or TnfnRAA and/or PP1. D, Confocal images of SW480-α9 cells to determine co-localization of α9β1 (red) with β-catenin (green, top panel) or E-cadherin (green, bottom panel); nuclei stained blue with DAPI. E, Immunoblots, of immunoprecipitates from SW480-α9 grown on plastic (no matrix) or TnfnRAA (right panel), to determine co-association of α9 with β-catenin or E-cadherin. F, Left panel, Immunoblots for β-catenin, src, E-cadherin or vimentin in lysates from SW480-α9 cells transfected with non-targeted siRNA or siRNA targeted to src or β-catenin; Right panel: Migration (left), invasion (middle) or proliferation (right) assays in SW480-α9 cells following transfection with siRNA as indicated.

Mentions: Having recently shown src is essential for α9β1 signaling28 and knowing src is implicated in EMT29 we investigated its role in α9β1-associated EMT. Fig 2A shows that association of src with integrin α9β1 is facilitated by α9β1 ligand and depends on the α9 subunit and not β1 (Fig 2B). During EMT there is loss of cell-cell contact resulting from β-catenin phosphorylation and disruption of the β-catenin/E-cadherin complex at the adherens junction.13Fig 2C shows that ligation of α9β1 induces phosphorylation of src-Y416 and also β-catenin-Y654, the tyrosine phosphorylation site associated with EMT.14 Src also mediated α9β1-induced β-catenin phosphorylation and changes in E-cadherin and vimentin expression consistent with EMT (Suppl. Fig S2-A). In non-α9 expressing SW480 neither src nor β-catenin phosphorylation were significantly induced by pFN, suggesting other β1-integrins are not playing a role (Fig 2C).


Integrin α9β1 promotes malignant tumor growth and metastasis by potentiating epithelial-mesenchymal transition.

Gupta SK, Oommen S, Aubry MC, Williams BP, Vlahakis NE - Oncogene (2012)

α9β1 co-associates with E-cadherin and β-catenin inducing EMT through src signalingA, Immunoblots for α9 or src from immunoprecipitates of SW480 cells cultured with or without the α9β1 specific ligand, TnfnRAA and/or the src inhibitor, PP1. B, Immunoblots for β1, α9 or Src from immunoprecipitates of matrix activated SW-480-mock (pFN) or SW480-α9 cells (TnfnRAA). C, Immunoblots for α9, Src or β-catenin in lysates from SW480-mock or α9 cells grown with pFN or TnfnRAA and/or PP1. D, Confocal images of SW480-α9 cells to determine co-localization of α9β1 (red) with β-catenin (green, top panel) or E-cadherin (green, bottom panel); nuclei stained blue with DAPI. E, Immunoblots, of immunoprecipitates from SW480-α9 grown on plastic (no matrix) or TnfnRAA (right panel), to determine co-association of α9 with β-catenin or E-cadherin. F, Left panel, Immunoblots for β-catenin, src, E-cadherin or vimentin in lysates from SW480-α9 cells transfected with non-targeted siRNA or siRNA targeted to src or β-catenin; Right panel: Migration (left), invasion (middle) or proliferation (right) assays in SW480-α9 cells following transfection with siRNA as indicated.
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Figure 2: α9β1 co-associates with E-cadherin and β-catenin inducing EMT through src signalingA, Immunoblots for α9 or src from immunoprecipitates of SW480 cells cultured with or without the α9β1 specific ligand, TnfnRAA and/or the src inhibitor, PP1. B, Immunoblots for β1, α9 or Src from immunoprecipitates of matrix activated SW-480-mock (pFN) or SW480-α9 cells (TnfnRAA). C, Immunoblots for α9, Src or β-catenin in lysates from SW480-mock or α9 cells grown with pFN or TnfnRAA and/or PP1. D, Confocal images of SW480-α9 cells to determine co-localization of α9β1 (red) with β-catenin (green, top panel) or E-cadherin (green, bottom panel); nuclei stained blue with DAPI. E, Immunoblots, of immunoprecipitates from SW480-α9 grown on plastic (no matrix) or TnfnRAA (right panel), to determine co-association of α9 with β-catenin or E-cadherin. F, Left panel, Immunoblots for β-catenin, src, E-cadherin or vimentin in lysates from SW480-α9 cells transfected with non-targeted siRNA or siRNA targeted to src or β-catenin; Right panel: Migration (left), invasion (middle) or proliferation (right) assays in SW480-α9 cells following transfection with siRNA as indicated.
Mentions: Having recently shown src is essential for α9β1 signaling28 and knowing src is implicated in EMT29 we investigated its role in α9β1-associated EMT. Fig 2A shows that association of src with integrin α9β1 is facilitated by α9β1 ligand and depends on the α9 subunit and not β1 (Fig 2B). During EMT there is loss of cell-cell contact resulting from β-catenin phosphorylation and disruption of the β-catenin/E-cadherin complex at the adherens junction.13Fig 2C shows that ligation of α9β1 induces phosphorylation of src-Y416 and also β-catenin-Y654, the tyrosine phosphorylation site associated with EMT.14 Src also mediated α9β1-induced β-catenin phosphorylation and changes in E-cadherin and vimentin expression consistent with EMT (Suppl. Fig S2-A). In non-α9 expressing SW480 neither src nor β-catenin phosphorylation were significantly induced by pFN, suggesting other β1-integrins are not playing a role (Fig 2C).

Bottom Line: In addition, we found that α9β1 forms a tri-partite protein complex with β-catenin and E-cadherin, which dissociates following integrin activation and subsequent src and β-catenin phosphorylation.These in vitro results are biologically significant as α9β1-expressing cancer cells induce greater tumor growth and metastases in mice as compared to the cells without α9β1 expression or when integrin expression is suppressed.Furthermore, integrin α9β1 is expressed in primary human small cell lung cancer and patients having a high expression of α9β1 demonstrated significantly worse long-term survival compared with patients with low α9β1 expression.

View Article: PubMed Central - PubMed

Affiliation: Thoracic Disease Research Unit, Division of Pulmonary & Critical Care Medicine, Mayo Clinic, Rochester 55905, MN, USA.

ABSTRACT
The integrin α9β1 binds a number of extracellular matrix components to mediate cell adhesion, migration and tissue invasion. Although expressed in a variety of normal human cells including endothelium, it is also expressed in cancer cells. We have previously shown that α9β1 binds VEGF-A to facilitate angiogenesis, an important component of the tumor microenvironment. As α9β1 induces accelerated cancer cell migration, we wished to determine what role it played in cancer growth and metastasis. In this study, we show that α9β1 expression induces molecular changes consistent with epithelial-mesenchymal transition. In addition, we found that α9β1 forms a tri-partite protein complex with β-catenin and E-cadherin, which dissociates following integrin activation and subsequent src and β-catenin phosphorylation. These findings were consistent in cells in which: α9β1 was exogenously over-expressed, or when its expression was suppressed in cancer cells endogenously expressing α9β1. These in vitro results are biologically significant as α9β1-expressing cancer cells induce greater tumor growth and metastases in mice as compared to the cells without α9β1 expression or when integrin expression is suppressed. Furthermore, integrin α9β1 is expressed in primary human small cell lung cancer and patients having a high expression of α9β1 demonstrated significantly worse long-term survival compared with patients with low α9β1 expression. These findings highlight a novel mechanism of integrin α9β1 function in human cancer.

Show MeSH
Related in: MedlinePlus