RESUMEN
Tetraspanin protein CD151 on tumor cells supports invasion and metastasis. In the present study, we show that host animal CD151 also plays a critical role. CD151-null mice showed markedly diminished experimental lung metastasis after injection of Lewis lung carcinoma or B16F10 melanoma cells. Diminished tumor cell residence in the lungs was evident 6-24 hours after injection. Consistent with an endothelial cell deficiency, isolated CD151-null mouse lung endothelial cells showed diminished support for B16F10 adhesion and transendothelial migration, diminished B16F10-induced permeability, and diminished B16F10 adhesion to extracellular matrix deposited by CD151-null mouse lung endothelial cells. However, CD151 deletion did not affect the size of metastatic foci or subcutaneous primary B16F10 tumors, tumor aggregation, tumor clearance from the blood, or tumor-induced immune cell activation and recruitment. Therefore, the effects of host CD151 on metastasis do not involve altered local tumor growth or immune surveillance. VEGF-induced endothelial cell signaling through Src and Akt was diminished in CD151-null endothelial cells. However, deficient signaling was not accompanied by reduced endothelial permeability either in vitro (monolayer permeability assay) or in vivo (VEGF-stimulated Miles assay). In summary, diminished metastasis in CD151-null host animals may be due to impaired tumor-endothelial interactions, with underlying defects in mouse lung endothelial cell extracellular matrix production.
Asunto(s)
Antígenos CD/genética , Metástasis de la Neoplasia/genética , Animales , Antígenos CD/fisiología , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patología , Células Cultivadas , Regulación hacia Abajo/genética , Regulación hacia Abajo/fisiología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Endoteliales/fisiología , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Matriz Extracelular/fisiología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Trasplante de Neoplasias , Tetraspanina 24 , Migración Transcelular de la Célula/genética , Migración Transendotelial y Transepitelial/genéticaRESUMEN
The alpha 3 beta 1 integrin shows strong, stoichiometric, direct lateral association with the tetraspanin CD151. As shown here, an extracellular CD151 site (QRD(194-196)) is required for strong (i.e., Triton X-100-resistant) alpha 3 beta 1 association and for maintenance of a key CD151 epitope (defined by monoclonal antibody TS151r) that is blocked upon alpha 3 integrin association. Strong CD151 association with integrin alpha 6 beta 1 also required the QRD(194-196) site and masked the TS151r epitope. For both alpha 3 and alpha 6 integrins, strong QRD/TS151r-dependent CD151 association occurred early in biosynthesis and involved alpha subunit precursor forms. In contrast, weaker associations of CD151 with itself, integrins, or other tetraspanins (Triton X-100-sensitive but Brij 96-resistant) were independent of the QRD/TS151r site, occurred late in biosynthesis, and involved mature integrin subunits. Presence of the CD151-QRD(194-196)-->INF mutant disrupted alpha 3 and alpha 6 integrin-dependent formation of a network of cellular cables by Cos7 or NIH3T3 cells on basement membrane Matrigel and markedly altered cell spreading. These results provide definitive evidence that strong lateral CD151-integrin association is functionally important, identify CD151 as a key player during alpha 3 and alpha 6 integrin-dependent matrix remodeling and cell spreading, and support a model of CD151 as a transmembrane linker between extracellular integrin domains and intracellular cytoskeleton/signaling molecules.
