RESUMO
Genomic characterization is beginning to define a molecular taxonomy for breast cancer; however, the molecular basis of invasion and metastasis remains poorly understood. We report a pivotal role for the fibroblast growth factor-inducible 14 (Fn14) receptor in this process. We examined whether Fn14 and its ligand tumor necrosis factor-like weak inducer of apoptosis (TWEAK) were expressed in breast tumors and whether deregulation of Fn14 levels affected malignant behavior of breast cancer cell lines. Analysis of TWEAK and Fn14 in publicly available gene expression data indicated that high Fn14 expression levels significantly correlated with several poor prognostic indicators (P < 0.05). Fn14 expression was highest in the HER2-positive/estrogen receptor-negative (HER2(+)/ER(-)) intrinsic subtype (P = 0.0008). An association between Fn14 and HER2 expression in breast tumors was confirmed by immunohistochemistry. Fn14 levels were elevated in invasive, ER(-) breast cancer cell lines. Overexpression of Fn14 in weakly invasive MCF7 and T47D cells resulted in a marked induction of invasion and activation of nuclear factor-kappaB (NF-kappaB) signaling. Ectopic expression of Fn14tCT, a Fn14 deletion mutant that cannot activate NF-kappaB signaling, was not able to induce invasion. Moreover, ectopic expression of Fn14tCT in highly invasive MDA-MB-231 cells reduced their invasive capability. RNA interference-mediated inhibition of Fn14 expression in both MDA-MB-231 and MDA-MB-436 cells reduced invasion. Expression profiling of the Fn14-depleted cells revealed deregulation of NF-kappaB activity. Our findings support a role for Fn14-mediated NF-kappaB pathway activation in breast tumor invasion and metastasis.
Assuntos
Neoplasias da Mama/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptor ErbB-2/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Adenoviridae/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Imuno-Histoquímica , NF-kappa B/metabolismo , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Proteínas Recombinantes/química , Receptor de TWEAKRESUMO
Malignant gliomas are characterized by their ability to invade normal brain tissue. We have previously shown that the small GTPase Rac1 plays a role in both migration and invasion in gliomas. Here, we aim to identify Rac-activating guanine nucleotide exchange factors (GEFs) that mediate glioblastoma invasiveness. Using a brain tumor expression database, we identified three GEFs, Trio, Ect2, and Vav3, that are expressed at higher levels in glioblastoma versus low-grade glioma. The expression of these GEFs is also associated with poor patient survival. Quantitative real-time polymerase chain reaction and immunohistochemical analyses on an independent set of tumors confirmed that these GEFs are overexpressed in glioblastoma as compared with either nonneoplastic brain or low-grade gliomas. In addition, depletion of Trio, Ect2, and Vav3 by siRNA oligonucleotides suppresses glioblastoma cell migration and invasion. Depletion of either Ect2 or Trio also reduces the rate of cell proliferation. These results suggest that targeting GEFs may present novel strategies for anti-invasive therapy for malignant gliomas.
Assuntos
Glioblastoma , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Encéfalo/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células , Bases de Dados de Ácidos Nucleicos , Perfilação da Expressão Gênica , Glioblastoma/patologia , Glioblastoma/fisiopatologia , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Invasividade Neoplásica , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-vav/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Glioblastoma multiforme (GBM) is the most common primary intracranial tumor and despite recent advances in treatment regimens, prognosis for affected patients remains poor. Active cell migration and invasion of GBM cells ultimately lead to ubiquitous tumor recurrence and patient death. To further understand the genetic mechanisms underlying the ability of glioma cells to migrate, we compared the matched transcriptional profiles of migratory and stationary populations of human glioma cells. Using a monolayer radial migration assay, motile and stationary cell populations from seven human long term glioma cell lines and three primary GBM cultures were isolated and prepared for expression analysis. RESULTS: Gene expression signatures of stationary and migratory populations across all cell lines were identified using a pattern recognition approach that integrates a priori knowledge with expression data. Principal component analysis (PCA) revealed two discriminating patterns between migrating and stationary glioma cells: i) global down-regulation and ii) global up-regulation profiles that were used in a proband-based rule function implemented in GABRIEL to find subsets of genes having similar expression patterns. Genes with up-regulation pattern in migrating glioma cells were found to be overexpressed in 75% of human GBM biopsy specimens compared to normal brain. A 22 gene signature capable of classifying glioma cultures based on their migration rate was developed. Fidelity of this discovery algorithm was assessed by validation of the invasion candidate gene, connective tissue growth factor (CTGF). siRNA mediated knockdown yielded reduced in vitro migration and ex vivo invasion; immunohistochemistry on glioma invasion tissue microarray confirmed up-regulation of CTGF in invasive glioma cells. CONCLUSION: Gene expression profiling of migratory glioma cells induced to disperse in vitro affords discovery of genomic signatures; selected candidates were validated clinically at the transcriptional and translational levels as well as through functional assays thereby underscoring the fidelity of the discovery algorithm.