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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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Compensatory β3 integrin expression enhances TGF-β signaling in β1 integrin–deficient 4T1 cells. (A) Quiescent parental (scram) and β1 integrin-deficient 4T1 cells were stimulated with TGF-β1 (5 ng/ml) for 0–120 min as indicated, at which point the phosphorylation status of Smad2 and Smad3 was analyzed by immunoblotting. Data are representative of three independent analyses. (B) Smad2/3 immunofluorescence (200×) depicts the subcellular localization of Smad2/3 in basal and TGF-β1 (5 ng/ml; 30 min)–stimulated parental (scram) and β1 integrin–deficient 4T1 cells. Data are representative of three independent experiments. (C) Parental (scram) and β1 integrin–deficient 4T1 cells were transiently transfected with pCMV-β-gal and pSBE-luciferase reporter genes and subsequently stimulated with TGF-β1 (5 ng/ml) for 24 h. Data are mean (±SE) of four independent experiments completed in triplicate. (D) Parental (scram) and β1 integrin–deficient 4T1 cells were stimulated with TGF-β1 (5 ng/ml) for 48 h, at which point alterations in PAI-1 mRNA were analyzed by semiquantitative real-time PCR. Data are mean (±SE) of three independent experiments completed in triplicate. (E, F) Parental (scram) and β1 integrin–deficient 4T1 cells were stimulated for 0–120 min (E) or 24 h (F) with TGF-β1 (5 ng/ml) before monitoring the phosphorylation status and expression levels of p38 MAPK. Data are representative of three (E) or two (F) independent analyses.
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Figure 4: Compensatory β3 integrin expression enhances TGF-β signaling in β1 integrin–deficient 4T1 cells. (A) Quiescent parental (scram) and β1 integrin-deficient 4T1 cells were stimulated with TGF-β1 (5 ng/ml) for 0–120 min as indicated, at which point the phosphorylation status of Smad2 and Smad3 was analyzed by immunoblotting. Data are representative of three independent analyses. (B) Smad2/3 immunofluorescence (200×) depicts the subcellular localization of Smad2/3 in basal and TGF-β1 (5 ng/ml; 30 min)–stimulated parental (scram) and β1 integrin–deficient 4T1 cells. Data are representative of three independent experiments. (C) Parental (scram) and β1 integrin–deficient 4T1 cells were transiently transfected with pCMV-β-gal and pSBE-luciferase reporter genes and subsequently stimulated with TGF-β1 (5 ng/ml) for 24 h. Data are mean (±SE) of four independent experiments completed in triplicate. (D) Parental (scram) and β1 integrin–deficient 4T1 cells were stimulated with TGF-β1 (5 ng/ml) for 48 h, at which point alterations in PAI-1 mRNA were analyzed by semiquantitative real-time PCR. Data are mean (±SE) of three independent experiments completed in triplicate. (E, F) Parental (scram) and β1 integrin–deficient 4T1 cells were stimulated for 0–120 min (E) or 24 h (F) with TGF-β1 (5 ng/ml) before monitoring the phosphorylation status and expression levels of p38 MAPK. Data are representative of three (E) or two (F) independent analyses.

Mentions: To further characterize the interplay between β1 and β3 integrins in regulating TGF-β signaling, we surveyed the coupling of this cytokine to its canonical and noncanonical effectors in parental and β1 integrin–deficient 4T1 cells. We previously demonstrated that 4T1 cells have diminished Smad3/4 transcriptional activity as compared with their indolent 67NR counterparts, despite the fact that these cell lines harbor similar levels of phosphorylated Smad3 (Wendt et al., 2009). As shown in Figure 4A, parental (scram) and β1 integrin–depleted 4T1 cells similarly activated Smad2/3 by 30 min of TGF-β treatment; however, the magnitude of this response was more robust in β1 integrin–depleted cells than in their parental counterparts. Accordingly, Smad2/3 appeared to localize more readily in the nuclei of β1 integrin–deficient 4T1 cells (Figure 4B), which significantly enhanced their Smad3/4 transcriptional activity as compared with that in parental 4T1 cells (Figure 4C). Of interest, we observed that PAI-1 transcripts were similarly induced by TGF-β in both 4T1 derivatives (Figure 4D), suggesting involvement of noncanonical TGF-β in mediating this response (Song et al., 2012). In support of this supposition, β1 integrin–deficient 4T1 cells possessed elevated activation of p38 MAPK (Figure 4E) and FAK (Figure 4F) in 4T1 cells that harbored compensatory β3 integrin expression. Taken together, these findings indicate that inactivation of β1 integrin significantly enhances TGF-β signaling in metastatic breast cancer cells.


