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Targeted inactivation of β1 integrin induces β3 integrin switching, which drives breast cancer metastasis by TGF-β.

Parvani JG, Galliher-Beckley AJ, Schiemann BJ, Schiemann WP - Mol. Biol. Cell (2013)

Bottom Line: We demonstrate that inactivation of β1 integrin impairs TGF-β from stimulating the motility of normal and malignant mammary epithelial cells (MECs) and elicits robust compensatory expression of β3 integrin solely in malignant MECs, but not in their normal counterparts.Compensatory β3 integrin expression also 1) enhances the growth of malignant MECs in rigid and compliant three-dimensional organotypic cultures and 2) restores the induction of the EMT phenotypes by TGF-β.Of importance, compensatory expression of β3 integrin rescues the growth and pulmonary metastasis of β1 integrin-deficient 4T1 tumors in mice, a process that is prevented by genetic depletion or functional inactivation of β3 integrin.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Case Western Reserve University, Cleveland, OH 44106 Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506 Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106.

ABSTRACT
Mammary tumorigenesis and epithelial-mesenchymal transition (EMT) programs cooperate in converting transforming growth factor-β (TGF-β) from a suppressor to a promoter of breast cancer metastasis. Although previous reports associated β1 and β3 integrins with TGF-β stimulation of EMT and metastasis, the functional interplay and plasticity exhibited by these adhesion molecules in shaping the oncogenic activities of TGF-β remain unknown. We demonstrate that inactivation of β1 integrin impairs TGF-β from stimulating the motility of normal and malignant mammary epithelial cells (MECs) and elicits robust compensatory expression of β3 integrin solely in malignant MECs, but not in their normal counterparts. Compensatory β3 integrin expression also 1) enhances the growth of malignant MECs in rigid and compliant three-dimensional organotypic cultures and 2) restores the induction of the EMT phenotypes by TGF-β. Of importance, compensatory expression of β3 integrin rescues the growth and pulmonary metastasis of β1 integrin-deficient 4T1 tumors in mice, a process that is prevented by genetic depletion or functional inactivation of β3 integrin. Collectively our findings demonstrate that inactivation of β1 integrin elicits metastatic progression via a β3 integrin-specific mechanism, indicating that dual β1 and β3 integrin targeting is necessary to alleviate metastatic disease in breast cancer patients.

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Inactivation of β1 integrin elicits compensatory expression of β3 integrin in human MDA-MB-231 cells. Parental (scram) and β1 integrin–deficient (shβ1) MDA-MB-231 cells were stimulated with TGF-β1 (5 ng/ml) for 4 d before monitoring alterations in the extent of β1 integrin deficiency, as well as β3 integrin compensation and the expression of vimentin, ZO-2, and β-actin by immunoblotting (A), and to assess changes in their morphologies by light microscopy (100×; B). Data are representative of at least three independent analyses. (C) Parental (scram) and β1 integrin–deficient MDA-MB-231 cells were propagated in compliant 3D-organotypic cultures for 4 d. The growth of these organoids was monitored by longitudinal bioluminescence. Data are representational (±SE) of three independent experiments completed in triplicate (*p < 0.025).
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Figure 5: Inactivation of β1 integrin elicits compensatory expression of β3 integrin in human MDA-MB-231 cells. Parental (scram) and β1 integrin–deficient (shβ1) MDA-MB-231 cells were stimulated with TGF-β1 (5 ng/ml) for 4 d before monitoring alterations in the extent of β1 integrin deficiency, as well as β3 integrin compensation and the expression of vimentin, ZO-2, and β-actin by immunoblotting (A), and to assess changes in their morphologies by light microscopy (100×; B). Data are representative of at least three independent analyses. (C) Parental (scram) and β1 integrin–deficient MDA-MB-231 cells were propagated in compliant 3D-organotypic cultures for 4 d. The growth of these organoids was monitored by longitudinal bioluminescence. Data are representational (±SE) of three independent experiments completed in triplicate (*p < 0.025).

Mentions: To evaluate the extent to which β1 integrin inactivation elicits β3 integrin switching in human breast cancer cells, we depleted β1 integrin expression in metastatic human MDA-MB-231 breast cancer cells (Figure 5A). Consistent with what we observed in the 4T1 cells, β1 integrin–deficient MDA-MB-231 cells exhibited compensatory β3 integrin expression and acquired more epithelial-like morphologies and features (Figure 5, A and B). Indeed, compared to their parental (scram) counterparts, β1 integrin–deficient MDA-MB-231 cells simultaneously expressed elevated levels of 1) the mesenchymal marker vimentin, whose expression was further induced by TGF-β, and 2) the epithelial marker ZO-2, whose expression was suppressed by TGF-β (Figure 5A). These findings are consistent with the acquisition of an augmented EMT phenotype in β1 integrin–deficient MDA-MB-231 cells stimulated with TGF-β. Finally, we examined the functional implications of β1 → β3 integrin switching by monitoring the growth of parental and β1 integrin–deficient MDA-MB-231 organoids in three-dimensional (3D) organotypic cultures that mimic the elasticity of normal breast (Paszek et al., 2005) and lung microenvironments (Lopez et al., 2008). Strikingly, β1 integrin–deficient MDA-MB-231 cells exhibited significantly elevated organoid growth rates compared with their parental counterparts in compliant 3D-organotypic cultures (Figure 5C). Collectively these findings demonstrate the relevance of β1 → β3 integrin switching in human breast cancer cells, which enhances their growth in compliant 3D-organotypic microenvironments via compensatory β3 integrin expression.


