RESUMEN
AIMS: Muscle and adipose tissue-derived mesenchymal stem cells presented high osteogenic potentials, which modulate osteoblast function through releasing extracellular vesicles (EVs) containing miRNAs. Herein, this study evaluated the function of bone marrow mesenchymal stem cell-derived extracellular vesicles (BMSC-EVs) delivering miR-497-5p in ossification of the posterior longitudinal ligament (OPLL). MAIN METHODS: The expression level of miR-497-5p was validated in ossified posterior longitudinal ligament (PLL) tissues and BMSC-EVs. The uptake of BMSC-EVs by ligament fibroblasts was observed by immunofluorescence. miR-497-5p was overexpressed or downregulated to assess its role in osteogenic differentiation of ligament fibroblasts. Further, an OPLL rat model was established to substantiate the effect of BMSC-EVs enriched with miR-497-5p on OPLL. KEY FINDINGS: Ossified PLL tissues presented with high miR-497-5p expression. PLL fibroblasts were identified to endocytose BMSC-EVs. BMSC-EVs could upregulate miR-497-5p and shuttle it to ligament fibroblasts to accelerate the osteogenic differentiation. miR-497-5p targeted and inversely regulated RSPO2. Then, RSPO2 overexpression activated Wnt/ß-catenin pathway and repressed the osteogenic differentiation of ligament fibroblasts. In vivo experiments further showed that miR-497-5p-containing BMSC-EVs enhanced OPLL through diminishing RSPO2 and inactivating Wnt/ß-catenin pathway. SIGNIFICANCE: BMSC-EVs could deliver miR-497-5p to ligament fibroblasts and modulate RSPO2-mediated Wnt/ß-catenin pathway, thereby accelerating OPLL.
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Diferenciación Celular , Vesículas Extracelulares/patología , Regulación de la Expresión Génica , Células Madre Mesenquimatosas/patología , MicroARNs/genética , Osificación del Ligamento Longitudinal Posterior/patología , Trombospondinas/antagonistas & inhibidores , Animales , Vesículas Extracelulares/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Osificación del Ligamento Longitudinal Posterior/genética , Osificación del Ligamento Longitudinal Posterior/metabolismo , Ratas , Ratas Wistar , Trombospondinas/genética , Trombospondinas/metabolismoRESUMEN
BACKGROUND: Aquaporin (AQP) 1 expression has been linked with tumor malignancy but its role in glioblastoma (GBM), a lethal glioma, remains to be clarified. METHODS: AQP1 expression was examined in 33 human GBM specimens by immunohistochemistry. GBM cells (U251 and U87) that stably express AQP1 were established and used for cellular proliferation, migration, invasion, and vascular tube formation assays. The GeneChip assay was used to identify differentially expressed genes in AQP1-expressing cells. RESULTS: AQP1 was expressed only in tumor cells. AQP1 dose-dependently accelerated cell migration and invasion, but not proliferation, in GBM cell lines. AQP1 also upregulated cathepsin B, focal adhesion kinase and activities of matrix metalloproteinase 9. AQP1 in GBM cells induced wall thickness of ECV304, vascular endothelial cells, in a contact-dependent manner. Downregulation of thrombospondin type 1 domain containing 7A (THSD7A) was identified in AQP1-expressing GBM cells in vitro, and was negatively correlated with AQP1 expression in human GBM specimens. CONCLUSION: AQP1 is involved in tumor malignancy by facilitating the migration and invasion of GBM cells, and promoting the formation of vascular beds that are characteristic of GBM by downregulating THSD7A.