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Movimento Celular/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/patologia , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Componente Principal , Reprodutibilidade dos Testes , Taxa de SobrevidaRESUMO
Osteosarcoma is the most common malignant bone tumor in children. Osteosarcoma patients who respond poorly to chemotherapy are at a higher risk of relapse and adverse outcome. Therefore, it was the aim of this study to identify prognostic factors at the time of diagnosis to characterize the genes predictive of poor survival outcome and to identify potential novel therapeutic targets. Expression profiling of 30 osteosarcoma diagnostic biopsy samples, 15 with inferior necrosis following induction chemotherapy (Huvos I/II) and 15 with superior necrosis following induction chemotherapy (Huvos III/IV), was conducted using Affymetrix U95Av2 oligonucleotide microarrays. One hundred and four genes were found to be statistically significant and highly differentially expressed between Huvos I/II and III/IV patients. Statistically significant genes were validated on a small independent cohort comprised of osteosarcoma xenograft tumor samples. Markers of Huvos I/II response predominantly were gene products involved in extracellular matrix (ECM) microenvironment remodeling and osteoclast differentiation. A striking finding was the significant decrease in osteoprotegerin, an osteoclastogenesis inhibitory factor. Additional genes involved in osteoclastogenesis and bone resorption, which were statistically different, include annexin 2, SMAD, PLA2G2A, and TGFbeta1. ECM remodeling genes include desmoplakin, SPARCL1, biglycan, and PECAM. Gene expression of select genes involved in tumor progression, ECM remodeling, and osteoclastogenesis were validated via quantitative reverse transcription-PCR in an independent cohort. We propose that osteosarcoma tumor-driven changes in the bone microenvironment contribute to the chemotherapy-resistant phenotype and offer testable hypotheses to potentially enhance therapeutic response.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Perfilação da Expressão Gênica , Osteossarcoma/diagnóstico , Osteossarcoma/tratamento farmacológico , Animais , Biomarcadores Tumorais/metabolismo , Biópsia , Neoplasias Ósseas/diagnóstico , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Criança , Cisplatino/administração & dosagem , Doxorrubicina/administração & dosagem , Regulação Neoplásica da Expressão Gênica , Humanos , Metotrexato/administração & dosagem , Camundongos , Camundongos SCID , Necrose , Análise de Sequência com Séries de Oligonucleotídeos , Osteossarcoma/metabolismo , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Transdução de Sinais , Taxa de Sobrevida , Transplante Heterólogo , Resultado do TratamentoRESUMO
Eph receptor tyrosine kinases and their ligands, ephrins, mediate neurodevelopmental processes such as boundary formation, axon guidance, vasculogenesis, and cell migration. We determined the expression profiles of the Eph family members in five glioma cell lines under migrating and nonmigrating conditions. EphB2 mRNA was overexpressed in all five during migration (1.2-2.8-fold). We found abundant EphB2 protein as well as strong phosphorylation of EphB2 in migrating U87 cells. Confocal imaging showed EphB2 localized in lamellipodia of motile U87 cells. Treatment with ephrin-B1/Fc chimera stimulated migration and invasion of U87, whereas treatment with a blocking EphB2 antibody significantly inhibited migration and invasion. Forced expression of EphB2 in U251 cells stimulated cell migration and invasion and diminished adhesion concomitant with the tyrosine phosphorylation of EphB2. U251 stably transfected with EphB2 showed more scattered and more pronounced invasive growth in an ex vivo rat brain slice. In human brain tumor specimens, EphB2 expression was higher in glioblastomas than in low-grade astrocytomas or normal brain; patterns of phosphorylated EphB2 matched the expression levels. Laser capture microdissection of invading glioblastoma cells revealed elevated EphB2 mRNA (1.5-3.5-fold) in 7 of 7 biopsy specimens. Immunohistochemistry demonstrated EphB2 localization primarily in glioblastoma cells (56 of 62 cases) and not in normal brain. This is the first demonstration that migrating glioblastoma cells overexpress EphB2 in vitro and in vivo; glioma migration and invasion are promoted by activation of EphB2 or inhibited by blocking EphB2. Dysregulation of EphB2 expression or function may underlie glioma invasion.