Targeted inactivation of β1 integrin induces β3 integrin switching, which drives breast cancer metastasis by TGF-β.

Parvani JG, Galliher-Beckley AJ, Schiemann BJ, Schiemann WP - Mol. Biol. Cell (2013)

Inactivation of β1 integrin elicits compensatory expression of β3 integrin in human MDA-MB-231 cells. Parental (scram) and β1 integrin–deficient (shβ1) MDA-MB-231 cells were stimulated with TGF-β1 (5 ng/ml) for 4 d before monitoring alterations in the extent of β1 integrin deficiency, as well as β3 integrin compensation and the expression of vimentin, ZO-2, and β-actin by immunoblotting (A), and to assess changes in their morphologies by light microscopy (100×; B). Data are representative of at least three independent analyses. (C) Parental (scram) and β1 integrin–deficient MDA-MB-231 cells were propagated in compliant 3D-organotypic cultures for 4 d. The growth of these organoids was monitored by longitudinal bioluminescence. Data are representational (±SE) of three independent experiments completed in triplicate (*p < 0.025).
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Related In: Results  -  Collection

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Figure 5: Inactivation of β1 integrin elicits compensatory expression of β3 integrin in human MDA-MB-231 cells. Parental (scram) and β1 integrin–deficient (shβ1) MDA-MB-231 cells were stimulated with TGF-β1 (5 ng/ml) for 4 d before monitoring alterations in the extent of β1 integrin deficiency, as well as β3 integrin compensation and the expression of vimentin, ZO-2, and β-actin by immunoblotting (A), and to assess changes in their morphologies by light microscopy (100×; B). Data are representative of at least three independent analyses. (C) Parental (scram) and β1 integrin–deficient MDA-MB-231 cells were propagated in compliant 3D-organotypic cultures for 4 d. The growth of these organoids was monitored by longitudinal bioluminescence. Data are representational (±SE) of three independent experiments completed in triplicate (*p < 0.025).
Mentions: To evaluate the extent to which β1 integrin inactivation elicits β3 integrin switching in human breast cancer cells, we depleted β1 integrin expression in metastatic human MDA-MB-231 breast cancer cells (Figure 5A). Consistent with what we observed in the 4T1 cells, β1 integrin–deficient MDA-MB-231 cells exhibited compensatory β3 integrin expression and acquired more epithelial-like morphologies and features (Figure 5, A and B). Indeed, compared to their parental (scram) counterparts, β1 integrin–deficient MDA-MB-231 cells simultaneously expressed elevated levels of 1) the mesenchymal marker vimentin, whose expression was further induced by TGF-β, and 2) the epithelial marker ZO-2, whose expression was suppressed by TGF-β (Figure 5A). These findings are consistent with the acquisition of an augmented EMT phenotype in β1 integrin–deficient MDA-MB-231 cells stimulated with TGF-β. Finally, we examined the functional implications of β1 → β3 integrin switching by monitoring the growth of parental and β1 integrin–deficient MDA-MB-231 organoids in three-dimensional (3D) organotypic cultures that mimic the elasticity of normal breast (Paszek et al., 2005) and lung microenvironments (Lopez et al., 2008). Strikingly, β1 integrin–deficient MDA-MB-231 cells exhibited significantly elevated organoid growth rates compared with their parental counterparts in compliant 3D-organotypic cultures (Figure 5C). Collectively these findings demonstrate the relevance of β1 → β3 integrin switching in human breast cancer cells, which enhances their growth in compliant 3D-organotypic microenvironments via compensatory β3 integrin expression.

Bottom Line: We demonstrate that inactivation of β1 integrin impairs TGF-β from stimulating the motility of normal and malignant mammary epithelial cells (MECs) and elicits robust compensatory expression of β3 integrin solely in malignant MECs, but not in their normal counterparts.Compensatory β3 integrin expression also 1) enhances the growth of malignant MECs in rigid and compliant three-dimensional organotypic cultures and 2) restores the induction of the EMT phenotypes by TGF-β.Of importance, compensatory expression of β3 integrin rescues the growth and pulmonary metastasis of β1 integrin-deficient 4T1 tumors in mice, a process that is prevented by genetic depletion or functional inactivation of β3 integrin.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Case Western Reserve University, Cleveland, OH 44106 Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506 Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106.

ABSTRACT
Mammary tumorigenesis and epithelial-mesenchymal transition (EMT) programs cooperate in converting transforming growth factor-β (TGF-β) from a suppressor to a promoter of breast cancer metastasis. Although previous reports associated β1 and β3 integrins with TGF-β stimulation of EMT and metastasis, the functional interplay and plasticity exhibited by these adhesion molecules in shaping the oncogenic activities of TGF-β remain unknown. We demonstrate that inactivation of β1 integrin impairs TGF-β from stimulating the motility of normal and malignant mammary epithelial cells (MECs) and elicits robust compensatory expression of β3 integrin solely in malignant MECs, but not in their normal counterparts. Compensatory β3 integrin expression also 1) enhances the growth of malignant MECs in rigid and compliant three-dimensional organotypic cultures and 2) restores the induction of the EMT phenotypes by TGF-β. Of importance, compensatory expression of β3 integrin rescues the growth and pulmonary metastasis of β1 integrin-deficient 4T1 tumors in mice, a process that is prevented by genetic depletion or functional inactivation of β3 integrin. Collectively our findings demonstrate that inactivation of β1 integrin elicits metastatic progression via a β3 integrin-specific mechanism, indicating that dual β1 and β3 integrin targeting is necessary to alleviate metastatic disease in breast cancer patients.

Show MeSH
Related in: MedlinePlus