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Acuaporina 1/metabolismo , Biomarcadores de Tumor/metabolismo , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Glioblastoma/irrigación sanguínea , Neovascularización Patológica/patología , Trombospondinas/antagonistas & inhibidores , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis , Acuaporina 1/genética , Biomarcadores de Tumor/genética , Proliferación Celular , Regulación hacia Abajo , Femenino , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Masculino , Persona de Mediana Edad , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Pronóstico , Tasa de Supervivencia , Trombospondinas/genética , Trombospondinas/metabolismo , Células Tumorales CultivadasRESUMEN
Primary membranous nephropathy (PMN) is a renal-specific autoimmune disease caused by circulating autoantibodies that target glomerular podocyte antigens (PLA2R/THSD7A). However, very little is known on the molecular mechanisms controlling B cell response in this nephropathy. The present study was aimed at correlating the serum levels of B cell activators BAFF/BLyS and APRIL with the presence of anti-PLA2R antibodies in PMN patients and with long-term clinical outcome. To this aim, 51 patients with anti-PLA2R-positive biopsy-proven PMN and nephrotic range proteinuria (>3.5 g/24 hours) were enrolled between January 2009 and December 2015 and treated with conventional 6-month immunosuppressive therapy. After 6 months, 29 patients (56.9%) cleared circulating anti-PLA2R, while in remaining 22 (43.1%), they persisted. Intriguingly, in the first group, baseline serum levels of BAFF/BLyS and APRIL were significantly lower than those in the second one. Moreover, after 6 months of immunosuppressive therapy, an overall reduction in both cytokine serum levels was observed. However, in PMN patients with anti-PLA2R clearance, this reduction was more prominent, as compared with those with anti-PLA2R persistence. When related to clinical outcome, lower baseline BAFF/BLyS (<6.05 ng/mL) and APRIL (<4.20 ng/mL) serum levels were associated with significantly higher probability to achieve complete or partial remission after 24-month follow-up. After dividing the entire study cohort into three groups depending on both cytokine baseline serum levels, patients with both BAFF/BLyS and APRIL below the cut-off showed a significantly higher rate of complete or partial remission as compared with patients with only one cytokine above the cut-off, while the composite endpoint was achieved in a very low rate of patients with both cytokines above the cut-off. Taken together, these results provide new insights into the role of BAFF/BLyS and APRIL in both the pathogenesis of anti-PLA2R-positive PMN and the response to immunosuppressive therapy.
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Factor Activador de Células B/sangre , Glomerulonefritis Membranosa/sangre , Glomerulonefritis Membranosa/tratamiento farmacológico , Inmunosupresores/uso terapéutico , Receptores de Fosfolipasa A2/antagonistas & inhibidores , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/sangre , Adulto , Anciano , Biomarcadores , Biopsia , Femenino , Glomerulonefritis Membranosa/inmunología , Glomerulonefritis Membranosa/metabolismo , Humanos , Inmunoensayo , Inmunosupresores/farmacología , Incidencia , Masculino , Persona de Mediana Edad , Curva ROC , Trombospondinas/antagonistas & inhibidoresRESUMEN
A Disintegrin and Metalloproteinase (ADAM) is a family of proteolytic enzymes that possess sheddase function and regulate shedding of membrane-bound proteins, growth factors, cytokines, ligands and receptors. Typically, ADAMs have a pro-domain, and a metalloproteinase, disintegrin, cysteine-rich and a characteristic transmembrane domain. Most ADAMs are activated by proprotein convertases, but can also be regulated by G-protein coupled receptor agonists, Ca2+ ionophores and protein kinase C activators. A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) is a family of secreted enzymes closely related to ADAMs. Like ADAMs, ADAMTS members have a pro-domain, and a metalloproteinase, disintegrin, and cysteine-rich domain, but they lack a transmembrane domain and instead have characteristic thrombospondin motifs. Activated ADAMs perform several functions and participate in multiple cardiovascular processes including vascular smooth muscle cell proliferation and migration, angiogenesis, vascular cell apoptosis, cell survival, tissue repair, and wound healing. ADAMs may also be involved in pathological conditions and cardiovascular diseases such as atherosclerosis, hypertension, aneurysm, coronary artery disease, myocardial infarction and heart failure. Like ADAMs, ADAMTS have a wide-spectrum role in vascular biology and cardiovascular pathophysiology. ADAMs and ADAMTS activity is naturally controlled by endogenous inhibitors such as tissue inhibitors of metalloproteinases (TIMPs), and their activity can also be suppressed by synthetic small molecule inhibitors. ADAMs and ADAMTS can serve as important diagnostic biomarkers and potential therapeutic targets for cardiovascular disorders. Natural and synthetic inhibitors of ADAMs and ADAMTS could be potential therapeutic tools for the management of cardiovascular diseases.
