RESUMO
Wnt proteins are critical to mammalian brain development and function. The canonical Wnt signaling pathway involves the stabilization and nuclear translocation of ß-catenin; however, Wnt also signals through alternative, noncanonical pathways. To gain a systems-level, genome-wide view of Wnt signaling, we analyzed Wnt1-stimulated changes in gene expression by transcriptional microarray analysis in cultured human neural progenitor (hNP) cells at multiple time points over a 72-hour time course. We observed a widespread oscillatory-like pattern of changes in gene expression, involving components of both the canonical and the noncanonical Wnt signaling pathways. A higher-order, systems-level analysis that combined independent component analysis, waveform analysis, and mutual information-based network construction revealed effects on pathways related to cell death and neurodegenerative disease. Wnt effectors were tightly clustered with presenilin1 (PSEN1) and granulin (GRN), which cause dominantly inherited forms of Alzheimer's disease and frontotemporal dementia (FTD), respectively. We further explored a potential link between Wnt1 and GRN and found that Wnt1 decreased GRN expression by hNPs. Conversely, GRN knockdown increased WNT1 expression, demonstrating that Wnt and GRN reciprocally regulate each other. Finally, we provided in vivo validation of the in vitro findings by analyzing gene expression data from individuals with FTD. These unbiased and genome-wide analyses provide evidence for a connection between Wnt signaling and the transcriptional regulation of neurodegenerative disease genes.
Assuntos
Doença de Alzheimer/metabolismo , Demência Frontotemporal/metabolismo , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/biossíntese , Transcrição Gênica , Via de Sinalização Wnt , Proteína Wnt1/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Células Cultivadas , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas do Tecido Nervoso/genética , Análise de Sequência com Séries de Oligonucleotídeos , Presenilina-1/biossíntese , Presenilina-1/genética , Progranulinas , Proteína Wnt1/genéticaRESUMO
Autism spectrum disorder (ASD) is a highly heritable, behaviorally defined, heterogeneous disorder of unknown pathogenesis. Several genetic risk genes have been identified, including the gene encoding the receptor tyrosine kinase MET, which regulates neuronal differentiation and growth. An ASD-associated polymorphism disrupts MET gene transcription, and there are reduced levels of MET protein expression in the mature temporal cortex of subjects with ASD. To address the possible neurodevelopmental contribution of MET to ASD pathogenesis, we examined the expression and transcriptional regulation of MET by a transcription factor, FOXP2, which is implicated in regulation of cognition and language, two functions altered in ASD. MET mRNA expression in the midgestation human fetal cerebral cortex is strikingly restricted, localized to portions of the temporal and occipital lobes. Within the cortical plate of the temporal lobe, the pattern of MET expression is highly complementary to the expression pattern of FOXP2, suggesting the latter may play a role in repression of gene expression. Consistent with this, MET and FOXP2 also are reciprocally expressed by differentiating normal human neuronal progenitor cells (NHNPs) in vitro, leading us to assess whether FOXP2 transcriptionally regulates MET. Indeed, FOXP2 binds directly to the 5' regulatory region of MET, and overexpression of FOXP2 results in transcriptional repression of MET. The expression of MET in restricted human neocortical regions, and its regulation in part by FOXP2, is consistent with genetic evidence for MET contributing to ASD risk.
Assuntos
Transtorno Autístico/genética , Transtornos Cognitivos/genética , Transtornos Cognitivos/metabolismo , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Proto-Oncogênicas c-met/biossíntese , Receptores de Fatores de Crescimento/biossíntese , Regiões 5' não Traduzidas/genética , Transtorno Autístico/metabolismo , Transtorno Autístico/patologia , Transtornos Globais do Desenvolvimento Infantil/genética , Transtornos Globais do Desenvolvimento Infantil/metabolismo , Transtornos Globais do Desenvolvimento Infantil/patologia , Transtornos Cognitivos/patologia , Feminino , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/metabolismo , Humanos , Recém-Nascido , Masculino , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/genética , Receptores de Fatores de Crescimento/antagonistas & inibidores , Receptores de Fatores de Crescimento/genética , Fatores de RiscoRESUMO
Glioblastoma multiforme (GBM) is a devastating disease, and the current therapies have only palliative effect. Evidence is mounting to indicate that brain tumor stem cells (BTSCs) are a minority of tumor cells that are responsible for cancer initiation, propagation, and maintenance. Therapies that fail to eradicate BTSCs may ultimately lead to regrowth of residual BTSCs. However, BTSCs are relatively resistant to the current treatments. Development of novel therapeutic strategies that effectively eradicate BTSC are, therefore, essential. In a previous study, we used patient-derived GBM sphere cells (stemlike GBM cells) to enrich for BTSC and identified maternal embryonic leucine-zipper kinase (MELK) as a key regulator of survival of stemlike GBM cells in vitro. Here, we demonstrate that a thiazole antibiotic, siomycin A, potently