RESUMO
We demonstrate a physiological role for tumstatin, a cleavage fragment of the alpha3 chain of type IV collagen (Col IValpha3), which is present in the circulation. Mice with a genetic deletion of Col IValpha3 show accelerated tumor growth associated with enhanced pathological angiogenesis, while angiogenesis associated with development and tissue repair are unaffected. Supplementing Col IValpha3-deficient mice with recombinant tumstatin to a normal physiological concentration abolishes the increased rate of tumor growth. The suppressive effects of tumstatin require alphaVbeta3 integrin expressed on pathological, but not on physiological, angiogenic blood vessels. Mice deficient in matrix metalloproteinase-9, which cleaves tumstatin efficiently from Col IValpha3, have decreased circulating tumstatin and accelerated growth of tumor. These results indicate that MMP-generated fragments of basement membrane collagen can have endogenous function as integrin-mediated suppressors of pathologic angiogenesis and tumor growth.
Assuntos
Autoantígenos/fisiologia , Carcinoma Pulmonar de Lewis/prevenção & controle , Colágeno Tipo IV/fisiologia , Integrina alfaVbeta3/metabolismo , Neovascularização Patológica , Animais , Autoantígenos/farmacologia , Membrana Basal/química , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Divisão Celular , Colágeno/metabolismo , Colágeno Tipo IV/farmacologia , Combinação de Medicamentos , Endotélio Vascular/metabolismo , Epitopos , Feminino , Heterozigoto , Homozigoto , Humanos , Laminina/metabolismo , Regeneração Hepática , Pulmão/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout/embriologia , Camundongos Knockout/crescimento & desenvolvimento , Neovascularização Fisiológica , Gravidez , Prenhez , Proteoglicanas/metabolismo , Proteínas Recombinantes/farmacologia , Taxa de Sobrevida , Células Tumorais Cultivadas , CicatrizaçãoRESUMO
Inhibition of alphavbeta3 or alphavbeta5 integrin function has been reported to suppress neovascularization and tumor growth, suggesting that these integrins are critical modulators of angiogenesis. Here we report that mice lacking beta3 integrins or both beta3 and beta5 integrins not only support tumorigenesis, but have enhanced tumor growth as well. Moreover, the tumors in these integrin-deficient mice display enhanced angiogenesis, strongly suggesting that neither beta3 nor beta5 integrins are essential for neovascularization. We also observed that angiogenic responses to hypoxia and vascular endothelial growth factor (VEGF) are augmented significantly in the absence of beta3 integrins. We found no evidence that the expression or functions of other integrins were altered as a consequence of the beta3 deficiency, but we did observe elevated levels of VEGF receptor-2 (also called Flk-1) in beta3-null endothelial cells. These data indicate that alphavbeta3 and alphavbeta5 integrins are not essential for vascular development or pathological angiogenesis and highlight the need for further evaluation of the mechanisms of action of alphav-integrin antagonists in anti-angiogenic therapeutics.
Assuntos
Cadeias beta de Integrinas , Integrinas/deficiência , Neovascularização Patológica/etiologia , Glicoproteínas da Membrana de Plaquetas/deficiência , Animais , Antígenos CD/genética , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/efeitos dos fármacos , Integrina beta3 , Integrinas/genética , Integrinas/metabolismo , Linfocinas/farmacologia , Camundongos , Camundongos Mutantes , Neoplasias Experimentais/irrigação sanguínea , Glicoproteínas da Membrana de Plaquetas/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Receptores de Vitronectina/metabolismo , Neovascularização Retiniana , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Tumstatin is a 28-kilodalton fragment of type IV collagen that displays both anti-angiogenic and proapoptotic activity. Here we show that tumstatin functions as an endothelial cell-specific inhibitor of protein synthesis. Through a requisite interaction with alphaVbeta3 integrin, tumstatin inhibits activation of focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3-kinase), protein kinase B (PKB/Akt), and mammalian target of rapamycin (mTOR), and it prevents the dissociation of eukaryotic initiation factor 4E protein (eIF4E) from 4E-binding protein 1. These results establish a role for integrins in mediating cell-specific inhibition of cap-dependent protein synthesis and suggest a potential mechanism for tumstatin's selective effects on endothelial cells.