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Proteínas ADAM/metabolismo , Desintegrinas/metabolismo , Endotelio Vascular/metabolismo , Trombospondinas/metabolismo , Enfermedades Vasculares/metabolismo , Proteínas ADAM/antagonistas & inhibidores , Secuencias de Aminoácidos/efectos de los fármacos , Secuencias de Aminoácidos/fisiología , Animales , Desintegrinas/antagonistas & inhibidores , Endotelio Vascular/efectos de los fármacos , Humanos , Inhibidores de la Metaloproteinasa de la Matriz/administración & dosificación , Trombospondinas/antagonistas & inhibidores , Enfermedades Vasculares/tratamiento farmacológicoRESUMEN
BACKGROUND/AIMS: Fibroblast growth factor receptor 2 (FGFR2) has attracted considerable interest as a therapeutic target in gastric cancer (GC). There is growing evidence to suggest that the bioavailability of the potent pro-tumor function of FGFR2 is associated with thrombospondins (TSPs). As a follow-on from our previous study, here we evaluated the potential clinical significance and mechanism of the relationship between FGFR2 and TSP4 in GC. METHODS: Expression levels of FGFR2 and TSP4 were detected by immunohistochemistry in GC tissue microarray slides. SGC7901 and MKN28 cell lines were used to confirm the relationship between FGFR2 and TSP4. In vitro cell viability, colony formation, and invasion and migration assays were performed to evaluate the effect of FGFR2-TSP4 axis on tumor cell activities. The mechanism of TSP4 regulated by FGFG2 was explored via small molecular inhibitors in vitro and a xenograft model. RESULTS: FGFR2 was shown to be markedly overexpressed in GC tissues and was correlated with a high risk of lymph node metastasis, late clinical stage, and poor prognosis. Low TSP4 expression was associated with shorter overall survival (OS) and advanced stage in GC patients. Interestingly, correlation analysis indicated that FGFR2 was negatively associated with TSP4. Indeed, in vitro and in vivo experiments suggested FGFR2 activation could downregulate TSP4 expression, which played an important role in the proliferation, invasion and migration of GC cells. We also found involvement of the PI3K-AKT-mTOR pathway in the FGFR2-TSP4 axis. CONCLUSION: The FGFR2 signal promotes human GC progression through the downregulation of TSP4 via PI3K-AKT-mTOR pathway. Our findings provide a foundation for further investigating promising therapeutic strategies for GC overexpressing FGFR2.
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Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Neoplasias Gástricas/patología , Serina-Treonina Quinasas TOR/metabolismo , Trombospondinas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Everolimus/uso terapéutico , Femenino , Humanos , Estimación de Kaplan-Meier , Metástasis Linfática , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Transducción de Señal , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidad , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Trombospondinas/antagonistas & inhibidoresRESUMEN
To investigate whether the human anti-thrombospondin type 1 domain-containing 7A (THSD7A) antibody-induced membranous nephropathy (MN) is mediated by activating lectin complement pathway. Automatic biochemical apparatus was used to assess renal function of mice. The serum levels of anti-THSD7A antibodies and complement were tested by using ELISA. The expression level of THSD7A and mannose-binding lectin (MBL) in clinical tissue, and the histological features of MN in mice were examined by immunochemical methods. We found that THSD7A, MBL, and complement expression level from patients with circulating anti-THSD7A antibodies were significantly higher than that in normal group. Furthermore, difference of renal function in anti-THSD7A antibody-containing serum treatment groups and control groups was significant. Meanwhile, human anti-THSD7A autoantibodies activated the complement system and induced the histological features of MN in mice. In conclusion, human anti-THSD7A antibodies induce MN through activating MBL lectin complement pathway in mice.
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Autoanticuerpos/administración & dosificación , Lectina de Unión a Manosa de la Vía del Complemento/genética , Proteínas del Sistema Complemento/inmunología , Glomerulonefritis Membranosa/inmunología , Trombospondinas/inmunología , Adulto , Anciano , Animales , Autoanticuerpos/biosíntesis , Estudios de Casos y Controles , Proteínas del Sistema Complemento/genética , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Glomerulonefritis Membranosa/etiología , Glomerulonefritis Membranosa/genética , Glomerulonefritis Membranosa/patología , Humanos , Sueros Inmunes/administración & dosificación , Pruebas de Función Renal , Masculino , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/genética , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/inmunología , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Trombospondinas/antagonistas & inhibidores , Trombospondinas/genéticaRESUMEN
Activating mutations in the Wnt pathway are a characteristic feature of colorectal cancer (CRC). The R-spondin (RSPO) family is a group of secreted proteins that enhance Wnt signaling and RSPO2 and RSPO3 gene fusions have been reported in CRC. We have previously shown that Wnt pathway blockers exhibit potent combinatorial activity with taxanes to inhibit tumor growth. Here we show that RSPO3 antagonism synergizes with paclitaxel based chemotherapies in patient-derived xenograft models (PDX) with RSPO3 fusions and in tumors with common CRC mutations such as APC, ß-catenin, or RNF43. In these latter types of tumors that represent over 90% of CRC, RSPO3 is produced by stromal cells in the tumor microenvironment and the activating mutations appear to sensitize the tumors to Wnt-Rspo synergy. The combination of RSPO3 inhibition and taxane treatment provides an approach to effectively target oncogenic WNT signaling in a significant number of patients with colorectal and other intestinal cancers.
