RESUMEN
Human Cripto-1 (CR-1) is overexpressed in approximately 80% of human breast, colon and lung carcinomas. Mouse Cr-1 upregulation is also observed in a number of transgenic (Tg) mouse mammary tumors. To determine whether CR-1 can alter mammary gland development and/or may contribute to tumorigenesis in vivo, we have generated Tg mouse lines that express human CR-1 under the transcriptional control of the mouse mammary tumor virus (MMTV). Stable Tg MMTV/CR-1 FVB/N lines expressing different levels of CR-1 were analysed. Virgin female MMTV/CR-1 Tg mice exhibited enhanced ductal branching, dilated ducts, intraductal hyperplasia, hyperplastic alveolar nodules and condensation of the mammary stroma. Virgin aged MMTV/CR-1 Tg mice also possessed persistent end buds. In aged multiparous MMTV/CR-1 mice, the hyperplastic phenotype was most pronounced with multifocal hyperplasias. In the highest CR-1-expressing subline, G4, 38% (12/31) of the multiparous animals aged 12-20 months developed hyperplasias and approximately 33% (11/31) developed papillary adenocarcinomas. The long latency period suggests that additional genetic alterations are required to facilitate mammary tumor formation in conjunction with CR-1. This is the first in vivo study that shows hyperplasia and tumor growth in CR-1-overexpressing animals.
Asunto(s)
Adenocarcinoma/genética , Factor de Crecimiento Epidérmico/genética , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Animales/genética , Glicoproteínas de Membrana/genética , Proteínas de Neoplasias/genética , Animales , División Celular , Cartilla de ADN , ADN Complementario/genética , Femenino , Proteínas Ligadas a GPI , Sustancias de Crecimiento/genética , Hiperplasia , Péptidos y Proteínas de Señalización Intercelular , Neoplasias Mamarias Animales/patología , Ratones , Ratones TransgénicosRESUMEN
BACKGROUND: Human cripto-1 (CR-1) promotes cell transformation and increases migration and invasion of various mouse and human epithelial cell lines. We investigated whether CR-1 also stimulates angiogenesis. METHODS: We used human umbilical vein endothelial cells (HUVECs) to measure in vitro migration with fibronectin-coated Boyden chambers, invasion with Matrigel-coated Boyden chambers, proliferation with a tetrazolium salt, and differentiation with an in vitro Matrigel assay. We investigated new blood vessel formation in vivo by use of Matrigel-filled silicone cylinders implanted under the skin of nude mice and by use of a breast cancer xenograft model with CR-1-transfected or control Neo-transfected MCF-7 human breast cancer cells. We also used a blocking anti-CR-1 monoclonal antibody to investigate the role of CR-1 in angiogenesis in vivo and in vitro. All statistical tests were two-sided. RESULTS: CR-1 stimulated HUVEC proliferation, migration, and invasion and induced HUVEC differentiation into vascular-like structures on Matrigel. In vivo, recombinant CR-1 protein induced microvessel formation in Matrigel-filled silicone cylinders, and microvessel formation was statistically significantly inhibited with a blocking anti-CR-1 monoclonal antibody (CR-1 and antibody = 127% of microvessel formation compared with that in untreated control cylinders and CR-1 alone = 259%; difference = 132%, 95% confidence interval [CI] = 123% to 140%; P<.001). Tumors formed by CR-1-transfected MCF-7 cells in the cleared mammary fat pad of nude mice had higher microvessel density than tumors formed by control Neo-transfected MCF-7 cells (CR-1-transfected cells = 4.66 vessels per field and Neo-transfected cells = 2.33 vessels per field; difference = 2.33 vessels per field, 95% CI = 1.2 to 2.8; P = .004). CONCLUSION: CR-1 appears to have an important role in the multistep process of angiogenesis.
Asunto(s)
Neoplasias de la Mama/irrigación sanguínea , Células Endoteliales/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neovascularización Patológica/metabolismo , Animales , Anticuerpos Monoclonales , Biomarcadores de Tumor/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Colágeno , Combinación de Medicamentos , Factor de Crecimiento Epidérmico/inmunología , Factor de Crecimiento Epidérmico/farmacología , Femenino , Factor 2 de Crecimiento de Fibroblastos/análisis , Proteínas Ligadas a GPI , Sustancias de Crecimiento/metabolismo , Humanos , Inmunoensayo , Péptidos y Proteínas de Señalización Intercelular , Laminina , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/farmacología , Ratones , Ratones Desnudos , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/farmacología , Proteoglicanos , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Trasplante Heterólogo , Venas Umbilicales , Factor A de Crecimiento Endotelial Vascular/análisisRESUMEN
Epithelial-mesenchymal transition (EMT) facilitates migration and invasion of epithelial tumor cells. Cripto-1 (CR-1), a member of the epidermal growth factor-CFC protein family increases migration of cells in vitro. Here the expression of molecular markers and signaling molecules characteristic of EMT were assessed in mammary gland hyperplasias and tumors from mice expressing the human CR-1 transgene by the MMTV promoter (MMTV-CR-1) and in mouse mammary epithelial cell line HC-11 overexpressing CR-1 (HC-11/CR-1). Western blot analysis showed decreased expression of E-cadherin in MMTV-CR-1 tumors and in HC-11/CR-1 cells. The expression of N-cadherin, vimentin, cyclin-D1, and of the zinc-finger transcription factor, snail, was increased in MMTV-CR-1 tumors. Increased snail mRNA was also found in HC-11/CR-1 cells. Expression of phosphorylated (P)-c-Src, P-focal adhesion kinase (FAK), P-Akt, P-glycogen synthease kinase 3beta (GSK-3beta), dephosphorylated (DP)-beta-catenin, and various integrins such as, alpha 3, alpha v, beta 1, beta 3, and beta 4 was also increased in MMTV-CR-1 tumors. Immunohistochemistry showed positive staining for vimentin, N-cadherin, cyclin-D1, smooth muscle actin, fibronectin, snail, and beta-catenin in MMTV-CR-1 tumor sections. HC-11/CR-1 cells treated with the c-Src inhibitor PP2 reduced the expression of P-c-Src and of P-FAK, P-Akt, P-GSK-3beta, DP-beta-catenin all known to be activated by c-Src. Migration of HC-11/CR-1 cells was also reduced by PP2 treatment. These results suggest that CR-1 may play a significant role in promoting the increased expression of markers and signaling molecules associated with EMT.