RESUMO
There is an unmet need for the identification of biomarkers to aid in the diagnosis, clinical management, prognosis and follow-up of meningiomas. There is currently no consensus on the optimum management of WHO grade II meningiomas. In this study, we identified the calcium binding extracellular matrix glycoprotein, Fibulin-2, via mass-spectrometry-based proteomics, assessed its expression in grade I and II meningiomas and explored its potential as a grade II biomarker. A total of 87 grade I and 91 grade II different meningioma cells, tissue and plasma samples were used for the various experimental techniques employed to assess Fibulin-2 expression. The tumours were reviewed and classified according to the 2016 edition of the Classification of the Tumours of the central nervous system (CNS). Mass spectrometry proteomic analysis identified Fibulin-2 as a differentially expressed protein between grade I and II meningioma cell cultures. Fibulin-2 levels were further evaluated in meningioma cells using Western blotting and Real-time Quantitative Polymerase Chain Reaction (RT-qPCR); in meningioma tissues via immunohistochemistry and RT-qPCR; and in plasma via Enzyme-Linked Immunosorbent Assay (ELISA). Proteomic analyses (p < 0.05), Western blotting (p < 0.05) and RT-qPCR (p < 0.01) confirmed significantly higher Fibulin-2 (FBLN2) expression levels in grade II meningiomas compared to grade I. Fibulin-2 blood plasma levels were also significantly higher in grade II meningioma patients compared to grade I patients. This study suggests that elevated Fibulin-2 might be a novel grade II meningioma biomarker, when differentiating them from the grade I tumours. The trend of Fibulin-2 expression observed in plasma may serve as a useful non-invasive biomarker.
Assuntos
Biomarcadores Tumorais/sangue , Proteínas de Ligação ao Cálcio/sangue , Proteínas da Matriz Extracelular/sangue , Neoplasias Meníngeas/sangue , Meningioma/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/patologia , Meningioma/genética , Meningioma/patologia , Pessoa de Meia-Idade , Gradação de Tumores , Prognóstico , ProteômicaAssuntos
Antineoplásicos/farmacologia , Neurofibromatose 2/metabolismo , Sorafenibe/farmacologia , Adulto , Antineoplásicos/metabolismo , Western Blotting , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Ciclina D1/efeitos dos fármacos , Ciclina D1/metabolismo , Humanos , Imuno-Histoquímica , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Terapia de Alvo Molecular , Neurofibromatose 2/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/efeitos dos fármacos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteína S6 Ribossômica/efeitos dos fármacos , Proteína S6 Ribossômica/metabolismo , Sorafenibe/metabolismoRESUMO
Meningioma and schwannoma are common tumours of the nervous system. They occur sporadically or as part of the hereditary NF2-related schwannomatosis syndrome. There is an unmet need for new effective drug treatments for both tumour types. In this paper, we demonstrate overexpression/activation of TAM (TYRO3/AXL/MERTK) receptors (TAMs) and overexpression/release of ligand GAS6 in patient-derived meningioma tumour cells and tissue. For the first time, we reveal the formation of MERTK/TYRO3 heterocomplexes in meningioma and schwannoma tissue. We demonstrate the dependence of AXL and TYRO3 expression on MERTK in both tumour types, as well as interdependency of MERTK and AXL expression in meningioma. We show that MERTK and AXL contribute to increased proliferation and survival of meningioma and schwannoma cells, which we inhibited in vitro using the MERTK/FLT3 inhibitor UNC2025 and the AXL inhibitor BGB324. UNC2025 was effective in both tumour types with superior efficacy over BGB324. Finally, we found that TAMs are expressed by tumour-associated macrophages in meningioma and schwannoma tumours and that UNC2025 strongly depleted macrophages in both tumour types.
RESUMO
AIMS: Schwann cells myelinate axons of the peripheral nervous system. This process of myelination is regulated by various transcription factors. c-Jun and Sox-2 are negative regulators of myelination and control Schwann cell differentiation and plasticity. Schwannoma cells within tumours no longer express myelin markers, and show increased proliferation and decreased apoptosis. We have shown previously that several signalling pathways are activated in schwannoma cells in situ, in particular the c-Jun N-terminal kinase (JNK) pathway. Both in vitro and in vivo we have demonstrated that c-Jun and Sox-2 are co-regulated in Schwann cells and evidence shows that both these proteins regulate myelination negatively. In this study, we aimed to characterize the expression of c-Jun and Sox-2 in schwannoma and traumatic neuroma. METHODS AND RESULTS: Immunohistochemistry using antibodies to c-Jun and Sox-2 was applied to six schwannomas, and the results were compared with those seen in traumatic neuroma and normal nerve. Increased expression of c-Jun and Sox-2 was seen in schwannoma. CONCLUSIONS: We have demonstrated increased expression of c-Jun and Sox-2 in schwannoma compared to traumatic neuroma. There was no expression of c-Jun and Sox-2 in a histologically normal peripheral nerve.
