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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 is essential for the growth and metastasis of β1 integrin-–deficient 4T1 tumors in mice. (A) Parental (scram) and β1 integrin–deficient 4T1 cells (12,000 cells/mouse) were engrafted into the fat pads of female BALB/c mice. Tumor growth was monitored using digital calipers on the indicated days postengraftment. Data are mean (±SE; n = 5) tumor volumes. (B) Primary tumors from A were excised and weighed at the time of killing. Data are mean tumor weights (±SE; n = 5). (C) Bioluminescence imaging of pulmonary metastasis from parental (scram) and β1 integrin–deficient 4T1 tumors from A at weeks 1 and 4 postengraftment. Inset, representative bioluminescence images of parental (scram) and β1 integrin–deficient 4T1 lung metastases. Data are mean (±SE) pulmonary area flux units detected at the indicated time points. (D) Parental (scram) and dual β1/β3 integrin (shβ1/shβ3 Int)–deficient 4T1 cells (10,000 cells/mouse) were engrafted into the fat pads of female BALB/c mice. Tumor growth was monitored using digital calipers on the indicated days postengraftment. Data are mean tumor volumes (±SE; n = 5). (E) Primary tumors from D were excised and weighed at the time of killing. Data are mean tumor weights (±SE; n = 5). (F) Bioluminescence imaging of pulmonary metastasis from parental (scram) and shβ1/shβ3 integrin–deficient 4T1 tumors from D at weeks 1 and 4 postengraftment. Inset, representative bioluminescence images of parental (scram) and β1 integrin–deficient 4T1 lung metastases. Data are mean (±SE) pulmonary area flux units detected at the indicated time points. *p < 0.05, **p < 0.0005, ***p < 0.00005.
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Figure 7: Compensatory β3 integrin expression is essential for the growth and metastasis of β1 integrin-–deficient 4T1 tumors in mice. (A) Parental (scram) and β1 integrin–deficient 4T1 cells (12,000 cells/mouse) were engrafted into the fat pads of female BALB/c mice. Tumor growth was monitored using digital calipers on the indicated days postengraftment. Data are mean (±SE; n = 5) tumor volumes. (B) Primary tumors from A were excised and weighed at the time of killing. Data are mean tumor weights (±SE; n = 5). (C) Bioluminescence imaging of pulmonary metastasis from parental (scram) and β1 integrin–deficient 4T1 tumors from A at weeks 1 and 4 postengraftment. Inset, representative bioluminescence images of parental (scram) and β1 integrin–deficient 4T1 lung metastases. Data are mean (±SE) pulmonary area flux units detected at the indicated time points. (D) Parental (scram) and dual β1/β3 integrin (shβ1/shβ3 Int)–deficient 4T1 cells (10,000 cells/mouse) were engrafted into the fat pads of female BALB/c mice. Tumor growth was monitored using digital calipers on the indicated days postengraftment. Data are mean tumor volumes (±SE; n = 5). (E) Primary tumors from D were excised and weighed at the time of killing. Data are mean tumor weights (±SE; n = 5). (F) Bioluminescence imaging of pulmonary metastasis from parental (scram) and shβ1/shβ3 integrin–deficient 4T1 tumors from D at weeks 1 and 4 postengraftment. Inset, representative bioluminescence images of parental (scram) and β1 integrin–deficient 4T1 lung metastases. Data are mean (±SE) pulmonary area flux units detected at the indicated time points. *p < 0.05, **p < 0.0005, ***p < 0.00005.

Mentions: The foregoing findings clearly demonstrate the essential role of compensatory β3 integrin expression in rescuing the growth of 4T1 organoids in 3D-organotypic culture systems. As such, we extended these analyses to assess the function of β1 → β3 integrin switching in mediating the growth and metastasis of β1 integrin–deficient 4T1 tumors produced in syngeneic BALB/c mice. In doing so, we observed that both 4T1 derivatives exhibited similar rates of tumor formation and growth upon engraftment in the mammary fat pad (Figure 7, A and B). In accord with Figure 6D, we also observed that parental and β1 integrin–deficient 4T1 cells exhibited similar kinetics and extent of pulmonary metastasis in BALB/c mice (Figure 7C). Thus these findings suggest that compensatory expression of β3 integrin renders β1 integrin–deficient 4T1 tumors competent to undergo metastatic progression. Indeed, ex vivo isolation and propagation of pulmonary metastases derived from β1 integrin–deficient 4T1 tumors confirmed the retention of compensatory β3 integrin expression by these metastatic isolates, as well as showed that these same cells more robustly up-regulated their expression of β3 integrin in response to TGF-β than their parental counterparts (Supplemental Figure S5). To demonstrate that compensatory β3 integrin expression was indeed responsible for driving the development and metastatic progression of β1 integrin–deficient 4T1 tumors, we engineered dual β1/β3 integrin–deficient 4T1 cells that exhibited reduced capacity to undergo β1 → β3 integrin switching (Supplemental Figure S5). Indeed, compared to parental (scram) 4T1 tumors, those formed by dual β1/β3 integrin–deficient 4T1 cells were significantly smaller (Figure 7, D and E) and possessed significantly reduced capacity to metastasize to the lungs of BALB/c mice (Figure 7F). Thus these findings demonstrate the ability of compensatory β1 → β3 integrin switching to rescue the progression of β1 integrin–deficient triple-negative breast cancers in vivo.