Asunto(s)
Antígenos CD/metabolismo , Adhesión Celular/fisiología , Integrina alfa3beta1/metabolismo , Integrina alfa6beta1/metabolismo , Animales , Células Cultivadas , Epítopos/metabolismo , Espacio Extracelular , Humanos , Immunoblotting , Mutagénesis Sitio-Dirigida , Octoxinol/química , Fragmentos de Péptidos/metabolismo , Plásmidos , Pruebas de Precipitina , Unión Proteica , Eliminación de Secuencia , Tetraspanina 24RESUMEN
Upon plating on basement membrane Matrigel, NIH3T3 cells formed an anastomosing network of cord-like structures, inhibitable by anti-alpha6beta1 integrin antibodies. For NIH3T3 cells transfected with human CD151 protein, the formation of a cord-like network was also inhibitable by anti-CD151 antibodies. Furthermore, CD151 and alpha6beta1 were physically associated within NIH3T3 cells. On removal of the short 8-amino acid C-terminal CD151 tail (by deletion or exchange), exogenous CD151 exerted a dominant negative effect, as it almost completely suppressed alpha6beta1-dependent cell network formation and NIH3T3 cell spreading on laminin-1 (an alpha6beta1 ligand). Importantly, mutant CD151 retained alpha6beta1 association and did not alter alpha6beta1-mediated cell adhesion to Matrigel. In conclusion, the CD151-alpha6beta1 integrin complex acts as a functional unit that markedly influences cellular morphogenesis, with the CD151 tail being of particular importance in determining the "outside-in" functions of alpha6beta1-integrin that follow ligand engagement. Also, antibodies to alpha6beta1 and CD151 inhibited formation of endothelial cell cord-like networks, thus pointing to possible relevance of CD151-alpha6beta1 complexes during angiogenesis.
Asunto(s)
Antígenos CD/metabolismo , Diferenciación Celular/fisiología , Integrinas/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Adhesión Celular , Células Cultivadas , Colágeno/metabolismo , Combinación de Medicamentos , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Eliminación de Gen , Humanos , Integrina alfa6beta1 , Integrina beta1/genética , Integrina beta1/inmunología , Integrina beta1/metabolismo , Laminina/metabolismo , Sustancias Macromoleculares , Ratones , Microscopía por Video , Morfogénesis , Proteoglicanos/metabolismo , Tetraspanina 24 , TransfecciónRESUMEN
EWI-2, a cell surface IgSF protein, is highly expressed in normal human brain but is considerably diminished in glioblastoma tumors and cell lines. Moreover, loss of EWI-2 expression correlated with a shorter survival time in human glioma patients, suggesting that EWI-2 might be a natural inhibitor of glioblastoma. In support of this idea, EWI-2 expression significantly impaired both ectopic and orthotopic tumor growth in nude mice in vivo. In vitro assays provided clues regarding EWI-2 functions. Expression of EWI-2 in T98G and/or U87-MG malignant glioblastoma cell lines failed to alter two-dimensional cell proliferation but inhibited glioblastoma colony formation in soft agar and caused diminished cell motility and invasion. At the biochemical level, EWI-2 markedly affects the organization of four molecules (tetraspanin proteins CD9 and CD81 and matrix metalloproteinases MMP-2 and MT1-MMP), which play key roles in the biology of astrocytes and gliomas. EWI-2 causes CD9 and CD81 to become more associated with each other, whereas CD81 and other tetraspanins become less associated with MMP-2 and MT1-MMP. We propose that EWI-2 inhibition of glioblastoma growth in vivo is at least partly explained by the capability of EWI-2 to inhibit growth and/or invasion in vitro. Underlying these functional effects, EWI-2 causes a substantial molecular reorganization of multiple molecules (CD81, CD9, MMP-2, and MT1-MMP) known to affect proliferation and/or invasion of astrocytes and/or glioblastomas.
Asunto(s)
Antígenos CD/fisiología , Genes Supresores de Tumor/fisiología , Glioblastoma/genética , Proteínas de la Membrana/fisiología , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Astrocitos/metabolismo , Astrocitos/patología , Encéfalo/metabolismo , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Glioblastoma/patología , Células HeLa , Humanos , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Inmunoglobulinas/fisiología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Desnudos , Unión Proteica , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CD151, a master regulator of laminin-binding integrins (alpha(6)beta(4), alpha(6)beta(1), and alpha(3)beta(1)), assembles these integrins into complexes called tetraspanin-enriched microdomains. CD151 protein expression is elevated in 31% of human breast cancers and is even more elevated in high-grade (40%) and estrogen receptor-negative (45%) subtypes. The latter includes triple-negative (estrogen receptor, progesterone receptor, and HER2 negative) basal-like tumors. CD151 ablation markedly reduced basal-like mammary cell migration, invasion, spreading, and signaling (through FAK, Rac1, and lck) while disrupting epidermal growth factor receptor (EGFR)-alpha(6) integrin collaboration. Underlying these defects, CD151 ablation redistributed alpha(6)beta(4) integrins subcellularly and severed molecular links between integrins and tetraspanin-enriched microdomains. In a prototypical basal-like mammary tumor line, CD151 ablation notably delayed tumor progression in ectopic and orthotopic xenograft models. These results (a) establish that CD151-alpha(6) integrin complexes play a functional role in basal-like mammary tumor progression; (b) emphasize that alpha(6) integrins function via CD151 linkage in the context of tetraspanin-enriched microdomains; and (c) point to potential relevance of CD151 as a high-priority therapeutic target, with relative selectivity (compared with laminin-binding integrins) for pathologic rather than normal physiology.