Assuntos
Neurilemoma/metabolismo , Neuroma/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Biomarcadores Tumorais/metabolismo , Humanos , Neurilemoma/patologia , Neuroma/etiologia , Neuroma/patologia , Traumatismos dos Nervos Periféricos/complicações , Traumatismos dos Nervos Periféricos/patologia , Nervos Periféricos/metabolismo , Nervos Periféricos/patologiaRESUMO
Loss of the tumor suppressor merlin causes development of the tumors of the nervous system, such as schwannomas, meningiomas, and ependymomas occurring spontaneously or as part of a hereditary disease Neurofibromatosis Type 2 (NF2). Current therapies, (radio) surgery, are not always effective. Therefore, there is a need for drug treatments for these tumors. Schwannomas are the most frequent of merlin-deficient tumors and are hallmark for NF2. Using our in vitro human schwannoma model, we demonstrated that merlin-deficiency leads to increased proliferation, cell-matrix adhesion, and survival. Increased proliferation due to strong activation of extracellular-signal-regulated kinase 1/2 (ERK1/2) is caused by overexpression/activation of platelet-derived growth factor receptor-ß (PDGFR-ß) and ErbB2/3 which we successfully blocked with AZD6244, sorafenib, or lapatinib. Schwannoma basal proliferation is, however, only partly dependent on PDGFR-ß and is completely independent of ErbB2/3. Moreover, the mechanisms underlying pathological cell-matrix adhesion and survival of schwannoma cells are still not fully understood. Here, we demonstrate that insulin-like growth factor-I receptor (IGF-IR) is strongly overexpressed and activated in human primary schwannoma cells. IGF-I and -II are overexpressed and released from schwannoma cells. We show that ERK1/2 is relevant for IGF-I-mediated increase in proliferation and cell-matrix adhesion, c-Jun N-terminal kinases for increased proliferation and AKT for survival. We demonstrate new mechanisms involved in increased basal proliferation, cell-matrix adhesion, and survival of schwannoma cells. We identified therapeutic targets IGF-IR and downstream PI3K for treatment of schwannoma and other merlin-deficient tumors and show usefulness of small molecule inhibitors in our model. PI3K is relevant for both IGF-IR and previously described PDGFR-ß signaling in schwannoma.
Assuntos
Neoplasias Encefálicas/metabolismo , Neurilemoma/metabolismo , Neurofibromatose 2/metabolismo , Transdução de Sinais/fisiologia , Somatomedinas/metabolismo , Neoplasias Encefálicas/patologia , Proliferação de Células , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neurilemoma/patologia , Neurofibromatose 2/patologia , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Deficiency of the tumor suppressor Merlin causes development of schwannoma, meningioma, and ependymoma tumors, which can occur spontaneously or in the hereditary disease neurofibromatosis type 2 (NF2). Merlin mutations are also relevant in a variety of other tumors. Surgery and radiotherapy are current first-line treatments; however, tumors frequently recur with limited treatment options. Here, we use human Merlin-negative schwannoma and meningioma primary cells to investigate the involvement of the endogenous retrovirus HERV-K in tumor development. HERV-K proteins previously implicated in tumorigenesis were overexpressed in schwannoma and all meningioma grades, and disease-associated CRL4DCAF1 and YAP/TEAD pathways were implicated in this overexpression. In normal Schwann cells, ectopic overexpression of HERV-K Env increased proliferation and upregulated expression of c-Jun and pERK1/2, which are key components of known tumorigenic pathways in schwannoma, JNK/c-Jun, and RAS/RAF/MEK/ERK. Furthermore, FDA-approved retroviral protease inhibitors ritonavir, atazanavir, and lopinavir reduced proliferation of schwannoma and grade I meningioma cells. These results identify HERV-K as a critical regulator of progression in Merlin-deficient tumors and offer potential strategies for therapeutic intervention. SIGNIFICANCE: The endogenous retrovirus HERV-K activates oncogenic signaling pathways and promotes proliferation of Merlin-deficient schwannomas and meningiomas, which can be targeted with antiretroviral drugs and TEAD inhibitors.