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 is essential for the growth and metastasis of β1 integrin-–deficient 4T1 tumors in mice. (A) Parental (scram) and β1 integrin–deficient 4T1 cells (12,000 cells/mouse) were engrafted into the fat pads of female BALB/c mice. Tumor growth was monitored using digital calipers on the indicated days postengraftment. Data are mean (±SE; n = 5) tumor volumes. (B) Primary tumors from A were excised and weighed at the time of killing. Data are mean tumor weights (±SE; n = 5). (C) Bioluminescence imaging of pulmonary metastasis from parental (scram) and β1 integrin–deficient 4T1 tumors from A at weeks 1 and 4 postengraftment. Inset, representative bioluminescence images of parental (scram) and β1 integrin–deficient 4T1 lung metastases. Data are mean (±SE) pulmonary area flux units detected at the indicated time points. (D) Parental (scram) and dual β1/β3 integrin (shβ1/shβ3 Int)–deficient 4T1 cells (10,000 cells/mouse) were engrafted into the fat pads of female BALB/c mice. Tumor growth was monitored using digital calipers on the indicated days postengraftment. Data are mean tumor volumes (±SE; n = 5). (E) Primary tumors from D were excised and weighed at the time of killing. Data are mean tumor weights (±SE; n = 5). (F) Bioluminescence imaging of pulmonary metastasis from parental (scram) and shβ1/shβ3 integrin–deficient 4T1 tumors from D at weeks 1 and 4 postengraftment. Inset, representative bioluminescence images of parental (scram) and β1 integrin–deficient 4T1 lung metastases. Data are mean (±SE) pulmonary area flux units detected at the indicated time points. *p < 0.05, **p < 0.0005, ***p < 0.00005.
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Figure 7: Compensatory β3 integrin expression is essential for the growth and metastasis of β1 integrin-–deficient 4T1 tumors in mice. (A) Parental (scram) and β1 integrin–deficient 4T1 cells (12,000 cells/mouse) were engrafted into the fat pads of female BALB/c mice. Tumor growth was monitored using digital calipers on the indicated days postengraftment. Data are mean (±SE; n = 5) tumor volumes. (B) Primary tumors from A were excised and weighed at the time of killing. Data are mean tumor weights (±SE; n = 5). (C) Bioluminescence imaging of pulmonary metastasis from parental (scram) and β1 integrin–deficient 4T1 tumors from A at weeks 1 and 4 postengraftment. Inset, representative bioluminescence images of parental (scram) and β1 integrin–deficient 4T1 lung metastases. Data are mean (±SE) pulmonary area flux units detected at the indicated time points. (D) Parental (scram) and dual β1/β3 integrin (shβ1/shβ3 Int)–deficient 4T1 cells (10,000 cells/mouse) were engrafted into the fat pads of female BALB/c mice. Tumor growth was monitored using digital calipers on the indicated days postengraftment. Data are mean tumor volumes (±SE; n = 5). (E) Primary tumors from D were excised and weighed at the time of killing. Data are mean tumor weights (±SE; n = 5). (F) Bioluminescence imaging of pulmonary metastasis from parental (scram) and shβ1/shβ3 integrin–deficient 4T1 tumors from D at weeks 1 and 4 postengraftment. Inset, representative bioluminescence images of parental (scram) and β1 integrin–deficient 4T1 lung metastases. Data are mean (±SE) pulmonary area flux units detected at the indicated time points. *p < 0.05, **p < 0.0005, ***p < 0.00005.
Mentions: The foregoing findings clearly demonstrate the essential role of compensatory β3 integrin expression in rescuing the growth of 4T1 organoids in 3D-organotypic culture systems. As such, we extended these analyses to assess the function of β1 → β3 integrin switching in mediating the growth and metastasis of β1 integrin–deficient 4T1 tumors produced in syngeneic BALB/c mice. In doing so, we observed that both 4T1 derivatives exhibited similar rates of tumor formation and growth upon engraftment in the mammary fat pad (Figure 7, A and B). In accord with Figure 6D, we also observed that parental and β1 integrin–deficient 4T1 cells exhibited similar kinetics and extent of pulmonary metastasis in BALB/c mice (Figure 7C). Thus these findings suggest that compensatory expression of β3 integrin renders β1 integrin–deficient 4T1 tumors competent to undergo metastatic progression. Indeed, ex vivo isolation and propagation of pulmonary metastases derived from β1 integrin–deficient 4T1 tumors confirmed the retention of compensatory β3 integrin expression by these metastatic isolates, as well as showed that these same cells more robustly up-regulated their expression of β3 integrin in response to TGF-β than their parental counterparts (Supplemental Figure S5). To demonstrate that compensatory β3 integrin expression was indeed responsible for driving the development and metastatic progression of β1 integrin–deficient 4T1 tumors, we engineered dual β1/β3 integrin–deficient 4T1 cells that exhibited reduced capacity to undergo β1 → β3 integrin switching (Supplemental Figure S5). Indeed, compared to parental (scram) 4T1 tumors, those formed by dual β1/β3 integrin–deficient 4T1 cells were significantly smaller (Figure 7, D and E) and possessed significantly reduced capacity to metastasize to the lungs of BALB/c mice (Figure 7F). Thus these findings demonstrate the ability of compensatory β1 → β3 integrin switching to rescue the progression of β1 integrin–deficient triple-negative breast cancers in vivo.

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