Assuntos
Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/patologia , Movimento Celular/fisiologia , Glioma/enzimologia , Glioma/patologia , Receptor EphB2/fisiologia , Animais , Astrocitoma/enzimologia , Astrocitoma/patologia , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Glioblastoma/enzimologia , Glioblastoma/patologia , Humanos , Masculino , Fosforilação , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Wistar , Receptor EphB2/biossíntese , Receptor EphB2/genética , Receptor EphB2/metabolismoRESUMO
P311, an 8-kDa polypeptide, was previously shown to be highly expressed in invasive glioma cells. Here, we report the functional characteristics of P311 with regard to influencing glioma cell migration. P311 is constitutively serine-phosphorylated; decreased phosphorylation is observed in migration-activated glioma cells. The primary amino acid sequence of P311 indicates a putative serine phosphorylation site (S59) near the PEST domain. Site-directed mutagenesis of S59A retarded P311 degradation and induced glioma cell motility. In contrast, S59D mutation resulted in the rapid degradation of P311 and reduced glioma cell migration. Coimmunoprecipitation coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis identified Filamin A as a binding partner of P311, and immunofluorescence studies showed that both proteins colocalized at the cell periphery. Moreover, P311-induced cell migration was abrogated by inhibition of beta1 integrin function using TACbeta1A, a dominant-negative inhibitor of beta1 integrin signaling, suggesting that P311 acts downstream of beta1 signaling. Finally, overexpression of P311 or P311 S59A mutant protein activates Rac1 GTPase; small interfering RNA-mediated depletion of Rac1 suppresses P311-induced motility. Collectively, these results suggest a role for levels of P311 in regulating glioma motility and invasion through the reorganization of actin cytoskeleton at the cell periphery.
Assuntos
Movimento Celular , Glioma/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Oncogênicas/metabolismo , Serina/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Proteínas Contráteis/metabolismo , Filaminas , Glioma/patologia , Humanos , Proteínas dos Microfilamentos/metabolismo , Dados de Sequência Molecular , Invasividade Neoplásica , Proteínas do Tecido Nervoso/química , Proteínas Oncogênicas/química , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The invasive phenotype of glioblastoma multiforme (GBM) is a hallmark of malignant process, yet molecular mechanisms that dictate this locally invasive behavior remain poorly understood. Gene expression profiles of human glioma cells were assessed from laser capture-microdissected GBM cells collected from paired patient tumor cores and white matter-invading cell populations. Changes in gene expression in invading GBM cells were validated by quantitative reverse transcription polymerase chain reaction (QRT-PCR) and immunohistochemistry in an independent sample set. QRT-PCR confirmed the differential expression in 19 of 21 genes tested. Immunohistochemical analyses of autotaxin (ATX), ephrin B3, B-cell lymphoma-w (BCLW), and protein tyrosine kinase 2 beta showed them to be expressed in invasive glioma cells. The known GBM markers, insulin-like growth factor binding protein 2 and vimentin, were robustly expressed in the tumor core. A glioma invasion tissue microarray confirmed the expression of ATX and BCLW in invasive cells of tumors of various grades. GBM phenotypic and genotypic heterogeneity is well documented. In this study, we show an additional layer of complexity: transcriptional differences between cells of tumor core and invasive cells located in the brain parenchyma. Gene products supporting invasion may be novel targets for manipulation of brain tumor behavior with consequences on treatment outcome.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Invasividade Neoplásica/genética , Proteínas de Neoplasias/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Lasers , Invasividade Neoplásica/patologia , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , RNA Mensageiro/metabolismoRESUMO