Assuntos
Antirretrovirais/farmacologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Retrovirus Endógenos/metabolismo , Neoplasias Meníngeas/metabolismo , Meningioma/metabolismo , Neurilemoma/metabolismo , Neurofibromina 2/metabolismo , Proteínas Virais/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Células HEK293 , Humanos , Neoplasias Meníngeas/complicações , Neoplasias Meníngeas/patologia , Neoplasias Meníngeas/virologia , Meningioma/complicações , Meningioma/patologia , Meningioma/virologia , Neurilemoma/complicações , Neurilemoma/patologia , Neurilemoma/virologia , Neurofibromatose 2/complicações , Neurofibromina 2/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transfecção , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genéticaRESUMO
Deficiency of the tumor suppressor merlin leads to the development of multiple tumors of the nervous system, such as schwannomas, meningiomas, and ependymomas. Due to the benign character of these tumors, classical chemotherapy is ineffective. Current therapies, surgery, and radiosurgery are local and quite invasive, thus new systemic treatments are required. We have previously described the Raf/mitogen-activated kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway activation and its role in schwannoma growth. Here, we targeted MEK1/2 known as a convergence point for multiple cascades towards ERK1/2 activation and cell proliferation, using MEK1/2 inhibitor AZD6244 (ARRY-142886; Astra Zeneca). We show that AZD6244 at low concentration completely abolished platelet-derived growth factor-DD-mediated ERK1/2 activation and cell proliferation in human primary schwannoma cells. Moreover, this drug was not toxic for either schwannoma or Schwann cells and has been reported to be safe with tolerable side effects. Thus, AZD6244 can be considered as a drug candidate for schwannoma treatment.
Assuntos
Benzimidazóis/farmacologia , Inibidores Enzimáticos/farmacologia , MAP Quinase Quinase 2/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neurilemoma/tratamento farmacológico , Neurilemoma/fisiopatologia , Antineoplásicos/farmacologia , Bromodesoxiuridina , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Immunoblotting , Imuno-Histoquímica , MAP Quinase Quinase 2/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Células de Schwann/efeitos dos fármacos , Células de Schwann/fisiologiaRESUMO
Activation of OX1 orexin receptors heterologously expressed in Chinese hamster ovary (CHO) cells led to a rapid, strong, and long-lasting increase in ERK phosphorylation (activation). Dissection of the signal pathways to ERK using multiple inhibitors and dominant-negative constructs indicated involvement of Ras, protein kinase C, phosphoinositide-3-kinase, and Src. Most interestingly, Ca2+ influx appeared central for the ERK response in CHO cells, and the same was indicated in recombinant neuro-2a cells and cultured rat striatal neurons. Detailed investigations in CHO cells showed that inhibition of the receptor- and store-operated Ca2+ influx pathways could fully attenuate the response, whereas inhibition of the store-operated Ca2+ influx pathway alone or the Ca2+ release was ineffective. If the receptor-operated pathway was blocked, an exogenously activated store-operated pathway could take its place and restore the coupling of OX1 receptors to ERK. Further experiments suggested that Ca2+ influx, as such, may not be required for ERK phosphorylation, but that Ca2+, elevated via influx, acts as a switch enabling OX1 receptors to couple to cascades leading to ERK phosphorylation, cAMP elevation, and phospholipase C activation. In conclusion, the data suggest that the primary coupling of orexin receptors to Ca2+ influx allows them to couple to other signal pathways; in the absence of coupling to Ca2+ influx, orexin receptors can act as signal integrators by taking advantage of other Ca2+ influx pathways.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Receptores de Neuropeptídeos/fisiologia , Animais , Células Cultivadas , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Receptores de Orexina , Orexinas , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Ratos , Receptores Acoplados a Proteínas G , Transdução de Sinais , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismoRESUMO
Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder characterized by the development of multiple tumors in the central nervous system, most notably schwannomas, and meningiomas. Mutational inactivation of the NF2 gene encoding the protein Merlin is found in most sporadic and inherited schwannomas, but the molecular mechanisms underlying neoplastic changes in schwannoma cells remain unclear. We report here that Nf2-deficient cells display elevated expression levels of key enzymes involved in lipogenesis and that this upregulation is caused by increased activity of Torc1. Inhibition or knockdown of fatty acid synthase (FASN), the enzyme that catalyzes the formation of palmitic acid from malonyl-CoA, drove NF2-deficient cells into apoptosis. Treatment of NF2-mutant cells with agents that inhibit the production of malonyl-CoA reduced their sensitivity to FASN inhibitors. Collectively, these results suggest that the altered lipid metabolism found in NF2-mutant cells renders them sensitive to elevated levels of malonyl-CoA, as occurs following blockade of FASN, suggesting new targeted strategies in the treatment of NF2-deficient tumors. Cancer Res; 77(18); 5026-38. ©2017 AACR.
