RESUMEN
Neuraminidases (NEUs) also called sialidases are glycosidases which catalyze the removal of terminal sialic acid residues from glycoproteins, glycolipids, and oligosaccharides. Mammalian NEU-1 participates in regulation of cell surface receptors such as insulin receptor (IR), epithelial growth factor receptor, low-density lipoprotein receptor, and toll-like receptor 4. At the plasma membrane, NEU-1 can be associated with the elastin-binding protein and the carboxypeptidase protective protein/cathepsin A to constitute the elastin receptor complex. In this complex, NEU-1 is essential for elastogenesis, signal transduction through this receptor and for biological effects of the elastin-derived peptides on atherosclerosis, thrombosis, insulin resistance, nonalcoholic steatohepatitis, and cancers. This is why research teams are developing inhibitors targeting this sialidase. Previously, we developed interfering peptides to inhibit the dimerization and the activation of NEU-1. In this study, we investigated the effects of these peptides on IR activation in vitro and in vivo. Using cellular overexpression and endogenous expression models of NEU-1 and IR (COS-7 and HepG2 cells, respectively), we have shown that interfering peptides inhibit NEU-1 dimerization and sialidase activity which results in a reduction of IR phosphorylation. These results demonstrated that NEU-1 positively regulates IR phosphorylation and activation in our conditions. In vivo, biodistribution study showed that interfering peptides are well distributed in mice. Treatment of C57Bl/6 mice during 8 weeks with interfering peptides induces a hyperglycemic effect in our experimental conditions. Altogether, we report here that inhibition of NEU-1 sialidase activity by interfering peptides decreases IR activity in vitro and glucose homeostasis in vivo.
Asunto(s)
Neuraminidasa , Receptor de Insulina , Neuraminidasa/metabolismo , Neuraminidasa/antagonistas & inhibidores , Animales , Receptor de Insulina/metabolismo , Humanos , Ratones , Células Hep G2 , Chlorocebus aethiops , Homeostasis/efectos de los fármacos , Péptidos/farmacología , Péptidos/química , Masculino , Glucosa/metabolismo , Ratones Endogámicos C57BL , Receptores de Superficie CelularRESUMEN
The composition and physical properties of the extracellular matrix (ECM) critically influence tumor progression, but the molecular mechanisms underlying ECM layering are poorly understood. Tumor-stroma interaction critically depends on cell communication mediated by exosomes, small vesicles generated within multivesicular bodies (MVBs). We show that caveolin-1 (Cav1) centrally regulates exosome biogenesis and exosomal protein cargo sorting through the control of cholesterol content at the endosomal compartment/MVBs. Quantitative proteomics profiling revealed that Cav1 is required for exosomal sorting of ECM protein cargo subsets, including Tenascin-C (TnC), and for fibroblast-derived exosomes to efficiently deposit ECM and promote tumor invasion. Cav1-driven exosomal ECM deposition not only promotes local stromal remodeling but also the generation of distant ECM-enriched stromal niches in vivo. Cav1 acts as a cholesterol rheostat in MVBs, determining sorting of ECM components into specific exosome pools and thus ECM deposition. This supports a model by which Cav1 is a central regulatory hub for tumor-stroma interactions through a novel exosome-dependent ECM deposition mechanism.
Asunto(s)
Caveolina 1/fisiología , Exosomas/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Cuerpos Multivesiculares/metabolismo , Proteoma/metabolismo , Tenascina/fisiología , Animales , Fibroblastos/citología , Ratones , Ratones NoqueadosRESUMEN
Current treatments in multiple sclerosis (MS) are modulating the inflammatory component of the disease, but no drugs are currently available to repair lesions. Our study identifies in MS patients the overexpression of Plexin-A1, the signalling receptor of the oligodendrocyte inhibitor Semaphorin 3A. Using a novel type of peptidic antagonist, we showed the possibility to counteract the Sema3A inhibitory effect on oligodendrocyte migration and differentiation in vitro when antagonizing Plexin-A1. The use of this compound in vivo demonstrated a myelin protective effect as shown with DTI-MRI and confirmed at the histological level in the mouse cuprizone model of induced demyelination/remyelination. This effect correlated with locomotor performances fully preserved in chronically treated animals. The administration of the peptide also showed protective effects, leading to a reduced severity of demyelination in the context of experimental autoimmune encephalitis (EAE). Hence, the disruption of the inhibitory microenvironmental molecular barriers allows normal myelinating cells to exert their spontaneous remyelinating capacity. This opens unprecedented therapeutic opportunity for patients suffering a disease for which no curative options are yet available.