reduced MELK expression and inhibited tumor growth in vivo. Treatment of stemlike GBM cells with siomycin A resulted in arrested self-renewal, decreased invasion, and induced apoptosis but had little effect on growth of the nonstem cells of matched tumors or normal neural stem/progenitor cells. MELK overexpression partially rescued the phenotype of siomycin A-treated stemlike GBM cells. In vivo, siomycin A pretreatment abraded the sizes of stemlike GBM cell-derived tumors in immunodeficient mice. Treatment with siomycin A of mice harboring intracranial tumors significantly prolonged their survival period compared with the control mice. Together, this study may be the first model to partially target stemlike GBM cells through a MELK-mediated pathway with siomycin A to pave the way for effective treatment of GBM.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Peptídeos/uso terapêutico , Proteínas Serina-Treonina Quinases/genética , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Glioblastoma/patologia , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia , Células-Tronco/metabolismo , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
BACKGROUND: Angiogenesis is a complex process involving endothelial cell migration, proliferation, invasion, and tube formation. Inhibition of these processes might have implications in various angiogenesis-mediated disorders. MATERIALS AND METHODS: The antiangiogenic efficacy of the novel alphavbeta3 antagonist TA138 was examined using in vivo and in vitro model systems. RESULTS: The in vitro studies demonstrated the ability of TA138 and RP747 (conjugated TA138) to inhibit endothelial cell migration toward vitronectin, with an IC50=0.04 and 0.045 microM, respectively. Furthermore, utilizing the chick chorioallantoic membrane models, TA138 inhibited basic fibroblast growth factor-induced neovascularization. CONCLUSION: TA138 might be a useful tool for the inhibition of angiogenesis associated with human tumor growth, or other pathological neovascularization processes. RP747 demonstrated antitumor efficacy in 1 spontaneous tumor model (c-neu oncomouse model, alphavbeta3 positive cells) and in 1 xenograft model (HCT116 human tumor colon carcinoma, alphavbeta3 negative cells) injected subcutaneously into nude mice.
Assuntos
Adenocarcinoma/tratamento farmacológico , Inibidores da Angiogênese/farmacologia , Neoplasias do Colo/tratamento farmacológico , Compostos Heterocíclicos com 1 Anel/farmacologia , Integrina alfaVbeta3/antagonistas & inibidores , Neoplasias Mamárias Experimentais/tratamento farmacológico , Sulfonamidas/farmacologia , Adenocarcinoma/irrigação sanguínea , Adenocarcinoma/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Feminino , Genes erbB-2 , Células HCT116 , Humanos , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Alternative splicing of the human beta-aspartyl (asparaginyl) hydroxylase (BAH) gene results in the expression of humbug, a truncated form of BAH that lacks the catalytic domain of the enzyme. Overexpression of BAH and humbug has been associated with a variety of human cancers, and although humbug lacks enzymatic activity, it is expressed at levels comparable with that of BAH in various cancer cell lines. Phosphorothioate antisense oligonucleotides (ONs) were designed to dissect out the function of these hydroxylase protein isoforms. In A549 cells, these ONs differentially down-regulated BAH and humbug at the mRNA and protein level. Phosphorothioate ON uptake and antisense studies were conducted in parallel in nude mice bearing A549 tumor xenografts. Microscopic examination of the tumor after administration of a fluorescein-labeled ON showed strong labeling of the outer layers of the tumor connective tissue but cells within the interior of the tumor were sparsely labeled. A modest but significant effect on tumor growth was observed in animals treated with an antisense ON directed against both BAH and humbug transcripts. However, Northern analysis of tumor RNA did not indicate a down-regulation of the targeted mRNA species. These results demonstrate the successful development of antisense ONs that selectively differentiate between the closely related beta-hydroxylase protein isoforms. However, determination of the biological function of these proteins in vivo was limited by the poor uptake properties of phosphorothioate ONs in A549 tumors.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Oxigenases de Função Mista/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Humanos , Isoenzimas/genética , Neoplasias Pulmonares , Deleção de Sequência , Células Tumorais CultivadasRESUMO
During the summer of 2001, Americans were treated to high political drama courtesy of the debate over embryonic and adult stem cell research. The popular press was flush with predictions about how neural stem cells would reverse, almost by magic, the devastation caused by diseases such as Alzheimer's, Parkinson's, stroke or spinal cord injury. Unfortunately, this promise remains unfulfilled because we have such a poor understanding of how stem cells function. With regard to adult stem cells, we are not even completely sure where they are, or how or when they got there. A provocative study by Ourednik et al. published in Science suggests that in primates, adult neural stem cells are allocated during early corticogenesis. The study also provides evidence for the existence of stem cells dispersed throughout the frontal cortex and striatum.