Assuntos
Biomarcadores Tumorais/metabolismo , Ácido Graxo Sintase Tipo I/metabolismo , Ácidos Graxos/metabolismo , Meningioma/patologia , Complexos Multiproteicos/metabolismo , Neurilemoma/patologia , Neurofibromina 2/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Ácido Graxo Sintase Tipo I/genética , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Lipogênese , Alvo Mecanístico do Complexo 1 de Rapamicina , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/metabolismo , Neoplasias Meníngeas/patologia , Meningioma/genética , Meningioma/metabolismo , Camundongos , Camundongos Nus , Complexos Multiproteicos/genética , Neurilemoma/genética , Neurilemoma/metabolismo , Neurofibromina 2/genética , Ratos , Taxa de Sobrevida , Serina-Treonina Quinases TOR/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Deficiency of the tumour suppressor merlin leads to the development of schwannomas, meningiomas and ependymomas occurring spontaneously or as a part of the hereditary disease Neurofibromatosis type 2 (NF2). Merlin loss is also found in a proportion of other cancers like mesothelioma, melanoma, breast cancer and glioblastoma. The tumour suppressor/transcription factor p53 regulates proliferation, survival and differentiation and its deficiency plays a role in the development of many tumours. 53 can be negatively regulated by FAK, PI3K/AKT and MDM2 and possibly positively regulated by merlin in different cell lines. In this study we investigated the role of p53 in merlin-deficient tumours. Using our in vitro model of primary human schwannoma cells we have previously demonstrated that FAK is overexpressed/activated and localises into the nucleus of schwannoma cells increasing proliferation. AKT is strongly activated via platelet-derived growth factor (PDGF) - and insulin-like growth factor 1 (IGF1) - receptors increasing survival. Here we investigated p53 regulation and its role in proliferation and survival of human primary schwannoma cells using western blotting, immunocytochemistry, immunohistochemistry and proliferation, survival and transcription factor assays. In human primary schwannoma cells p53 was found to be downregulated while MDM2 was upregulated leading to increased cell proliferation and survival. p53 is regulated by merlin involving FAK, AKT and MDM2. Merlin reintroduction into schwannoma cells increased p53 levels and activity, and treatment with Nutlin-3, a drug which increases p53 stability by disrupting the p53/MDM2 complex, decreased tumour growth and reduced cell survival. These findings are important to dissect the mechanisms responsible for the development of merlin-deficient tumours and to identify new therapeutic targets. We suggest that Nutlin-3, possibly in combination with FAK or PI3K inhibitors, can be employed as a novel treatment for schwannoma and other merlin-deficient tumours.
Assuntos
Complexos Multiproteicos/metabolismo , Neurilemoma/metabolismo , Neurofibromina 2/deficiência , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Humanos , Complexos Multiproteicos/genética , Neurilemoma/genética , Neurilemoma/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Deficiency of the tumor suppressor protein merlin leads to the development of benign tumors of the nervous system such as schwannomas, ependymomas and meningiomas. These tumors can occur spontaneously or as part of a tumor predisposition syndrome called neurofibromatosis type 2 (NF2), which involves multiple tumors. Schwannomas are the hallmark tumors of NF2 and are the most frequent and well-characterized of the merlin-deficient tumors. Surgery or radiotherapy are used to treat single tumors and can leave the patient with substantial morbidity. Limitations of other treatment options for merlin-deficient tumors, such as the lack of effectiveness of chemotherapy, have led to an urgent requirement for new pharmaceutical therapies. Merlin-deficient tumors are genetically well-defined, which allows rational testing of new molecular therapies that have been developed and successfully used to treat various cancers in the past few years. This Review centers on four key families of receptor tyrosine kinases-the ErbB family, platelet-derived growth factor receptor ß, insulin-like growth factor 1 receptor, and vascular endothelial growth factor receptors-focusing on their role in schwannoma pathobiology and the therapeutic potential of targeting these receptors and their downstream signaling pathways.
