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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 promotes phenotypic and functional changes of EMTA, Top: Phase contrast photomicrographs comparing SW480-Mock and SW480-α9 cells following 36h of serum depletion; 2nd-4th panels: immunoflourescent photomicrographs of SW480-Mock and α9 cells for E-cadherin, α-SMA or cytoskeletal F-actin. Cell nuclei stained blue with DAPI. B, Immunoblots of SW480-mock and α9 lysates for α9 and markers of EMT: E-cadherin, α-SMA and vimentin. C, Immunoblots of SW480-mock and α9 lysates grown in the absence or presence of activating matrix, as indicated, demonstrating the relative expression of the α9 integrin subunit and markers of EMT: E-cadherin, N-cadherin, α-SMA, vimentin and Snail. D, Chemotaxis and haptotaxis assays with activating matrices: pFN or TnfnRAA; using SW480-mock (grey) or SW480-α9 (black). E, Invasion assays: Left panel, SW480-mock or α9 cells after 24h or 36h growth; Right panel, SW480-α9 cells in absence (grey) or presence of TnfnRAA (black), with or without α9β1 inhibitors Y9A2 or VLO5.
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Figure 1: α9β1 promotes phenotypic and functional changes of EMTA, Top: Phase contrast photomicrographs comparing SW480-Mock and SW480-α9 cells following 36h of serum depletion; 2nd-4th panels: immunoflourescent photomicrographs of SW480-Mock and α9 cells for E-cadherin, α-SMA or cytoskeletal F-actin. Cell nuclei stained blue with DAPI. B, Immunoblots of SW480-mock and α9 lysates for α9 and markers of EMT: E-cadherin, α-SMA and vimentin. C, Immunoblots of SW480-mock and α9 lysates grown in the absence or presence of activating matrix, as indicated, demonstrating the relative expression of the α9 integrin subunit and markers of EMT: E-cadherin, N-cadherin, α-SMA, vimentin and Snail. D, Chemotaxis and haptotaxis assays with activating matrices: pFN or TnfnRAA; using SW480-mock (grey) or SW480-α9 (black). E, Invasion assays: Left panel, SW480-mock or α9 cells after 24h or 36h growth; Right panel, SW480-α9 cells in absence (grey) or presence of TnfnRAA (black), with or without α9β1 inhibitors Y9A2 or VLO5.

Mentions: We have previously used SW480 colon carcinoma cells7 transfected with integrin α9β125 and observed that, compared to mock cells, α9-transfected SW480 (Fig 1A) displayed a more spindle shape, suggestive of a mesenchymal phenotype. Immunoflourescent staining (Fig 1A) and immunoblots of cell lysates (Fig 1B) showed that compared to mock, SW480-α9 expressed less E-cadherin, more α-SMA and vimentin and demonstrated loss of cortical F-actin; all molecular changes consistent with EMT. These molecular changes increased significantly with α9β1 activation, and were associated with increased expression of the transcriptional repressor Snail (Fig. 1C), further supporting the notion that α9β1-induces EMT. In contrast, SW480-mock did not demonstrate these changes when activated by the common integrin ligand, plasma fibronectin (pFN). Transient α9-overexpression in other cancer cell lines also resulted in similar EMT changes measured by immunoblotting (Suppl. Fig-S1A) and immunocytochemistry (Suppl. Fig-S1B); helping to rule out the confounding effect of cell type.


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 promotes phenotypic and functional changes of EMTA, Top: Phase contrast photomicrographs comparing SW480-Mock and SW480-α9 cells following 36h of serum depletion; 2nd-4th panels: immunoflourescent photomicrographs of SW480-Mock and α9 cells for E-cadherin, α-SMA or cytoskeletal F-actin. Cell nuclei stained blue with DAPI. B, Immunoblots of SW480-mock and α9 lysates for α9 and markers of EMT: E-cadherin, α-SMA and vimentin. C, Immunoblots of SW480-mock and α9 lysates grown in the absence or presence of activating matrix, as indicated, demonstrating the relative expression of the α9 integrin subunit and markers of EMT: E-cadherin, N-cadherin, α-SMA, vimentin and Snail. D, Chemotaxis and haptotaxis assays with activating matrices: pFN or TnfnRAA; using SW480-mock (grey) or SW480-α9 (black). E, Invasion assays: Left panel, SW480-mock or α9 cells after 24h or 36h growth; Right panel, SW480-α9 cells in absence (grey) or presence of TnfnRAA (black), with or without α9β1 inhibitors Y9A2 or VLO5.
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Figure 1: α9β1 promotes phenotypic and functional changes of EMTA, Top: Phase contrast photomicrographs comparing SW480-Mock and SW480-α9 cells following 36h of serum depletion; 2nd-4th panels: immunoflourescent photomicrographs of SW480-Mock and α9 cells for E-cadherin, α-SMA or cytoskeletal F-actin. Cell nuclei stained blue with DAPI. B, Immunoblots of SW480-mock and α9 lysates for α9 and markers of EMT: E-cadherin, α-SMA and vimentin. C, Immunoblots of SW480-mock and α9 lysates grown in the absence or presence of activating matrix, as indicated, demonstrating the relative expression of the α9 integrin subunit and markers of EMT: E-cadherin, N-cadherin, α-SMA, vimentin and Snail. D, Chemotaxis and haptotaxis assays with activating matrices: pFN or TnfnRAA; using SW480-mock (grey) or SW480-α9 (black). E, Invasion assays: Left panel, SW480-mock or α9 cells after 24h or 36h growth; Right panel, SW480-α9 cells in absence (grey) or presence of TnfnRAA (black), with or without α9β1 inhibitors Y9A2 or VLO5.
Mentions: We have previously used SW480 colon carcinoma cells7 transfected with integrin α9β125 and observed that, compared to mock cells, α9-transfected SW480 (Fig 1A) displayed a more spindle shape, suggestive of a mesenchymal phenotype. Immunoflourescent staining (Fig 1A) and immunoblots of cell lysates (Fig 1B) showed that compared to mock, SW480-α9 expressed less E-cadherin, more α-SMA and vimentin and demonstrated loss of cortical F-actin; all molecular changes consistent with EMT. These molecular changes increased significantly with α9β1 activation, and were associated with increased expression of the transcriptional repressor Snail (Fig. 1C), further supporting the notion that α9β1-induces EMT. In contrast, SW480-mock did not demonstrate these changes when activated by the common integrin ligand, plasma fibronectin (pFN). Transient α9-overexpression in other cancer cell lines also resulted in similar EMT changes measured by immunoblotting (Suppl. Fig-S1A) and immunocytochemistry (Suppl. Fig-S1B); helping to rule out the confounding effect of cell type.

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