Asunto(s)
Esclerosis Múltiple/fisiopatología , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/fisiología , Receptores de Superficie Celular/metabolismo , Remielinización , Semaforina-3A/metabolismo , Transducción de Señal , Animales , Encéfalo/diagnóstico por imagen , Línea Celular , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Imagen por Resonancia Magnética , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Receptores de Superficie Celular/antagonistas & inhibidoresRESUMEN
Components with self-assembly properties derived from plant viruses provide the opportunity to design biological nanoscaffolds for the ordered display of agents of diverse nature and with complementing functions. With the aim of designing a functionalized nanoscaffold to target cancer, the coat protein (CP) of Tobacco mosaic virus (TMV) was tested as nanocarrier for an insoluble, highly hydrophobic peptide that targets the transmembrane domain of the Neuropilin-1 (NRP1) receptor in cancer cells. The resulting construct CPL-K (CP-linker-"Kill") binds to NRP1 in cancer cells and disrupts NRP1 complex formation with PlexA1 as well as downstream Akt survival signaling. The application of CPL-K also inhibits angiogenesis and cell migration. CP was also fused to a peptide that targets the extracellular domain of NRP1 and this fusion protein (CPL-F, CP-Linker-"Find") is shown to bind to cultured cancer cells and to inhibit NRP1-dependent angiogenesis as well. CPL-K and CPL-F maintain their anti-angiogenic properties upon co-assembly to oligomers/nanoparticles together with CPL. The observations show that the CP of TMV can be employed to generate a functionalized nanoparticle with biological activity. Remarkably, fusion to CPL allowed us to solubilize the highly insoluble transmembrane NRP1 peptide and to retain its anti-angiogenic effect.
RESUMEN
Metastasis is a major cause of death in cancer patients. The extracellular matrix molecule tenascin-C is a known promoter of metastasis, however the underlying mechanisms are not well understood. To further analyze the impact of tenascin-C on cancer progression we generated MMTV-NeuNT mice that develop spontaneous mammary tumors, on a tenascin-C knockout background. We also developed a syngeneic orthotopic model in which tumor cells derived from a MMTV-NeuNT tumor. Tumor cells were transfected with control shRNA or with shRNA to knockdown tenascin-C expression and, were grafted into the mammary gland of immune competent, wildtype or tenascin-C knockout mice. We show that stromal-derived tenascin-C increases metastasis by reducing apoptosis and inducing the cellular plasticity of cancer cells located in pulmonary blood vessels invasions (BVI), before extravasation. We characterized BVI as organized structures of tightly packed aggregates of proliferating tumor cells with epithelial characteristics, surrounded by Fsp1+ cells, internally located platelets and, a luminal monolayer of endothelial cells. We found extracellular matrix, in particular, tenascin-C, between the stromal cells and the tumor cell cluster. In mice lacking stromal-derived tenascin-C, the organization of pulmonary BVI was significantly affected, revealing novel functions of host-derived tenascin-C in supporting the integrity of the endothelial cell coat, increasing platelet abundance, tumor cell survival, epithelial plasticity, thereby promoting overall lung metastasis. Many effects of tenascin-C observed in BVI including enhancement of cellular plasticity, survival and migration, could be explained by activation of TGF-ß signaling. Finally, in several human cancers, we also observed BVI to be surrounded by an endothelial monolayer and to express tenascin-C. Expression of tenascin-C is specific to BVI and is not observed in lymphatic vascular invasions frequent in breast cancer, which lack an endothelial lining. Given that BVI have prognostic significance for many tumor types, such as shorter cancer patient survival, increased metastasis, vessel occlusion, and organ failure, our data revealing a novel mechanism by which stromal tenascin-C promotes metastasis in human cancer, may have potential for diagnosis and therapy.