Assuntos
Neoplasias do Sistema Nervoso/metabolismo , Neoplasias do Sistema Nervoso/terapia , Neurilemoma/metabolismo , Neurilemoma/terapia , Neurofibromina 2/deficiência , Humanos , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais/genéticaRESUMO
Loss of the tumor suppressor merlin is a cause of frequent tumors of the nervous system, such as schwannomas, meningiomas, and ependymomas, which occur spontaneously or as part of neurofibromatosis type 2 (NF2). Because there is medical need for drug therapies for these tumors, our aim is to find therapeutic targets. We have studied the pathobiology of schwannomas, because they are the most common merlin-deficient tumors and are a model for all merlin-deficient tumors. With use of a human schwannoma in vitro model, we previously described strong overexpression/activation of platelet-derived growth factor receptor-ß (PDGFR-ß) leading to strong, long-lasting activation of extracellular-signal-regulated kinase (ERK1/2) and AKT and increased schwannoma growth, which we successfully inhibited using the PDGFR/Raf inhibitor sorafenib. However, the benign character of schwannomas may require long-term treatment; thus, drug tolerability is an issue. With the use of Western blotting, proliferation assays, viability assays, and a primary human schwannoma cell in vitro model, we tested the PDGFR/c-KIT inhibitors imatinib (Glivec(;) Novartis) and nilotinib (Tasigna(;) Novartis). Imatinib and nilotinib inhibited PDGF-DD-mediated ERK1/2 activation, basal and PDGF-DD-mediated activation of PDGFR-ß and AKT, and schwannoma proliferation. Nilotinib is more potent than imatinib, exerting its maximal inhibitory effect at concentrations lower than steady-state trough plasma levels. In addition, nilotinib combined with the MEK1/2 inhibitor selumetinib (AZD6244) at low concentrations displayed stronger efficiency toward tumor growth inhibition, compared with nilotinib alone. We suggest that therapy with nilotinib or combinational therapy that simultaneously inhibits PDGFR and the downstream Raf/MEK1/2/ERK1/2 pathway could represent an effective treatment for schwannomas and other merlin-deficient tumors.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzimidazóis/farmacologia , Neurilemoma/tratamento farmacológico , Neurofibromatose 2/tratamento farmacológico , Benzamidas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Mesilato de Imatinib , Immunoblotting , Sistema de Sinalização das MAP Quinases , Neurilemoma/metabolismo , Neurofibromatose 2/metabolismo , Neurofibromina 2/deficiência , Fosforilação , Piperazinas/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/administração & dosagem , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de SinaisRESUMO
Neurofibromatosis type 2 (NF2) is an inherited predisposition cancer syndrome characterized by the development of multiple benign tumors in the nervous system including schwannomas, meningiomas, and ependymomas. Using a disease model comprising primary human schwannoma cells, we previously demonstrated that adherens junctions (AJs) are impaired in schwannoma cells because of a ubiquitous, upregulated Rac activity. However, the mechanism by which loss of contact inhibition leads to proliferation remains obscure in merlin-deficient tumors. In this study, we show that proliferative Wnt/ß-catenin signaling is elevated as active ß-catenin (dephosphorylated at serine 37 and threoine 41) localizes to the nucleus and the Wnt targets genes c-myc and cyclin D1 are upregulated in confluent human schwannoma cells. We demonstrate that Rac effector p21-activated kinase 2 (PAK2) is essential for the activation of Wnt/ß-catenin signaling because depletion of PAK2 suppressed active ß-catenin, c-myc, and cyclin D1. Most importantly, the link between the loss of the AJ complex and the increased proliferation in human schwannoma cells is connected by Src and platelet-derived growth factor receptor-induced tyrosine 654 phosphorylation on ß-catenin and associated with degradation of N-cadherin. We also demonstrate that active merlin maintains ß-catenin and N-cadherin complex at the plasma membrane through direct regulation. Finally, we demonstrate that phosphorylation of tyrosine 654 is critical for the increased proliferation in human schwannoma cells because overexpression of a Y654F mutant ß-catenin reduces hyperproliferation of schwannoma cells. We suggest a model that these pathways are coordinated and relevant for proliferation in merlin-deficient tumors.