Asunto(s)
Hidrocarburos Aromáticos con Puentes/farmacología , Neoplasias Colorrectales , Mutación , Proteínas de Neoplasias , Paclitaxel/farmacología , Taxoides/farmacología , Trombospondinas , Microambiente Tumoral/efectos de los fármacos , Vía de Señalización Wnt , Animales , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Trombospondinas/antagonistas & inhibidores , Trombospondinas/genética , Trombospondinas/metabolismo , Microambiente Tumoral/genética , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND/AIMS: We recently discovered that harmful variants in THSD1 (Thrombospondin type-1 domain-containing protein 1) likely cause intracranial aneurysm and subarachnoid hemorrhage in a subset of both familial and sporadic patients with supporting evidence from two vertebrate models. The current study seeks to elucidate how THSD1 and patient-identified variants function molecularly in focal adhesions. METHODS: Co-immunostaining and co-immunoprecipitation were performed to define THSD1 subcellular localization and interacting partners. Transient expression of patient-identified THSD1 protein variants and siRNA-mediated loss-of-function THSD1 were used to interrogate gene function in focal adhesion and cell attachment to collagen I in comparison to controls. RESULTS: THSD1 is a novel nascent adhesion protein that co-localizes with several known markers such as FAK, talin, and vinculin, but not with mature adhesion marker zyxin. Furthermore, THSD1 forms a multimeric protein complex with FAK/talin/vinculin, wherein THSD1 promotes talin binding to FAK but not to vinculin, a key step in nascent adhesion assembly. Accordingly, THSD1 promotes mature adhesion formation and cell attachment, while its rare variants identified in aneurysm patients show compromised ability. Interestingly, THSD1 also localizes at different stages of endosomes. Clathrin-mediated but not caveolae-mediated endocytosis pathway is involved in THSD1 intracellular trafficking, which positively regulates THSD1-induced focal adhesion assembly, in contrast to the traditional role of endosomes in termination of integrin signals. CONCLUSIONS: The data suggest that THSD1 functions at the interface between endosome dynamics and nascent focal adhesion assembly that is impaired by THSD1 rare variants identified from intracranial aneurysm patients.
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Endosomas/metabolismo , Adhesiones Focales/metabolismo , Trombospondinas/metabolismo , Clatrina/metabolismo , Endocitosis , Quinasa 1 de Adhesión Focal/metabolismo , Adhesiones Focales/química , Células HEK293 , Células HeLa , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inmunoprecipitación , Aneurisma Intracraneal/genética , Aneurisma Intracraneal/patología , Microscopía Fluorescente , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Talina/metabolismo , Trombospondinas/antagonistas & inhibidores , Trombospondinas/genética , Vinculina/metabolismoRESUMEN
The four R-spondins (RSPOs) and their three related receptors, LGR4, 5 and 6, have emerged as a major ligand-receptor system with critical roles in development and stem cell survival. However, the exact roles of the RSPO-LGR system in osteogenesis remain largely unknown. In the present study, we showed that RSPO3-shRNA increased the osteogenic potential of human adipose-derived stem cells (hASCs) significantly. Mechanistically, we demonstrated that RSPO3 is a negative regulator of ERK/FGF signalling. We confirmed that inhibition of the ERK1/2 signalling pathway blocked osteogenic differentiation in hASCs, and the increased osteogenic capacity observed after RSPO3 knockdown in hASCs was reversed by inhibition of ERK signalling. Further, silencing of LGR4 inhibited the activity of ERK signalling and osteogenic differentiation of hASCs. Most importantly, we found that loss of LGR4 abrogated RSPO3-regulated osteogenesis and RSPO3-induced ERK1/2 signalling inhibition. Collectively, our data show that ERK signalling works downstream of LGR4 and RSPO3 regulates osteoblastic differentiation of hASCs possibly via the LGR4-ERK signalling.
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Diferenciación Celular , Factores de Crecimiento de Fibroblastos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Osteogénesis , Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/metabolismo , Tejido Adiposo/citología , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Butadienos/farmacología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Humanos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/genética , Nitrilos/farmacología , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogénesis/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genética , Transducción de Señal , Células Madre/citología , Células Madre/metabolismo , Trombospondinas/antagonistas & inhibidores , Trombospondinas/genéticaRESUMEN
BACKGROUND: We have previously identified TAX2 peptide as an orthosteric antagonist for thrombospondin-1 (TSP-1) interaction with the cell-surface receptor CD47. TAX2 displays exciting antiangiogenic, antitumor, and antimetastatic properties in both allograft and xenograft models of melanoma as well as pancreatic carcinoma. Here, TAX2 therapeutic potential was investigated in two distinct preclinical mouse models of neuroblastoma. METHODS: SK-N-BE(2) (MYCN-amplified) and SK-N-SH (MYCN-negative) human neuroblastoma cells have been implanted in outbred NMRI nude mice prior to systemic administrations of TAX2, and then tumor growth as well as intratumoral blood flow were longitudinally monitored. At study termination, subcutaneous xenografts were macroscopically and histopathologically examined. RESULTS: In both models, TAX2 induced a significant inhibition of tumor burden in mice engrafted with large pre-established neuroblastoma tumors. Indeed, TAX2 administered at biologically relevant doses sharply alters xenograft vascularization as well as multiple features of tumor progression. CONCLUSION: Altogether, our results present TAX2 peptide specifically targeting TSP-1:CD47 interaction as a new putative therapeutic approach for treating neuroblastoma, whether utilized alone or in combination with existing chemotherapy drugs.
