RESUMO
The microenvironment hosting a tumor actively participates in regulating tumor cell proliferation, migration, and invasion. Among the extracellular matrix proteins enriched in the stroma of carcinomas are the tenascin family members tenascin-C and tenascin-W. Whereas tenascin-C overexpression in gliomas is known to correlate with poor prognosis, the status of tenascin-W in brain tumors has not been investigated so far. In the present study, we analyzed protein levels of tenascin-W in 38 human gliomas and found expression of tenascin-W in 80% of the tumor samples, whereas no tenascin-W could be detected in control, nontumoral brain tissues. Double immunohistochemical staining of tenascin-W and von Willebrand factor revealed that tenascin-W is localized around blood vessels, exclusively in tumor samples. In vitro, the presence of tenascin-W increased the proportion of elongated human umbilical vein endothelial cells (HUVECs) and augmented the mean speed of cell migration. Furthermore, tenascin-W triggered sprouting of HUVEC spheroids to a similar extent as the proangiogenic factor tenascin-C. In conclusion, our study identifies tenascin-W as a candidate biomarker for brain tumor angiogenesis that could be used as a molecular target for therapy irrespective of the glioma subtype.-Martina, E., Degen, M., Rüegg, C., Merlo, A., Lino, M. M., Chiquet-Ehrismann, R., Brellier, F. Tenascin-W is a specific marker of glioma-associated blood vessels and stimulates angiogenesis in vitro.
Assuntos
Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/farmacologia , Vasos Sanguíneos/metabolismo , Glioma/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Tenascina/metabolismo , Tenascina/farmacologia , Western Blotting , Crescimento Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Imunofluorescência , Humanos , Técnicas In Vitro , Esferoides Celulares/efeitos dos fármacos , Veias Umbilicais/citologiaRESUMO
BACKGROUND: Neurogenesis in the hippocampal dentate gyrus and the subventricular zone occurs throughout the life of mammals and newly generated neurons can integrate functionally into established neuronal circuits. Neurogenesis levels in the dentate gyrus are modulated by changes in the environment (enrichment, exercise), hippocampal-dependent tasks, NMDA receptor (NMDAR) activity, sonic hedgehog (SHH) and/or other factors. RESULTS: previously, we showed that Protease Nexin-1 (PN-1), a potent serine protease inhibitor, regulates the NMDAR availability and activity as well as SHH signaling. Compared with wild-type (WT), we detected a significant increase in BrdU-labeled cells in the dentate gyrus of mice lacking PN-1 (PN-1 -/-) both in controls and after running exercise. Patched homologue 1 (Ptc1) and Gli1 mRNA levels were higher and Gli3 down-regulated in mutant mice under standard conditions and to a lesser extent after running exercise. However, the number of surviving BrdU-positive cells did not differ between WT and PN-1 -/- animals. NMDAR availability was altered in the hippocampus of mutant animals after exercise. CONCLUSION: All together our results indicate that PN-1 controls progenitors proliferation through an effect on the SHH pathway and suggest an influence of the serpin on the survival of newly generated neurons through modulation of NMDAR availability.
Assuntos
Giro Denteado/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Condicionamento Físico Animal/fisiologia , Serpinas/genética , Animais , Western Blotting , Contagem de Células , Proliferação de Células , Sobrevivência Celular/fisiologia , Imuno-Histoquímica , Hibridização In Situ , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores Patched , Receptor Patched-1 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serpina E2 , Serpinas/metabolismo , Proteína GLI1 em Dedos de Zinco , Proteína Gli3 com Dedos de ZincoRESUMO
Tenascin-C is an extracellular matrix protein over-expressed in a large variety of cancers. In the present study, we aimed at identifying new interactors of tenascin-C by purifying secreted proteins on a tenascin-C affinity column. Analysis of eluates by mass spectrometry revealed phosphoglycerate kinase 1, clusterin, fibronectin, SPARC-related modular calcium-binding protein 1 (SMOC1) and nidogen-2 as potential interactors of tenascin-C. The interaction between tenascin-C and SMOC1 was confirmed by co-immunoprecipitation and further analyzed by Surface Plasmon Resonance Spectroscopy, which revealed an apparent dissociation constant (K(D)) value of 2.59∗10(-9)M. Further analyses showed that this binding is reduced in the presence of EDTA. To investigate whether SMOC1 itself could be over-expressed in the context of tumorigenesis, we analyzed data of two independent RNA profiling studies and found that mRNA levels of SMOC1 are significantly increased in oligodendrogliomas compared to control brain samples. In support of these data, western blot analysis of protein extracts from 12 oligodendrogliomas, 4 astrocytomas and 13 glioblastomas revealed elevated levels compared to healthy brain extract. Interestingly, cell migration experiments revealed that SMOC1 can counteract the chemo-attractive effect of tenascin-C on U87 glioma cells. The present study thus identified SMOC1 as a new cancer-associated protein capable of interacting with tenascin-C in vitro.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Osteonectina/metabolismo , Tenascina/metabolismo , Regulação para Cima , Adulto , Idoso , Neoplasias Encefálicas/patologia , Estudos de Casos e Controles , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Glioma/patologia , Humanos , Imunoprecipitação , Pessoa de Meia-Idade , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
GBM, the most common and malignant primary tumor of the CNS, is characterized by exponential growth and diffuse invasiveness. Although the diverse causative genotypes that give rise to a inhomogeneous histological phenotype are well defined, effective therapy inducing tumor cell apoptosis has not been established so far. Following surgery, billions of invasive tumor cells remain to be targeted by systemic and local therapies. Targeting non-overlapping pathways, rather than a single agent approach, is more likely to be effective. The potential of local drug application has not been exploited yet. Systemically, novel drug combinations have to be developed that not only target key molecules at the signaling crossroads but also exploit energy demand and the epigenetic cancer program of GBM.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Análise Mutacional de DNA , Resistencia a Medicamentos Antineoplásicos , Epigênese Genética , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Tenascin-C (TNC) expression is known to correlate with malignancy in glioblastoma (GBM), a highly invasive and aggressive brain tumor that shows limited response to conventional therapies. In these malignant gliomas as well as in GBM cell lines, we found Notch2 protein to be strongly expressed. In a GBM tumor tissue microarray, RBPJk protein, a Notch2 cofactor for transcription, was found to be significantly coexpressed with TNC. We show that the TNC gene is transactivated by Notch2 in an RBPJk-dependent manner mediated by an RBPJk binding element in the TNC promoter. The transactivation is abrogated by a Notch2 mutation, which we detected in the glioma cell line Hs683 that does not express TNC. This L1711M mutation resides in the RAM domain, the site of interaction between Notch2 and RBPJk. In addition, transfection of constructs encoding activated Notch2 or Notch1 increased endogenous TNC expression identifying TNC as a novel Notch target gene. Overexpression of a dominant negative form of the transcriptional coactivator MAML1 or knocking down RBPJk in LN319 cells led to a dramatic decrease in TNC protein levels accompanied by a significant reduction of cell migration. Because addition of purified TNC stimulated glioma cell migration, this represents a mechanism for the invasive properties of glioma cells controlled by Notch signaling and defines a novel oncogenic pathway in gliomagenesis that may be targeted for therapeutic intervention in GBM patients.