Assuntos
Inibição de Contato/genética , Neurilemoma/genética , Neurilemoma/metabolismo , Neurofibromina 2/deficiência , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Caderinas/metabolismo , Membrana Celular/metabolismo , Proliferação de Células , Inativação Gênica , Células HEK293 , Humanos , Modelos Biológicos , Neurofibromina 2/genética , Fosforilação , Ligação Proteica , RNA Interferente Pequeno/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , beta Catenina/genética , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Domínios de Homologia de src , Quinases da Família src/metabolismoRESUMO
Vestibular schwannomas (VS) arising sporadically or in patients with neurofibromatosis type 2 (NF2) consistently lack expression of Merlin, a tumor suppressor. Conventional treatment options include surgery and radiotherapy but there is no validated medical option. Recent evidence suggests that Merlin deficiency may result in abnormal activation of receptor tyrosine kinases (RTKs) and downstream signaling, promoting tumor growth. Although small-molecule RTK inhibitors are widely available for clinical use, no such therapy has been validated in patients with VS. To screen for RTK activation, surgical VS specimens from patients with and without NF2 were analyzed by phospho-RTK profiling arrays. Downstream signaling pathway activation was analyzed by phospho-MAPK arrays. Activated RTKs and downstream kinases were validated immunohistochemically in corresponding formalin-fixed, paraffin-embedded tissues. Phospho-RTK arrays and immunohistochemistry showed consistent overexpression and activation of EGFR family receptors and evidence of ERK1/2 downstream signaling was observed in all samples analyzed (n = 11). Based on the findings, the small-molecule EGFR/ErbB2 kinase inhibitor lapatinib was selected for evaluation of target inhibition and treatment efficacy in our in vitro human schwannoma model. EGFR/ErbB2 targeted therapy with lapatinib inhibited ErbB2 phosphorylation and survivin upregulation, as well as downstream ERK1/2 and AKT activation, resulting in decreased proliferation. We conclude that EGFR family receptor activation is a consistent feature of both sporadic and NF2-related VS. Molecular targeted therapy with lapatinib downregulates survivin and has antiproliferative activity in a preclinical VS model. Based on these findings, a clinical trial with lapatinib for the treatment of VS is currently underway.
Assuntos
Antineoplásicos/farmacologia , Neuroma Acústico/metabolismo , Quinazolinas/farmacologia , Receptor ErbB-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Receptores ErbB/metabolismo , Humanos , Imuno-Histoquímica , Lapatinib , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Análise Serial de Proteínas , Transdução de Sinais/fisiologiaRESUMO
Schwannomas are Schwann cell tumors of the nervous system that occur spontaneously and in patients with neurofibromatosis 2 (NF2) and lack the tumor suppressor merlin. Merlin is known to bind paxillin, beta1 integrin and focal adhesion kinase, members of focal contacts, multi-protein complexes that mediate cell adhesion to the extracellular matrix. Moreover, merlin-deficient Schwannomas show pathological adhesion to the extracellular matrix making the characterization of focal contacts indispensable. Using our Schwannoma in vitro model of human primary Schwann and Schwannoma cells, we here show that Schwannoma cells display an increased number of mature and stable focal contacts. In addition to an involvement of RhoA signaling via the Rho kinase ROCK, Rac1 plays a significant role in the pathological adhesion of Schwannoma cells. The Rac1 guanine exchange factor- beta-Pix, localizes to focal contacts in human primary Schwannoma cells, and we show that part of the Rac1 activation, an effect of merlin-deficiency, occurs at the level of focal contacts in human primary Schwannoma cells. Our results help explaining the pathological adhesion of Schwannoma cells, further strengthen the importance of RhoGTPase signaling in Schwannoma development, and suggest that merlin's role in tumor suppression is linked to focal contacts.