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Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Neuroblastoma/tratamiento farmacológico , Péptidos Cíclicos/farmacología , Trombospondinas/antagonistas & inhibidores , Animales , Antígeno CD47/química , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neovascularización Patológica , Neuroblastoma/patología , Péptidos/química , Trombospondinas/metabolismo , Resultado del Tratamiento , Carga TumoralRESUMEN
UNLABELLED: Aberrant WNT signaling is associated with the formation and growth of numerous human cancer types. The low-density lipoprotein receptor-related protein 6 (LRP6) is the least redundant component of the WNT receptor complex with two independent WNT ligand-binding sites. Using domain antibody (dAb) technology, a bispecific antibody (GSK3178022) to LRP6 was identified that is capable of blocking stimulation in the presence of a range of WNT and R-spondin (RSPO) ligands in vitro GSK3178022 was also efficacious in reducing WNT target gene expression in vivo, in both cancer cell line and patient-derived xenograft models, and delays tumor growth in a patient-derived RSPO fusion model of colorectal cancer. IMPLICATIONS: This article demonstrates the inhibition of a key oncogenic receptor, intractable to mAb inhibition due to multiple independent ligand interaction sites, using an innovative dAb approach. Mol Cancer Res; 14(9); 859-68. ©2016 AACR.
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Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/inmunología , Vía de Señalización Wnt/efectos de los fármacos , Animales , Anticuerpos Biespecíficos/farmacocinética , Línea Celular Tumoral , Femenino , Fibrosarcoma/inmunología , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Fibrosarcoma/terapia , Células HEK293 , Humanos , Ligandos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/antagonistas & inhibidores , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Trombospondinas/antagonistas & inhibidores , Trombospondinas/inmunología , Trombospondinas/metabolismo , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/inmunología , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Colorectal cancer remains a major unmet medical need, prompting large-scale genomics efforts in the field to identify molecular drivers for which targeted therapies might be developed. We previously reported the identification of recurrent translocations in R-spondin genes present in a subset of colorectal tumours. Here we show that targeting RSPO3 in PTPRK-RSPO3-fusion-positive human tumour xenografts inhibits tumour growth and promotes differentiation. Notably, genes expressed in the stem-cell compartment of the intestine were among those most sensitive to anti-RSPO3 treatment. This observation, combined with functional assays, suggests that a stem-cell compartment drives PTPRK-RSPO3 colorectal tumour growth and indicates that the therapeutic targeting of stem-cell properties within tumours may be a clinically relevant approach for the treatment of colorectal tumours.
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Diferenciación Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Terapia Molecular Dirigida , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Trombospondinas/metabolismo , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Anticuerpos/uso terapéutico , División Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Progresión de la Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mucosa Intestinal/metabolismo , Intestinos/citología , Intestinos/efectos de los fármacos , Intestinos/patología , Masculino , Ratones , Células Madre Neoplásicas/metabolismo , Células Madre/citología , Células Madre/metabolismo , Trombospondinas/antagonistas & inhibidores , Trombospondinas/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
During metastatic colonization, tumor cells must establish a favorable microenvironment or niche that will sustain their growth. However, both the temporal and molecular details of this process remain poorly understood. Here, we found that metastatic initiating cells (MICs) exhibit a high capacity for lung fibroblast activation as a result of Thrombospondin 2 (THBS2) expression. Importantly, inhibiting the mesenchymal phenotype of MICs by blocking the epithelial-to-mesenchymal transition (EMT)-associated kinase AXL reduces THBS2 secretion, niche-activating ability, and, consequently, metastatic competence. Subsequently, disseminated metastatic cells revert to an AXL-negative, more epithelial phenotype to proliferate and decrease the phosphorylation levels of TGF-ß-dependent SMAD2-3 in favor of BMP/SMAD1-5 signaling. Remarkably, newly activated fibroblasts promote this transition. In summary, our data reveal a crosstalk between cancer cells and their microenvironment whereby the EMT status initially triggers and then is regulated by niche activation during metastatic colonization.