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)

Compensatory β3 integrin expression enhances TGF-β signaling in β1 integrin–deficient 4T1 cells. (A) Quiescent parental (scram) and β1 integrin-deficient 4T1 cells were stimulated with TGF-β1 (5 ng/ml) for 0–120 min as indicated, at which point the phosphorylation status of Smad2 and Smad3 was analyzed by immunoblotting. Data are representative of three independent analyses. (B) Smad2/3 immunofluorescence (200×) depicts the subcellular localization of Smad2/3 in basal and TGF-β1 (5 ng/ml; 30 min)–stimulated parental (scram) and β1 integrin–deficient 4T1 cells. Data are representative of three independent experiments. (C) Parental (scram) and β1 integrin–deficient 4T1 cells were transiently transfected with pCMV-β-gal and pSBE-luciferase reporter genes and subsequently stimulated with TGF-β1 (5 ng/ml) for 24 h. Data are mean (±SE) of four independent experiments completed in triplicate. (D) Parental (scram) and β1 integrin–deficient 4T1 cells were stimulated with TGF-β1 (5 ng/ml) for 48 h, at which point alterations in PAI-1 mRNA were analyzed by semiquantitative real-time PCR. Data are mean (±SE) of three independent experiments completed in triplicate. (E, F) Parental (scram) and β1 integrin–deficient 4T1 cells were stimulated for 0–120 min (E) or 24 h (F) with TGF-β1 (5 ng/ml) before monitoring the phosphorylation status and expression levels of p38 MAPK. Data are representative of three (E) or two (F) independent analyses.
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Figure 4: Compensatory β3 integrin expression enhances TGF-β signaling in β1 integrin–deficient 4T1 cells. (A) Quiescent parental (scram) and β1 integrin-deficient 4T1 cells were stimulated with TGF-β1 (5 ng/ml) for 0–120 min as indicated, at which point the phosphorylation status of Smad2 and Smad3 was analyzed by immunoblotting. Data are representative of three independent analyses. (B) Smad2/3 immunofluorescence (200×) depicts the subcellular localization of Smad2/3 in basal and TGF-β1 (5 ng/ml; 30 min)–stimulated parental (scram) and β1 integrin–deficient 4T1 cells. Data are representative of three independent experiments. (C) Parental (scram) and β1 integrin–deficient 4T1 cells were transiently transfected with pCMV-β-gal and pSBE-luciferase reporter genes and subsequently stimulated with TGF-β1 (5 ng/ml) for 24 h. Data are mean (±SE) of four independent experiments completed in triplicate. (D) Parental (scram) and β1 integrin–deficient 4T1 cells were stimulated with TGF-β1 (5 ng/ml) for 48 h, at which point alterations in PAI-1 mRNA were analyzed by semiquantitative real-time PCR. Data are mean (±SE) of three independent experiments completed in triplicate. (E, F) Parental (scram) and β1 integrin–deficient 4T1 cells were stimulated for 0–120 min (E) or 24 h (F) with TGF-β1 (5 ng/ml) before monitoring the phosphorylation status and expression levels of p38 MAPK. Data are representative of three (E) or two (F) independent analyses.
Mentions: To further characterize the interplay between β1 and β3 integrins in regulating TGF-β signaling, we surveyed the coupling of this cytokine to its canonical and noncanonical effectors in parental and β1 integrin–deficient 4T1 cells. We previously demonstrated that 4T1 cells have diminished Smad3/4 transcriptional activity as compared with their indolent 67NR counterparts, despite the fact that these cell lines harbor similar levels of phosphorylated Smad3 (Wendt et al., 2009). As shown in Figure 4A, parental (scram) and β1 integrin–depleted 4T1 cells similarly activated Smad2/3 by 30 min of TGF-β treatment; however, the magnitude of this response was more robust in β1 integrin–depleted cells than in their parental counterparts. Accordingly, Smad2/3 appeared to localize more readily in the nuclei of β1 integrin–deficient 4T1 cells (Figure 4B), which significantly enhanced their Smad3/4 transcriptional activity as compared with that in parental 4T1 cells (Figure 4C). Of interest, we observed that PAI-1 transcripts were similarly induced by TGF-β in both 4T1 derivatives (Figure 4D), suggesting involvement of noncanonical TGF-β in mediating this response (Song et al., 2012). In support of this supposition, β1 integrin–deficient 4T1 cells possessed elevated activation of p38 MAPK (Figure 4E) and FAK (Figure 4F) in 4T1 cells that harbored compensatory β3 integrin expression. Taken together, these findings indicate that inactivation of β1 integrin significantly enhances TGF-β signaling in metastatic breast cancer cells.

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