Asunto(s)
Antígenos CD/fisiología , Neoplasias de la Mama/genética , Integrina alfa6/genética , Integrina alfa6/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Adhesión Celular/genética , Movimiento Celular/genética , Progresión de la Enfermedad , Factor de Crecimiento Epidérmico/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Integrina alfa6/fisiología , Glándulas Mamarias Humanas/efectos de los fármacos , Glándulas Mamarias Humanas/metabolismo , Ratones , Ratones Desnudos , Invasividad Neoplásica , Unión Proteica , Transducción de Señal/genética , Tetraspanina 24 , Distribución Tisular , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Tetraspanin protein CD151 is abundant on endothelial cells. To determine whether CD151 affects angiogenesis, Cd151-null mice were prepared. Cd151-null mice showed no vascular defects during normal development or during neonatal oxygen-induced retinopathy. However, Cd151-null mice showed impaired pathologic angiogenesis in other in vivo assays (Matrigel plug, corneal micropocket, tumor implantation) and in the ex vivo aortic ring assay. Cd151-null mouse lung endothelial cells (MLECs) showed normal adhesion and proliferation, but marked alterations in vitro, in assays relevant to angiogenesis (migration, spreading, invasion, Matrigel contraction, tube and cable formation, spheroid sprouting). Consistent with these functional impairments, and with the close, preferential association of CD151 with laminin-binding integrins, Cd151-null MLECs also showed selective signaling defects, particularly on laminin substrate. Adhesion-dependent activation of PKB/c-Akt, e-NOS, Rac, and Cdc42 was diminished, but Raf, ERK, p38 MAP kinase, FAK, and Src were unaltered. In Cd151-null MLECs, connections were disrupted between laminin-binding integrins and at least 5 other proteins. In conclusion, CD151 modulates molecular organization of laminin-binding integrins, thereby supporting secondary (ie, after cell adhesion) functions of endothelial cells, which are needed for some types of pathologic angiogenesis in vivo. Selective effects of CD151 on pathologic angiogenesis make it a potentially useful target for anticancer therapy.
Asunto(s)
Antígenos CD/fisiología , Neovascularización Patológica , Animales , Animales Recién Nacidos , Antígenos CD/genética , Adhesión Celular , Proliferación Celular , Células Endoteliales , Pulmón/citología , Ratones , Ratones Noqueados , Transducción de Señal , Tetraspanina 24RESUMEN
The tetraspanin CD151 molecule associates specifically with laminin-binding integrins, including alpha6beta1. To probe strength of alpha6beta1-dependent adhesion to laminin-1, defined forces (0-1.5 nN) were applied to magnetic laminin-coated microbeads bound to NIH 3T3 cells. For NIH 3T3 cells bearing wild-type CD151, adhesion strengthening was observed, as bead detachment became more difficult over time. In contrast, mutant CD151 (with the C-terminal region replaced) showed impaired adhesion strengthening. Static cell adhesion to laminin-1, and detachment of beads coated with fibronectin or anti-alpha6 antibody were all unaffected by CD151 mutation. Hence, CD151 plays a key role in selectively strengthening alpha6beta1 integrin-mediated adhesion to laminin-1.