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Células Madre Neoplásicas/metabolismo , Trombospondinas/metabolismo , Animales , Benzocicloheptenos/toxicidad , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Antígeno CD24/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal , Femenino , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Ratones , Ratones Desnudos , Ratones Transgénicos , Metástasis de la Neoplasia , Células Madre Neoplásicas/citología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Proteínas Smad/genética , Proteínas Smad/metabolismo , Trombospondinas/antagonistas & inhibidores , Trombospondinas/genética , Factor de Crecimiento Transformador beta/metabolismo , Trasplante Heterólogo , Triazoles/toxicidad , Tirosina Quinasa del Receptor AxlRESUMEN
The thrombospondin (TSP) family consists of five multimeric, multidomain calcium-binding glycoproteins that act as regulators of cell-cell and cell-matrix associations as well as interact with other extracellular matrix molecules affecting their function. Increasing interest on cardiac TSP-1, TSP-2 and TSP-4 has emerged, and they have been studied in cardiac hypertrophy, myocardial infarction, heart failure, atherosclerosis and aortic valve stenosis. The aim of this MiniReview is to summarize the current knowledge on each TSP in various cardiovascular pathologies. We specifically emphasize the role of TSPs in cardiac remodelling and evaluate TSPs as potential cardiovascular drug targets. Thrombospondin-1 (TSP-1) is the most studied TSP, being antiangiogenic and able to activate transforming growth factor-ß. The functions of TSP-2 and TSP-4 are linked in maintaining the composition of the matrix of the hypertrophied heart, whereas there is very little knowledge on cardiac TSP-3 and TSP-5. TSP-1, TSP-2 and TSP-4 have been shown to affect cardiac remodelling in vivo, for example, by modulating matrix metalloproteinase and transforming growth factor-ß activity, collagen synthesis, myofibroblast differentiation, cell death and stretch-mediated augmentation of cardiac contractility. The detrimental role for TSPs in cardiovascular pathophysiology has been clearly demonstrated in knockout mouse models, and augmentation of TSP signalling in the heart during stress and haemodynamic overload might be beneficial. In conclusion, the role of TSP-1, TSP-2 and TSP-4 in cardiac hypertrophy, remodelling after myocardial infarction, heart failure, atherosclerosis and aortic valve stenosis encourages further investigation to validate them as potential drug targets.
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Enfermedades Cardiovasculares/etiología , Trombospondinas/fisiología , Animales , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/etiología , Calcinosis/tratamiento farmacológico , Calcinosis/etiología , Enfermedades Cardiovasculares/tratamiento farmacológico , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/etiología , Enfermedades de las Válvulas Cardíacas/tratamiento farmacológico , Enfermedades de las Válvulas Cardíacas/etiología , Humanos , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/etiología , Trombospondinas/antagonistas & inhibidoresRESUMEN
Thrombospondins TSP-1 and TSP-2 are potent endogenous inhibitors of angiogenesis. They inhibit angiogenesis through direct effects on endothelial cell migration, proliferation, survival, and apoptosis and by antagonizing the activity of VEGF. Several of the membrane receptor systems and signal transduction molecules that mediate the effects of TSP-1 and TSP-2 have been elucidated. TSP-1 and TSP-2 exert their direct effects through CD36, CD47, and integrins. Recent data indicate that CD36 and ß1 integrins collaborate to transmit the signals that are initiated by TSP-1 and TSP-2. Furthermore, these receptors appear to associate with VEGFR2 to form a platform for the integration of positive and negative signals for angiogenesis. Cross talk between pro- and antiangiogenic signal transduction pathways may enable TSP-1 and TSP-2 to inhibit angiogenesis by antagonizing survival pathways while also activating apoptotic pathways. CD36 and CD47 are both involved in the suppression of nitric oxide (NO). Advances in understanding of the molecular regulation of angiogenesis by TSP have paved the way for innovations in experimental treatment of cancers and will likely continue to offer vast avenues for discovery in other disease processes as well.
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Inhibidores de la Angiogénesis/fisiología , Neoplasias/irrigación sanguínea , Neovascularización Patológica/fisiopatología , Trombospondina 1/fisiología , Trombospondinas/fisiología , Antígenos CD/fisiología , Antineoplásicos/uso terapéutico , Apoptosis/fisiología , Movimiento Celular/fisiología , Proliferación Celular , Células Endoteliales/fisiología , Predicción , Humanos , Neoplasias/tratamiento farmacológico , Óxido Nítrico/fisiología , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/fisiología , Transducción de Señal/fisiología , Trombospondina 1/antagonistas & inhibidores , Trombospondinas/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
Zn-metalloproteinases are an important class of hydrolytic enzymes that are characterized by the presence of a catalytic zinc(II) atom in their active center which is fundamental for proteolytic activity. Metzincins, a superfamily of Zn-metalloproteinases with many structural and functional commonalities among its members, are responsible for the fine tuning of key physiological functions in mammals and the deregulation of their activity is directly connected to numerous inflammatory and degenerative diseases such as arthritis or cancer. Development of small-molecule exogenous inhibitors of metzincins able to re-establish normal proteolytic activity in pathological conditions has been a field of intense research effort for many years but applications in the clinic were not always successful. One of the main reasons for this failure is the uncontrolled action of these inhibitors on target as well as anti-target metzincin family members. Current medicinal efforts have been shifted to the discovery of target-specific inhibitors that will help to improve our understanding of metzincins biological function and provide the basis for the development of safer pharmaceutical agents. This review focuses on the cases of certain medicinally important metzincins [matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs), ADAMs with thrombospondin motifs (ADAMTSs), and procollagen C-proteinase (PCP)] and summarizes the latest advances on the discovery of inhibitors of these enzymes that display improved selectivity profiles.