The propensity of malignant gliomas to invade surrounding brain tissue contributes to poor clinical outcome. Integrin-mediated adhesion to extracellular matrix regulates the migration and proliferation of many cell types, but its role in glioma progression is undefined. We investigated the role of the cytoplasmic tyrosine kinases FAK and Pyk2, potential integrin effectors, in the phenotypic determination of four different human glioblastoma cell lines. While FAK expression was similar between the four cell lines, increased FAK activity correlated with high proliferation and low migratory rates. In contrast, Pyk2 activity was significantly increased in migratory cell lines and depressed in proliferative cell lines. Overexpression of Pyk2 stimulated migration, whereas FAK overexpression inhibited cell migration and stimulated cellular proliferation. These data suggest that FAK and Pyk2 function as important signaling effectors in gliomas and indicate that their differential regulation may be determining factors in the temporal development of proliferative or migrational phenotypes.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Proteínas Tirosina Quinases/genética , Divisão Celular/fisiologia , Movimento Celular/fisiologia , Quinase 1 de Adesão Focal , Quinase 2 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Fenótipo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Células Tumorais CultivadasRESUMO
The invasive nature of malignant gliomas is a clinical problem rendering tumors incurable by conventional treatment modalities such as surgery, ionizing radiation, and temozolomide. Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1) is a multifunctional adaptor protein, recruiting cytoplasmic signaling proteins and membrane receptors/transporters into functional complexes. This study revealed that NHERF-1 expression is increased in highly invasive cells that reside in the rim of glioblastoma multiforme (GBM) tumors and that NHERF-1 sustains glioma migration and invasion. Gene expression profiles were evaluated from laser capture-microdissected human GBM cells isolated from patient tumor cores and corresponding invaded white matter regions. The role of NHERF-1 in the migration and dispersion of GBM cell lines was examined by reducing its expression with small-interfering RNA followed by radial migration, three-dimensional collagen dispersion, immunofluorescence, and survival assays. The in situ expression of NHERF-1 protein was restricted to glioma cells and the vascular endothelium, with minimal to no detection in adjacent normal brain tissue. Depletion of NHERF-1 arrested migration and dispersion of glioma cell lines and caused an increase in cell-cell cohesiveness. Glioblastoma multiforme cells with depleted NHERF-1 evidenced a marked decrease in stress fibers, a larger cell size, and a more rounded shape with fewer cellular processes. When NHERF-1 expression was reduced, glioma cells became sensitized to temozolomide treatment resulting in increased apoptosis. Taken together, these results provide the first evidence for NHERF-1 as a participant in the highly invasive phenotype of malignant gliomas and implicate NHERF-1 as a possible therapeutic target for treatment of GBM.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Invasividade Neoplásica/genética , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Western Blotting , Neoplasias Encefálicas/genética , Adesão Celular/genética , Movimento Celular/genética , Imunofluorescência , Perfilação da Expressão Gênica , Glioblastoma/genética , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Lasers , Microdissecção , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trocadores de Sódio-Hidrogênio/genética , Análise Serial de Tecidos , TransfecçãoRESUMO
Malignant gliomas are the most common primary brain tumors. Despite intensive clinical investigation and significant technical advances in surgical and radiation treatment, the impact on clinical outcome for patients with malignant gliomas is disappointing. We have previously shown that tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, can stimulate glioma cell survival via binding to the Fn14 receptor, activation of the NF-kappaB pathway, and upregulation of BCL-X(L) gene expression. Here, we show that TWEAK treatment of glioma cells leads to phosphorylation of Akt and BAD. TWEAK stimulation results in the phosphorylation of both Akt1 and Akt2. However, small interfering RNA (siRNA)-mediated depletion of either Akt1 or Akt2 showed that BAD serine 136 phosphorylation is dependent specifically on Akt2 function. Depletion of Akt2 expression by siRNA also abrogates TWEAK-stimulated glioma cell survival, whereas no effect on glioma cell survival was observed after siRNA-mediated depletion of Akt1 expression. Surprisingly, although siRNA-mediated depletion of BAD in glioma cells abrogates cytotoxic- and chemotherapy-induced apoptosis, TWEAK still displays a strong protective effect, suggesting that BAD serine 136 phosphorylation plays a minor role in TWEAK-Akt2-induced glioma cell survival. We also report here that AKT2 gene expression levels increased with glioma grade and inversely correlate with patient survival. Additionally, immunohistochemical analysis showed that Akt2 expression positively correlates with Fn14 expression in glioblastoma multiforme specimens. We hypothesize that the TWEAK-Fn14 signaling axis functions, in part, to enhance glioblastoma cell survival by activation of the Akt2 serine/threonine protein kinase.
Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioma/metabolismo , Glioma/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Necrose Tumoral/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Citocina TWEAK , Perfilação da Expressão Gênica , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioma/tratamento farmacológico , Glioma/genética , Humanos , Imuno-Histoquímica , Fosforilação , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Receptor de TWEAK , Transfecção , Fatores de Necrose Tumoral/metabolismo , Proteína de Morte Celular Associada a bcl/deficiência , Proteína de Morte Celular Associada a bcl/genéticaRESUMO
Glial tumors progress to malignant grades by heightened proliferation and relentless dispersion throughout the central nervous system. Understanding genetic and biochemical processes that foster these behaviors is likely to reveal specific and effective targets for therapeutic intervention. Our current report shows that the fibroblast growth factor-inducible 14 (Fn14), a member of the tumor necrosis factor (TNF) receptor superfamily, is expressed at high levels in migrating glioma cells in vitro and invading glioma cells in vivo. Forced Fn14 overexpression stimulates glioma cell migration and invasion, and depletion of Rac1 by small interfering RNA inhibits this cellular response. Activation of Fn14 signaling by the ligand TNF-like weak inducer of apoptosis (TWEAK) stimulates migration and up-regulates expression of Fn14; this TWEAK effect requires Rac1 and nuclear factor-kappaB (NF-kappaB) activity. The Fn14 promoter region contains NF-kappaB binding sites, which mediate positive feedback causing sustained overexpression of Fn14 and enduring glioma cell invasion. Furthermore, Fn14 gene expression levels increase with glioma grade and inversely correlate with patient survival. These results show that the Fn14 cascade operates as a positive feedback mechanism for elevated and sustained Fn14 expression. Such a feedback loop argues for aggressive targeting of the Fn14 axis as a unique and specific driver of glioma malignant behavior.
Assuntos
Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Glioma/patologia , NF-kappa B/fisiologia , Invasividade Neoplásica/fisiopatologia , Proteínas de Neoplasias/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Linhagem Celular Tumoral/fisiologia , Movimento Celular/genética , Movimento Celular/fisiologia , Retroalimentação Fisiológica , Regulação Neoplásica da Expressão Gênica/genética , Glioma/metabolismo , Glioma/mortalidade , Humanos , Quinase I-kappa B/fisiologia , Invasividade Neoplásica/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Prognóstico , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/farmacologia , Ratos , Receptores do Fator de Necrose Tumoral/biossíntese , Receptores do Fator de Necrose Tumoral/genética , Receptor de TWEAK , Transfecção , Resultado do TratamentoRESUMO
Eph receptor tyrosine kinases mediate neurodevelopmental processes such as boundary formation, vasculogenesis, and cell migration. Recently, we found that overexpression of EphB2 in glioma cells results in reduced cell adhesion and increased cell invasion. Since R-Ras has been shown to play a critical role in EphB2 regulation of integrin activity, we explored whether the biological role of EphB2 in glioma invasion is mediated by downstream R-Ras activation. On EphB2 activation, R-Ras associated with the receptor and became highly phosphorylated. Depletion of endogenous R-Ras expression by siRNA abrogated EphB2 effects on glioma cell adhesion, proliferation, and invasion in ex vivo rat brain slices. Anti-proliferative responses to EphB2 activation were consistent with suppressed mitogen-activated protein kinase activity. Moreover, R-Ras was highly phosphorylated in the invading glioma cells. In human brain tumor specimens, R-Ras expression and phosphorylation correlated with increasing grade of gliomas. Laser capture microdissection of invading glioblastoma cells revealed elevated R-Ras mRNA (1.5- to 26-fold) in 100% (eight of eight) of biopsy specimens, and immunohistochemistry revealed high R-Ras localization primarily in glioblastoma cells. The phosphorylation ratio of R-Ras positively correlated with the phosphorylation ratio of EphB2 in glioblastoma tissues. These results demonstrate that R-Ras plays an important role in glioma pathology, further suggesting the EphB2/R-Ras signaling pathway as a potential therapeutic target.
