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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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Functional disruption of β1 integrin attenuates TGF-β–mediated motility in normal NMuMG cells. (A) Confluent NMuMG cell monolayers were wounded and allowed to heal for 24 h in the absence (unstim) or presence of TGF-β1 (5 ng/ml), neutralizing β1 integrin antibodies (β1 N.A.; 5 μg/ml), or the p38 MAPK inhibitor SB203580 (p38 Inh; 10 μM) as indicated. Representative photomicrographs from a single experiment performed three times in triplicate. (B) Quantification of wounded NMuMG cultures at 24 h was conducted using ImageJ (v1.34S; National Institutes of Health, Bethesda, MD). Data are mean (±SE) percentage wound closure of three independent experiments completed in triplicate. (C, D) NMuMG cells were stimulated for 24 h with TGF-β1 (5 ng/ml) in the absence (diluent) or presence of either neutralizing β1 integrin antibodies (β1 N.A.; 5 μg/ml) or p38 MAPK inhibitor SB203580 (p38 Inh; 10 μM) as indicated. Afterward, total RNA was isolated to monitor changes in the expression of PAI-1 (C) or Cox-2 (D) by semiquantitative real-time PCR. Data are mean (±SE) of three independent experiments completed in triplicate. In B–D, *,#p < 0.05.
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Figure 1: Functional disruption of β1 integrin attenuates TGF-β–mediated motility in normal NMuMG cells. (A) Confluent NMuMG cell monolayers were wounded and allowed to heal for 24 h in the absence (unstim) or presence of TGF-β1 (5 ng/ml), neutralizing β1 integrin antibodies (β1 N.A.; 5 μg/ml), or the p38 MAPK inhibitor SB203580 (p38 Inh; 10 μM) as indicated. Representative photomicrographs from a single experiment performed three times in triplicate. (B) Quantification of wounded NMuMG cultures at 24 h was conducted using ImageJ (v1.34S; National Institutes of Health, Bethesda, MD). Data are mean (±SE) percentage wound closure of three independent experiments completed in triplicate. (C, D) NMuMG cells were stimulated for 24 h with TGF-β1 (5 ng/ml) in the absence (diluent) or presence of either neutralizing β1 integrin antibodies (β1 N.A.; 5 μg/ml) or p38 MAPK inhibitor SB203580 (p38 Inh; 10 μM) as indicated. Afterward, total RNA was isolated to monitor changes in the expression of PAI-1 (C) or Cox-2 (D) by semiquantitative real-time PCR. Data are mean (±SE) of three independent experiments completed in triplicate. In B–D, *,#p < 0.05.

Mentions: Previous studies demonstrated that administering neutralizing antibodies to β1 integrin prevents TGF-β from activating p38 MAPK and inducing EMT programs in NMuMG cells (Bhowmick et al., 2001), a well-established model for studying EMT and its regulation by TGF-β (Miettinen et al., 1994). Along these lines, we found a similar requirement for β3 integrin in mediating these same biological readouts in NMuMG cells stimulated by TGF-β (Galliher and Schiemann, 2006). These discrepant findings raised important questions as to whether the activities of β1 integrin lie upstream of β3 integrin in the TGF-β pathway, a notion that was speculated on previously (Galliher and Schiemann, 2006), or whether both β integrins lie in distinct branches of the TGF-β signaling system. As an initial attempt to address these questions, we inhibited the activities of β1 integrin and p38 MAPK to assess their function in coupling TGF-β to the motility of NMuMG cells. Under unstimulated conditions, wounded NMuMG cell monolayers exhibited minimal wound closure (∼10%), whereas inclusion of TGF-β during the healing process significantly stimulated the closure of NMuMG cell wounds (Figure 1, A and B). Addition of either neutralizing β1 integrin antibodies or the p38 MAPK inhibitor SB203580 to TGF-β–treated NMuMG cultures abrogated their ability to initiate wound closure in response to TGF-β (Figure 1, A and B). Previous studies demonstrated that 1) elevated expression of the classical TGF-β gene target, plasminogen-activator inhibitor-1 (PAI-1), promotes integrin internalization and subsequent cell detachment (Czekay and Loskutoff, 2009), and 2) depleted expression of β1 integrin reduces breast cancer invasion and cyclooxygenase 2 (Cox-2) expression (Mitchell et al., 2010). Thus we asked whether the diminished migration of NMuMG cells elicited by inactivating β1 integrin and p38 MAPK activity reflected alterations in PAI-1 expression stimulated by TGF-β. As shown in Figure 1C, PAI-1 expression induced by TGF-β was unaffected by the neutralization of β1 integrin activity but was significantly decreased in cells treated with the p38 MAPK inhibitor SB203580. Along these lines, the extent of cyclooxygenase 2 (Cox-2) expression induced by TGF-β was not significantly affected by β1 integrin inactivation, a cellular condition that did elicit diminished basal levels of Cox-2 expression (Figure 1D). Finally, inhibiting the activity of p38 MAPK significantly decreased the coupling of TGF-β to Cox-2 expression in NMuMG cells (Figure 1D). Collectively these findings suggest that β1 integrin and p38 MAPK both play important roles in promoting TGF-β stimulation of cell motility and that the coupling of TGF-β to PAI-1 and Cox-2 expression occurs through a p38 MAPK-dependent process that is independent of β1 integrin activity.