Assuntos
Autoantígenos/farmacologia , Colágeno Tipo IV/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Autoantígenos/química , Autoantígenos/metabolismo , Proteínas de Transporte/metabolismo , Bovinos , Proteínas de Ciclo Celular , Células Cultivadas , Colágeno Tipo IV/química , Colágeno Tipo IV/metabolismo , Endotélio Vascular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos , Fatores de Iniciação em Eucariotos , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/farmacologia , Fatores de Iniciação de Peptídeos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores de Proteínas Quinases , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Vitronectina/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR , Células Tumorais CultivadasRESUMO
As with many types of cancer, cell motility is an important factor in the progression and metastasis of hepatocellular carcinomas (HCC). HCC is associated with significant fibrosis in the liver. The fibrotic microenvironment in the liver is characterized by an altered composition of the extracellular matrix (ECM) and an abundance of growth factors that are likely conducive to migration of HCC cells. The purpose of this study was to delineate promigratory stimuli within the fibrotic microenvironment and to identify specific targets for prevention of HCC cell migration. We used a modified Boyden chamber system that allowed distinction between chemotactic (indirect stimulation) and haptotactic (direct stimulation) migration of two distinct HCC cell lines across the ECM-coated membrane. Fibrotic microenvironment-associated growth factors, such as transforming growth factor beta1 (TGF-beta1), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF), induced chemotactic and haptotactic migration of HepG2 and Chang cells. Neutralizing antibodies to individual growth factors significantly decreased chemotactic and haptotactic migration. Haptotactic stimulation, but not chemotactic stimulation of HCC cell lines with TGF-beta1, bFGF, and EGF, induced production of matrix metalloproteinase (MMP) 2, a potential mediator of migration. Inhibition of MMPs significantly decreased haptotactic migration induced by individual growth factors but had an insignificant effect on chemotactic migration, suggesting an MMP-independent migration in this setting. Inhibition of cell-ECM interactions with blocking antibodies to alpha1 and alpha2 integrins were sufficient to inhibit both haptotactic and chemotactic migration induced by individual growth factors, strongly suggesting that targeting these integrins to abrogate pathogenic cell-ECM interactions might be a promising tool for inhibiting growth factor-induced invasion and metastasis of HCC.
Assuntos
Carcinoma Hepatocelular/patologia , Matriz Extracelular/fisiologia , Integrina alfa1beta1/fisiologia , Integrina alfa2beta1/fisiologia , Neoplasias Hepáticas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Substâncias de Crescimento/farmacologia , Humanos , Metaloproteinases da Matriz/metabolismo , Invasividade NeoplásicaRESUMO
Tumor growth, tumor angiogenesis, and vascular endothelial growth factor (VEGF)-specific angiogenesis are all enhanced in beta(3)-integrin-null mice. Furthermore, endothelial cells isolated from beta(3)-null mice show elevated levels of Flk1 (VEGF receptor 2) expression, suggesting that beta(3)-integrin can control the amplitude of VEGF responses by controlling Flk1 levels or activity. We now show that Flk1 signaling is required for the enhanced tumor growth and angiogenesis seen in beta(3)-null mice. Moreover, beta(3)-null endothelial cells exhibit enhanced migration and proliferation in response to VEGF in vitro, and this phenotype requires Flk1 signaling. Upon VEGF stimulation, beta(3)-null endothelial cells exhibit higher levels of phosphorylated Flk1 and extracellular-related kinases 1 and 2 than wild-type endothelial cells. Furthermore, signaling via ERK1/2 is required to mediate the elevated responses to VEGF observed in beta(3)-null endothelial cells and aortic rings in vitro. These data confirm that VEGF signaling via Flk1 is enhanced in beta(3)-integrin-deficient mice and suggests that this increase may mediate the enhanced angiogenesis and tumor growth observed in these mice in vivo.