Assuntos
Adesões Focais/metabolismo , Neurofibromina 2/deficiência , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/metabolismo , Adenoviridae/genética , Amidas/farmacologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Adesões Focais/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Imuno-Histoquímica , Microscopia Confocal , Neurilemoma/genética , Neurilemoma/metabolismo , Neurilemoma/patologia , Neurofibromina 2/genética , Paxilina/metabolismo , Peptídeos/farmacologia , Piridinas/farmacologia , Fatores de Troca de Nucleotídeo Guanina Rho , Células de Schwann/citologia , Células de Schwann/metabolismo , Transfecção , Células Tumorais Cultivadas , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Merlin loss causes benign tumours of the nervous system, mainly schwannomas and meningiomas. Schwannomas show enhanced Rac1 and Cdc42 activity, the p21-activated kinase 2 (PAK2) activation and increased ruffling and cell adhesion. PAK regulates activation of merlin. PAK has been proposed as a potential therapeutic target in schwannomas. However where PAK stands in the Rac pathway is insufficiently characterised. We used a novel small-molecule PAK inhibitor, IPA-3, to investigate the role of PAK activation on Rac1/Cdc42 activity, cell spreading and adhesion in human primary schwannoma and Schwann cells. We show that IPA-3 blocks activation of PAK2 at Ser192/197 that antagonises PAK's interaction with Pix. Accordingly, Pix-mediated Rac1 activation is decreased in IPA-3 treated schwannoma cells, indicating that PAK acts upstream of Rac. We show that this Rac activation at the level of focal adhesions in schwannoma cells is essential for cell spreading and adhesion in Schwann and schwannoma cells.
Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Neurilemoma/enzimologia , Células de Schwann/efeitos dos fármacos , Quinases Ativadas por p21/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Dissulfetos/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Naftóis/farmacologia , Neurilemoma/patologia , Células de Schwann/citologia , Serina/metabolismo , Fatores de Tempo , Quinases Ativadas por p21/antagonistas & inibidoresRESUMO
Schwannomas are tumors of the nervous system that occur sporadically and in patients with the cancer predisposition syndrome neurofibromatosis type 2 (NF2). Schwannomas and all NF2-related tumors are caused by loss of the tumor suppressor merlin. Using our human in vitro model for schwannoma, we analyzed extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT signaling pathways, their upstream growth factor receptors, and their role in schwannoma cell proliferation and adhesion to find new systemic therapies for these tumors that, to date, are very difficult to treat. We show here that human primary schwannoma cells show an enhanced basal Raf/mitogen-activated protein/ERK kinase/ERK1/2 pathway activity compared with healthy Schwann cells. Due to a strong and prolonged activation of platelet-derived growth factor receptor beta (PDGFRbeta), which is highly overexpressed, ERK1/2 and AKT activation was further increased in schwannoma, leading to increased proliferation. Using specific inhibitors, we discovered that ERK1/2 activation involves the integrin/focal adhesion kinase/Src/Ras signaling cascades and PDGFRbeta-mediated ERK1/2 activation is triggered through the phosphatidylinositol 3-kinase/protein kinase C/Src/c-Raf pathway. Due to the complexity of signals leading to schwannoma cell proliferation, potential new therapeutic agents should target several signaling pathways. The PDGFR and c-Raf inhibitor sorafenib (BAY 43-9006; Bayer Pharmaceuticals), currently approved for treatment of advanced renal cell cancer, inhibits both basal and PDGFRbeta-mediated ERK1/2 and AKT activity and decreases cell proliferation in human schwannoma cells, suggesting that this drug constitutes a promising tool to treat schwannomas. We conclude that our schwannoma in vitro model can be used to screen for new therapeutic targets in general and that sorafenib is possible candidate for future clinical trials.
Assuntos
Sistemas de Liberação de Medicamentos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Neurilemoma/metabolismo , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Linfocinas/farmacologia , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Neurilemoma/enzimologia , Neurilemoma/etiologia , Neurilemoma/genética , Neurofibromatose 2/complicações , Proteína Oncogênica v-akt/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
We have investigated the signaling of OX(1) receptors to cell death using Chinese hamster ovary cells as a model system. OX(1) receptor stimulation with orexin-A caused a delayed cell death independently of cytosolic Ca(2+) elevation. The classical mitogen-activated protein kinase (MAPK) pathways, ERK and p38, were strongly activated by orexin-A. p38 was essential for induction of cell death, whereas the ERK pathway appeared protective. A pathway often implicated in the p38-mediated cell death, activation of p53, did not mediate the cell death, as there was no stabilization of p53 or increase in p53-dependent transcriptional activity, and dominant-negative p53 constructs did not inhibit cell demise. Under basal conditions, orexin-A-induced cell death was associated with compact chromatin condensation and it required de novo gene transcription and protein synthesis, the classical hallmarks of programmed (apoptotic) cell death. However, though the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethyl ketone (Z-VAD-fmk) fully inhibited the caspase activity, it did not rescue the cells from orexin-A-induced death. In the presence of Z-VAD-fmk, orexin-A-induced cell death was still dependent on p38 and de novo protein synthesis, but it no longer required gene transcription. Thus, caspase inhibition causes activation of alternative, gene transcription-independent death pathway. In summary, the present study points out mechanisms for orexin receptor-mediated cell death and adds to our general understanding of the role of G-protein-coupled receptor signaling in cell death by suggesting a pathway from G-protein-coupled receptors to cell death via p38 mitogen-/stress-activated protein kinase independent of p53 and caspase activation.