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)

Functional disruption of β1 integrin attenuates TGF-β–mediated motility in normal NMuMG cells. (A) Confluent NMuMG cell monolayers were wounded and allowed to heal for 24 h in the absence (unstim) or presence of TGF-β1 (5 ng/ml), neutralizing β1 integrin antibodies (β1 N.A.; 5 μg/ml), or the p38 MAPK inhibitor SB203580 (p38 Inh; 10 μM) as indicated. Representative photomicrographs from a single experiment performed three times in triplicate. (B) Quantification of wounded NMuMG cultures at 24 h was conducted using ImageJ (v1.34S; National Institutes of Health, Bethesda, MD). Data are mean (±SE) percentage wound closure of three independent experiments completed in triplicate. (C, D) NMuMG cells were stimulated for 24 h with TGF-β1 (5 ng/ml) in the absence (diluent) or presence of either neutralizing β1 integrin antibodies (β1 N.A.; 5 μg/ml) or p38 MAPK inhibitor SB203580 (p38 Inh; 10 μM) as indicated. Afterward, total RNA was isolated to monitor changes in the expression of PAI-1 (C) or Cox-2 (D) by semiquantitative real-time PCR. Data are mean (±SE) of three independent experiments completed in triplicate. In B–D, *,#p < 0.05.
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Related In: Results  -  Collection

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Figure 1: Functional disruption of β1 integrin attenuates TGF-β–mediated motility in normal NMuMG cells. (A) Confluent NMuMG cell monolayers were wounded and allowed to heal for 24 h in the absence (unstim) or presence of TGF-β1 (5 ng/ml), neutralizing β1 integrin antibodies (β1 N.A.; 5 μg/ml), or the p38 MAPK inhibitor SB203580 (p38 Inh; 10 μM) as indicated. Representative photomicrographs from a single experiment performed three times in triplicate. (B) Quantification of wounded NMuMG cultures at 24 h was conducted using ImageJ (v1.34S; National Institutes of Health, Bethesda, MD). Data are mean (±SE) percentage wound closure of three independent experiments completed in triplicate. (C, D) NMuMG cells were stimulated for 24 h with TGF-β1 (5 ng/ml) in the absence (diluent) or presence of either neutralizing β1 integrin antibodies (β1 N.A.; 5 μg/ml) or p38 MAPK inhibitor SB203580 (p38 Inh; 10 μM) as indicated. Afterward, total RNA was isolated to monitor changes in the expression of PAI-1 (C) or Cox-2 (D) by semiquantitative real-time PCR. Data are mean (±SE) of three independent experiments completed in triplicate. In B–D, *,#p < 0.05.
Mentions: Previous studies demonstrated that administering neutralizing antibodies to β1 integrin prevents TGF-β from activating p38 MAPK and inducing EMT programs in NMuMG cells (Bhowmick et al., 2001), a well-established model for studying EMT and its regulation by TGF-β (Miettinen et al., 1994). Along these lines, we found a similar requirement for β3 integrin in mediating these same biological readouts in NMuMG cells stimulated by TGF-β (Galliher and Schiemann, 2006). These discrepant findings raised important questions as to whether the activities of β1 integrin lie upstream of β3 integrin in the TGF-β pathway, a notion that was speculated on previously (Galliher and Schiemann, 2006), or whether both β integrins lie in distinct branches of the TGF-β signaling system. As an initial attempt to address these questions, we inhibited the activities of β1 integrin and p38 MAPK to assess their function in coupling TGF-β to the motility of NMuMG cells. Under unstimulated conditions, wounded NMuMG cell monolayers exhibited minimal wound closure (∼10%), whereas inclusion of TGF-β during the healing process significantly stimulated the closure of NMuMG cell wounds (Figure 1, A and B). Addition of either neutralizing β1 integrin antibodies or the p38 MAPK inhibitor SB203580 to TGF-β–treated NMuMG cultures abrogated their ability to initiate wound closure in response to TGF-β (Figure 1, A and B). Previous studies demonstrated that 1) elevated expression of the classical TGF-β gene target, plasminogen-activator inhibitor-1 (PAI-1), promotes integrin internalization and subsequent cell detachment (Czekay and Loskutoff, 2009), and 2) depleted expression of β1 integrin reduces breast cancer invasion and cyclooxygenase 2 (Cox-2) expression (Mitchell et al., 2010). Thus we asked whether the diminished migration of NMuMG cells elicited by inactivating β1 integrin and p38 MAPK activity reflected alterations in PAI-1 expression stimulated by TGF-β. As shown in Figure 1C, PAI-1 expression induced by TGF-β was unaffected by the neutralization of β1 integrin activity but was significantly decreased in cells treated with the p38 MAPK inhibitor SB203580. Along these lines, the extent of cyclooxygenase 2 (Cox-2) expression induced by TGF-β was not significantly affected by β1 integrin inactivation, a cellular condition that did elicit diminished basal levels of Cox-2 expression (Figure 1D). Finally, inhibiting the activity of p38 MAPK significantly decreased the coupling of TGF-β to Cox-2 expression in NMuMG cells (Figure 1D). Collectively these findings suggest that β1 integrin and p38 MAPK both play important roles in promoting TGF-β stimulation of cell motility and that the coupling of TGF-β to PAI-1 and Cox-2 expression occurs through a p38 MAPK-dependent process that is independent of β1 integrin activity.

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