RESUMO
Epithelial-mesenchymal transition (EMT), crucial during embryogenesis for new tissue and organ formation, is also considered to be a prerequisite to cancer metastasis. We report here that the protein tyrosine phosphatase Pez is expressed transiently in discrete locations in developing brain, heart, pharyngeal arches, and somites in zebrafish embryos. We also find that Pez knock-down results in defects in these organs, indicating a crucial role in organogenesis. Overexpression of Pez in epithelial MDCK cells causes EMT, with a drastic change in cell morphology and function that is accompanied by changes in gene expression typical of EMT. Transfection of Pez induced TGFbeta signaling, critical in developmental EMT with a likely role also in oncogenic EMT. In zebrafish, TGFbeta3 is co- expressed with Pez in a number of tissues and its expression was lost from these tissues when Pez expression was knocked down. Together, our data suggest Pez plays a crucial role in organogenesis by inducing TGFbeta and EMT.
Assuntos
Células Epiteliais/citologia , Mesoderma/citologia , Organogênese , Proteínas Tirosina Fosfatases/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Núcleo Celular/metabolismo , Cães , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Organogênese/efeitos dos fármacos , Fenótipo , Transporte Proteico/efeitos dos fármacos , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad4/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta3/genética , Fator de Crescimento Transformador beta3/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Factors secreted by tumor cells shape the local microenvironment to promote invasion and metastasis, as well as condition the premetastatic niche to enable secondary-site colonization and growth. In addition to this secretome, tumor cells have increased abundance of growth-promoting receptors at the cell surface. We found that the tyrosine phosphatase PTPN14 (also called Pez, which is mutated in various cancers) suppressed metastasis by reducing intracellular protein trafficking through the secretory pathway. Knocking down PTPN14 in tumor cells or injecting the peritoneum of mice with conditioned medium from PTPN14-deficient cell cultures promoted the growth and metastasis of breast cancer xenografts. Loss of catalytically functional PTPN14 increased the secretion of growth factors and cytokines, such as IL-8 (interleukin-8), and increased the abundance of EGFR (epidermal growth factor receptor) at the cell surface of breast cancer cells and of FLT4 (vascular endothelial growth factor receptor 3) at the cell surface of primary lymphatic endothelial cells. We identified RIN1 (Ras and Rab interactor 1) and PRKCD (protein kinase C-δ) as binding partners and substrates of PTPN14. Similar to cells overexpressing PTPN14, receptor trafficking to the cell surface was inhibited in cells that lacked PRKCD or RIN1 or expressed a nonphosphorylatable RIN1 mutant, and cytokine secretion was decreased in cells treated with PRKCD inhibitors. Invasive breast cancer tissue had decreased expression of PTPN14, and patient survival was worse when tumors had increased expression of the genes encoding RIN1 or PRKCD. Thus, PTPN14 prevents metastasis by restricting the trafficking of both soluble and membrane-bound proteins.
Assuntos
Neoplasias da Mama/metabolismo , Metástase Neoplásica/fisiopatologia , Transporte Proteico/fisiologia , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Microambiente Tumoral/fisiologia , Animais , Western Blotting , Linhagem Celular Tumoral , Cromatografia Líquida , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Técnicas de Silenciamento de Genes , Xenoenxertos/metabolismo , Xenoenxertos/fisiopatologia , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Marcação por Isótopo , Camundongos , Invasividade Neoplásica/genética , Metástase Neoplásica/prevenção & controle , Proteína Quinase C-delta/antagonistas & inibidores , Proteína Quinase C-delta/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/farmacologia , Espectrometria de Massas em Tandem , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Pigs are currently considered the most likely source of organs for human xenotransplantation because of anatomical and physiological similarities to humans, and the relative ease with which they can be bred in large numbers. A severe form of rejection known as hyperacute rejection has been the major barrier to the use of xenografts. Generating transgenic pigs for organ transplantation is likely to involve precise genetic manipulation to ablate the alpha(1,3) galactosyltransferase (galT) gene. In contrast to the mouse, homologous recombination in livestock species to ablate genes is hampered by the inability to isolate functional embryonic stem cells. However, nuclear transfer using genetically targeted cultured somatic cells provides an alternative means to producing pigs deficient for galT. In this study we successfully produced galT+/- somatic porcine fetal fibroblasts using two approaches; positive negative selection (PNS) using an isogenic targeting construct, and with a promoterless vector using non-isogenic DNA.