Assuntos
Neoplasias Encefálicas/metabolismo , Adesão Celular , Proliferação de Células , Invasividade Neoplásica , Receptor EphB2/metabolismo , Transdução de Sinais , Proteínas ras/metabolismo , Animais , Astrocitoma/metabolismo , Astrocitoma/patologia , Neoplasias Encefálicas/patologia , Matriz Extracelular , Glioblastoma/metabolismo , Glioblastoma/patologia , Fosforilação , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Wistar , Receptor EphB2/genética , Proteínas ras/genéticaRESUMO
The Fn14 gene encodes a type Ia transmembrane protein that belongs to the tumor necrosis factor receptor superfamily. We recently showed that fibroblast growth factor-inducible 14 (Fn14) is overexpressed in migrating glioma cells in vitro and in glioblastoma multiforme clinical specimens in vivo. To determine the biological role of Fn14 in brain cancer progression, we examined the activity of Fn14 as a potential mediator of cell survival. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK)-stimulated glioma cells had increased cellular resistance to cytotoxic therapy-induced apoptosis. Either TWEAK treatment or Fn14 overexpression in glioma cells resulted in the activation of NFkappaB and subsequently the translocation of NFkappaB from the cytoplasm to the nucleus. In addition, Fn14 activation induced BCL-XL and BCL-W mRNA and protein levels, and this effect was dependent upon NFkappaB transcriptional activity. Substitution of a putative NFkappaB binding site identified in the BCL-X promoter significantly decreased Fn14-induced transactivation. Furthermore Fn14-induced transactivation of the BCL-X promoter was also diminished by the super-repressor IkappaBalpha mutant, which specifically inhibits NFkappaB activity, and by mutations in the NFkappaB binding motif of the BCL-X promoter. Additionally small interfering RNA-mediated depletion of either BCL-XL or BCL-W antagonized the TWEAK protective effect on glioma cells. Our results suggest that NFkappaB-mediated up-regulation of BCL-XL and BCL-W expression in glioma cells increases cellular resistance to cytotoxic therapy-induced apoptosis. We propose that the Fn14 protein functions, in part, through the NFkappaB signaling pathway to up-regulate BCL-XL and BCL-W expression to foster malignant glioblastoma cell survival. Targeted therapy against Fn14 as an adjuvant to surgery may improve management of invasive glioma cells and advance the outcome of this devastating cancer.
Assuntos
Astrocitoma , Neoplasias Encefálicas , Proteínas de Transporte/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/genética , Linhagem Celular , Sobrevivência Celular/fisiologia , Citocina TWEAK , Citotoxinas/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas I-kappa B/metabolismo , Ligantes , Inibidor de NF-kappaB alfa , Fosforilação , Proteínas , RNA Interferente Pequeno , Receptores do Fator de Necrose Tumoral/genética , Transdução de Sinais/fisiologia , Receptor de TWEAK , Fatores de Necrose Tumoral , Proteína bcl-XRESUMO
Glioblastoma multiforme comprises the majority of human brain tumors. Patients with glioblastoma multiforme have poor survival rates, with an average life expectancy of <1 year. To assess possible mechanisms and to potentially target invasive glioma cells, we previously measured the gene expression profiles of glioma cells under migration-activated or passive states. One of the genes identified was Fn14, which encodes a cell surface receptor for the tumor necrosis factor superfamily member named TWEAK. In this study, we show that Fn14 gene expression is induced in migration-activated glioma cells in vitro and significantly increases according to tumor grade in vivo (P < 0.01), with highest levels in glioblastoma tissue specimens. The in situ expression pattern of Fn14 mRNA and protein was confined to primary glioma cells and the vascular endothelium, with no detection in adjacent normal brain. Conversely, TWEAK mRNA levels are low in glioblastoma samples relative to normal brain tissue. In addition, activation of the Fn14 receptor by addition of recombinant TWEAK resulted in increased glioma cell migration in vitro. These results suggest a positive role for TWEAK and Fn14 in glioma progression and indicate that Fn14 gene expression may serve as a marker for invasive glioma cells.