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Quelantes/química , Inhibidores Enzimáticos/farmacología , Inhibidores de la Metaloproteinasa de la Matriz , Zinc/química , Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/química , Proteínas ADAM/metabolismo , Proteína ADAM17 , Animales , Proteína Morfogenética Ósea 1/antagonistas & inhibidores , Proteína Morfogenética Ósea 1/química , Proteína Morfogenética Ósea 1/metabolismo , Catálisis , Dominio Catalítico , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Metaloproteinasas de la Matriz/química , Metaloproteinasas de la Matriz/metabolismo , Relación Estructura-Actividad , Trombospondinas/antagonistas & inhibidores , Trombospondinas/química , Trombospondinas/metabolismoRESUMEN
BACKGROUND: Thrombospondin-1 (TSP-1) has been implicated in many different processes based in part on inhibitory activities of anti-TSP-1 monoclonal antibodies (mAbs). OBJECTIVE: To map epitopes of 13 anti-TSP-1 mAbs to individual modules or groups of modules spanning TSP-1 and the closely related TSP-2 homolog. RESULTS: The mapping has led to assignment or reassignment of the epitopes of four mAbs, refinement of the epitopes of six mAbs, and confirmation of the epitopes of the remaining three mAbs. ESTs10, P12, and MA-II map to the N-terminal domain; 5G11, TSP127.6, and ESTs12 to the third properdin module; C6.7, HB8432, and P10 to epidermal growth factor (EGF)-like modules 1 and/or 2; and A6.1, mAb133, MA-I, and D4.6 to the calcium-binding wire module. A6.1, which recognizes a region of the wire that is identical in mouse and human TSP-1, reacts with TSP-1 from both species, and also reacts weakly with human TSP-2. Two other mouse antihuman TSP-1 mAbs, A4.1 and D4.6, also react with mouse TSP-1. CONCLUSIONS: Consideration of previous literature and mapping of epitopes of inhibitory mAbs suggest that biological activities are present throughout TSP-1, including the EGF-like modules that have not been implicated in the past. Because the epitopes for 10 of the antibodies likely are within 18 nm of one another in calcium-replete TSP-1, some of the inhibitory effects may result from steric hindrance. Such seems to be the case for mAb133, which binds the calcium-binding wire but is still able to interfere with the activation of latent TGF-beta by the properdin modules.
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Anticuerpos Monoclonales/farmacología , Trombospondina 1/antagonistas & inhibidores , Trombospondina 1/inmunología , Secuencia de Aminoácidos , Animales , Bovinos , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Mapeo Epitopo , Humanos , Ratones , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Trombospondina 1/química , Trombospondina 1/genética , Trombospondinas/antagonistas & inhibidores , Trombospondinas/química , Trombospondinas/genética , Trombospondinas/inmunología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Tumour growth is dependent on angiogenesis. Antiangiogenic chemotherapy, i.e. continuous or metronomic low-dose chemotherapy, is a method for administrating cytostatics at a low and well-tolerated concentration without prolonged breaks. The target is the genetically stable endothelial cells playing a pivotal role in angiogenesis within the tumour. Different mediators could mediate the antiangiogenic effect of metronomic chemotherapy. One of these mediators could be thrombospondin (TSP). TSP is a potent inhibitor of angiogenesis and might therefore be important in controlling tumour growth. This study was designed to evaluate the effects of low-dose continuous or moderate-dose bolus chemotherapy on tumour growth and on tumour expression of TSP. MATERIALS AND METHODS: Rats bearing a malignant prostate tumour (Dunning AT-1) not expressing TSP were treated systemically with cyclophosphamide, doxorubicin or paclitaxel and the combination of cyclophosphamide and doxorubicin. Tumour growth and body weight were measured during the treatment. CD36, one of TSP's main receptors, was also analysed. The expression pattern of TSP-1, TSP-2 and CD36 was investigated using immunohistochemistry and Western blot analyses. Q-PCR was used to analyse TSP-1 mRNA expression. RESULTS: Low-dose cyclophosphamide and paclitaxel re-induced the expression of TSP in the tumours. However, following a bolus dose of doxorubicin, tumours showed no expression of TSP. Both cyclophosphamide and doxorubicin treatments decreased the tumour weight by more than 60% compared with vehicle controls. When cyclophosphamide and doxorubicin were combined the tumour weight was reduced by 47%, while paclitaxel reduced the tumour weight by 18% compared to the vehicle controls. CONCLUSIONS: Systemic low-dose continuous treatment of a rat prostate cancer model with cyclophosphamide and paclitaxel induced the expression of TSP in tumour tissue and inhibited tumour growth. These findings support the hypothesis that the anti-tumour effect of low-dose metronomic chemotherapy, at least with certain chemotherapeutics, is partly mediated by induction of endogenous antiangiogenic factors.