Asunto(s)
Vasos Sanguíneos/patología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/patología , Receptor ErbB-2/genética , Tenascina/genética , Animales , Vasos Sanguíneos/metabolismo , Línea Celular Tumoral , Femenino , Técnicas de Inactivación de Genes , Humanos , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/genética , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Transgénicos , Ratas , Transducción de Señal , Células del Estroma , Tenascina/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The sensory innervation of the dental pulp is essential for tooth function and protection. It is mediated by axons originating from the trigeminal ganglia and is spatio-temporally regulated. We have previously shown that the innervation of bioengineered teeth can be achieved only under immunosuppressive conditions. The aim of this study was to develop a model to determine the role of Semaphorin 3A (Sema3A) in the innervation of bioengineered teeth. We first analysed innervation of the dental pulp of mandibular first molars in newborn (postnatal day 0: PN0) mice deficient for Sema3A (Sema3A-/- ), a strong inhibitor of axon growth. While at PN0, axons detected by immunostaining for peripherin and NF200 were restricted to the peridental mesenchyme in Sema3A+/+ mice, they entered the dental pulp in Sema3A-/- mice. Then, we have implanted cultured teeth obtained from embryonic day-14 (E14) molar germs of Sema3A-/- mice together with trigeminal ganglia. The dental pulps of E14 cultured and implanted Sema3A-/- teeth were innervated, whereas the axons did not enter the pulp of E14 Sema3A+/+ cultured and implanted teeth. A "Membrane Targeting Peptide NRP1," suppressing the inhibitory effect of Sema3A, has been previously identified. The injection of this peptide at the site of implantation allowed the innervation of the dental pulp of bioengineered teeth obtained from E14 dental dissociated mesenchymal and epithelial cells reassociations of ICR mice. In conclusion, these data show that inhibition of only one axon repellent molecule, Sema3A, allows for pulp innervation of bioengineered teeth.
Asunto(s)
Pulpa Dental , Diente Molar , Receptores de Superficie Celular/metabolismo , Semaforina-3A/metabolismo , Ingeniería de Tejidos , Ganglio del Trigémino , Animales , Pulpa Dental/inervación , Pulpa Dental/metabolismo , Pulpa Dental/patología , Mandíbula/inervación , Mandíbula/metabolismo , Mandíbula/patología , Ratones , Ratones Endogámicos ICR , Ratones Mutantes , Diente Molar/inervación , Diente Molar/metabolismo , Diente Molar/patología , Receptores de Superficie Celular/genética , Semaforina-3A/genética , Ganglio del Trigémino/metabolismo , Ganglio del Trigémino/patologíaRESUMEN
The extracellular matrix (ECM) molecule tenascin-C (TNC) promotes tumor progression. This has recently been demonstrated in the stochastic murine RIP1-Tag2 insulinoma model, engineered to either express TNC abundantly or to be devoid of TNC. However, our knowledge about organization of the TNC microenvironment is scant. Here we determined the spatial distribution of TNC together with other ECM molecules in murine RIP1-Tag2 insulinoma and human cancer tissue (insulinoma and colorectal carcinoma). We found that TNC is organized in matrix tracks together with other ECM molecules of the AngioMatrix signature, a previously described gene expression profile that characterizes the angiogenic switch. Moreover, stromal cells including endothelial cells, fibroblasts and leukocytes were enriched in the TNC tracks. Thus, TNC tracks may provide niches for stromal cells and regulate their behavior. Given similarities of TNC rich niches for stromal cells in human insulinoma and colon cancer, we propose that the RIP1-Tag2 model may be useful for providing insights into the contribution of the tumor stroma specific ECM as promoter of cancer progression.