Assuntos
Caspases/metabolismo , Receptores de Neuropeptídeos/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Western Blotting , Bromodesoxiuridina/farmacologia , Células CHO , Cálcio/química , Cálcio/metabolismo , Proliferação de Células , Cromatina/química , Corantes/farmacologia , Cricetinae , Meios de Cultura Livres de Soro/química , Relação Dose-Resposta a Droga , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Vetores Genéticos , Humanos , Indóis/farmacologia , Sistema de Sinalização das MAP Quinases , Maleimidas/farmacologia , Receptores de Orexina , Proteína Quinase C/metabolismo , Receptores Acoplados a Proteínas G , Receptores de Neuropeptídeos/metabolismo , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Fatores de Tempo , Transcrição Gênica , Transfecção , Proteína Supressora de Tumor p53/metabolismoRESUMO
In this study, the mechanism of OX(1) orexin receptors to regulate adenylyl cyclase activity when recombinantly expressed in Chinese hamster ovary cells was investigated. In intact cells, stimulation with orexin-A led to two responses, a weak (21%), high potency (EC(50) approximately 1 nm) inhibition and a strong (4-fold), low potency (EC(50) = approximately 300 nm) stimulation. The inhibition was reversed by pertussis toxin, suggesting the involvement of G(i/o) proteins. Orexin-B was, surprisingly, almost equally as potent as orexin-A in elevating cAMP (pEC(50) = approximately 500 nm). cAMP elevation was not caused by Ca(2+) elevation or by Gbetagamma. In contrast, it relied in part on a novel protein kinase C (PKC) isoform, PKCdelta, as determined using pharmacological inhibitors. Yet, PKC stimulation alone only very weakly stimulated cAMP production (1.1-fold). In the presence of G(s) activity, orexins still elevated cAMP; however, the potencies were greatly increased (EC(50) of orexin-A = approximately 10 nm and EC(50) of orexin-B = approximately 100 nm), and the response was fully dependent on PKCdelta. In permeabilized cells, only a PKC-independent low potency component was seen. This component was sensitive to anti-Galpha(s) antibodies. We conclude that OX(1) receptors stimulate adenylyl cyclase via a low potency G(s) coupling and a high potency phospholipase C --> PKC coupling. The former or some exogenous G activation is essentially required for the PKC to significantly activate adenylyl cyclase. The results also suggest that orexin-B-activated OX(1) receptors couple to G(s) almost as efficiently as the orexin-A-activated receptors, in contrast to Ca(2+) elevation and phospholipase C activation, for which orexin-A is 10-fold more potent.
Assuntos
Adenilil Ciclases/metabolismo , Receptores de Neuropeptídeos/fisiologia , Animais , Células COS , Cálcio , AMP Cíclico/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Neuropeptídeos/fisiologia , Receptores de Orexina , Orexinas , Proteína Quinase C/metabolismo , Receptores Acoplados a Proteínas G , Receptores de Neuropeptídeos/genética , Proteínas Recombinantes , Transgenes , Fosfolipases Tipo C/metabolismoRESUMO
Orexin A and orexin B are hypothalamic peptides that act on their targets via two G protein-coupled receptors (OX1 and OX2 receptors). In the central nervous system, the cell bodies producing orexins are localized in a narrow region within the lateral hypothalamus and project mainly to regions involved in feeding, sleep, and autonomic functions. Via putative pre- and postsynaptic effects, orexins increase synaptic activity in these regions. In isolated neurons and cells expressing recombinant receptors orexins cause Ca2+ elevation, which is mainly dependent on influx. The activity of orexinergic cells appears to be controlled by feeding- and sleep-related signals via a variety of neurotransmitters/hormones from the brain and other tissues. Orexins and orexin receptors are also found outside the central nervous system, particularly in organs involved in feeding and energy metabolism, e.g., gastrointestinal tract, pancreas, and adrenal gland. In the present review we focus on the physiological properties of the cells that secrete or respond to orexins.