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Protocolos de Quimioterapia Combinada Antineoplásica , Neovascularización Patológica , Neoplasias de la Próstata , Trombospondinas/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Masculino , Trasplante de Neoplasias , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Neoplasias de la Próstata/irrigación sanguínea , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Ratas , Ratas Endogámicas , Trombospondinas/biosíntesis , Resultado del TratamientoRESUMEN
Hepatic stellate cells (HSC) cultured on plastic spontaneously transdifferentiate to a myofibroblast-like cell type (MFB). This model system of hepatic fibrogenesis is characterized by phenotypic changes of the cells and increased matrix synthesis. Here, we analyzed if transdifferentiation-dependent induction of ECM components, e.g., collagen type I and thrombospondin-2 (TSP-2), and phenotypic changes are coregulated events and if both processes are mediated via TGF-beta pathway(s). Blocking the TGF-beta-dependent p38 MAPK pathway in HSC with the specific inhibitor SB203580 strongly reduces collagen I and TSP-2 mRNA expression without inhibiting upregulation of the typical MFB-marker, alpha-smooth-muscle actin (alpha-SMA). Similarly, interference with the Smad2/3/4 pathway using dexamethasone also heavily decreased expression of collagen type I and TSP-2 whereas transdifferentiation of HSC to the typical morphology of MFB with loss of fat droplets and increasing alpha-SMA was unchanged. Further, p38 MAPK mediated induction of collagen I and TSP-2 expression by TGF-beta1 was still achieved in the presence of dexamethasone, showing that dexamethasone does not block p38 while it delays Smad2 phosphorylation and antagonizes stimulation of a Smad3/Smad4 dependent TGF-beta reporter construct. Interestingly, in contrast to SB203580 and dexamethasone, overexpression of the TGF-beta antagonist Smad7 reduced ECM expression and simultaneously inhibited morphologic transdifferentiation, indicating that Smad7 fulfills additional features in HSC. In conclusion, our data show that phenotypic changes of transdifferentiating HSC and induction of matrix synthesis are independent processes, the latter being stimulated by both, Smad dependent and MAPK dependent TGF-beta signaling.
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Actinas/metabolismo , Colágeno Tipo I/metabolismo , Hígado/citología , Hígado/metabolismo , Músculo Liso/metabolismo , Trombospondinas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Diferenciación Celular/fisiología , Colágeno Tipo I/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Dexametasona/farmacología , Matriz Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Glucocorticoides/farmacología , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Proteína Smad2 , Trombospondinas/antagonistas & inhibidores , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/fisiología , Factor de Crecimiento Transformador beta1 , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Thrombospondins (TSPs) 1 and 2 are extracellular modular glycoproteins that are best known for their anti-angiogenic properties and their ability to modulate cell-matrix interactions. However, these proteins, and in particular TSP2, are pleiotropic in function and affect processes as disparate as bone growth and hemostasis. In recognition of their ability to influence a wide variety of cell functions, and in the absence of convincing evidence for their participation as integral components of extracellular structures, the term 'matricellular' has been applied to these and a small group of functionally related proteins. In this review, we focus on the role of TSP1 and 2 in two forms of injury in mice, excisional skin wounds and subcutaneously implanted biomaterials, and take advantage of mice with targeted disruptions of one or both genes to identify likely biochemical mechanisms that could account for the characteristics of the injury response in these knockout mice. In work that stems largely from our own laboratory, we show that pericellular levels of the matrix metalloproteinase, MMP2, are controlled to a large extent by TSP2 (and potentially also by TSP1), and that elevated levels of MMP2 are likely to account in part for defects as diverse as reduced cellular adhesion, abnormal collagen fibril structure, and increased endothelial cell and vascular proliferation.