Asunto(s)
Movimiento Celular/fisiología , Neoplasias Colorrectales/metabolismo , Matriz Extracelular/metabolismo , Células del Estroma/patología , Tenascina/metabolismo , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Humanos , Ratones TransgénicosRESUMEN
Angiogenesis represents a rate-limiting step during tumor progression. Targeting angiogenesis is already applied in cancer treatment, yet limits of anti-angiogenic therapies have emerged, notably because tumors adapt and recur after treatment. Therefore, there is a strong need to better understand the molecular and cellular mechanisms underlying tumor angiogenesis. Using the RIP1-Tag2 transgenic murine model, we identified 298 genes that are deregulated during the angiogenic switch, revealing an ingression/expansion of specific stromal cell types including endothelial cells and pericytes, but also macrophages and perivascular mesenchymal cells. Canonical TGF-ß signaling is up-regulated during the angiogenic switch, especially in tumor-associated macrophages and fibroblasts. The matrisome, comprising extracellular matrix (ECM) and ECM-associated molecules, is significantly enriched, which allowed us to define the AngioMatrix signature as the 110 matrisomal genes induced during the RIP1-Tag2 angiogenic switch. Several AngioMatrix molecules were validated at expression level. Ablation of tenascin-C, one of the most highly induced ECM molecules during the switch, resulted in reduced angiogenesis confirming its important role. In human glioma and colorectal samples, the AngioMatrix signature correlates with the expression of endothelial cell markers, is increased with tumor progression and finally correlates with poor prognosis demonstrating its diagnostic and therapeutic potential.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Matriz Extracelular/metabolismo , Proteínas Activadoras de GTPasa/fisiología , Perfilación de la Expresión Génica , Glioma/genética , Neovascularización Patológica/genética , Animales , Biomarcadores de Tumor/metabolismo , Células Cultivadas , Neoplasias Colorrectales/irrigación sanguínea , Neoplasias Colorrectales/mortalidad , Fibroblastos/citología , Fibroblastos/metabolismo , Glioma/irrigación sanguínea , Glioma/mortalidad , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Células del Estroma/citología , Células del Estroma/metabolismo , Tasa de SupervivenciaRESUMEN
The extracellular matrix molecule tenascin-C (TNC) is a major component of the cancer-specific matrix, and high TNC expression is linked to poor prognosis in several cancers. To provide a comprehensive understanding of TNC's functions in cancer, we established an immune-competent transgenic mouse model of pancreatic ß-cell carcinogenesis with varying levels of TNC expression and compared stochastic neuroendocrine tumor formation in abundance or absence of TNC. We show that TNC promotes tumor cell survival, the angiogenic switch, more and leaky vessels, carcinoma progression, and lung micrometastasis. TNC downregulates Dickkopf-1 (DKK1) promoter activity through the blocking of actin stress fiber formation, activates Wnt signaling, and induces Wnt target genes in tumor and endothelial cells. Our results implicate DKK1 downregulation as an important mechanism underlying TNC-enhanced tumor progression through the provision of a proangiogenic tumor microenvironment.
Asunto(s)
Regulación hacia Abajo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Tenascina/metabolismo , Proteínas Wnt/metabolismo , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Modelos Animales de Enfermedad , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica , Tumores Neuroendocrinos/metabolismo , Tumores Neuroendocrinos/patología , Transducción de Señal , Tenascina/deficiencia , Tenascina/genética , Proteínas Wnt/antagonistas & inhibidoresRESUMEN
Tenascins are extracellular matrix glycoproteins associated with cell motility, proliferation and differentiation. Tenascin-C inhibits cell spreading by binding to fibronectin; tenascin-R and tenascin-X also have anti-adhesive properties in vitro. Here we have studied the adhesion modulating properties of the most recently characterized tenascin, tenascin-W. C2C12 cells, a murine myoblast cell line, will form broad lamellipodia with stress fibers and focal adhesion complexes after culture on fibronectin. In contrast, C2C12 cells cultured on tenascin-W fail to spread and form stress fibers or focal adhesion complexes, and instead acquire a multipolar shape with short, actin-tipped pseudopodia. The same stellate morphology is observed when C2C12 cells are cultured on a mixture of fibronectin and tenascin-W, or on fibronectin in the presence of soluble tenascin-W. Tenascin-W combined with fibronectin also inhibits the spreading of mouse embryo fibroblasts when compared with cells cultured on fibronectin alone. The similarity between the adhesion modulating effects of tenascin-W and tenascin-C in vitro led us to study the possibility of tenascin-W compensating for tenascin-C in tenascin-C knockout mice, especially during epidermal wound healing. Dermal fibroblasts harvested from a tenascin-C knockout mouse express tenascin-W, but dermal fibroblasts taken from a wild type mouse do not. However, there is no upregulation of tenascin-W in the dermis of tenascin-C knockout mice, or in the granulation tissue of skin wounds in tenascin-C knockout animals. Similarly, tenascin-X is not upregulated in early wound granulation tissue in the tenascin-C knockout mice. Thus, tenascin-W is able to inhibit cell spreading in vitro and it is upregulated in dermal fibroblasts taken from the tenascin-C knockout mouse, but neither it nor tenascin-X are likely to compensate for missing tenascin-C during wound healing.