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1.
Stem Cells ; 36(11): 1736-1751, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29999568

RESUMO

Adult neurogenesis in the brain continuously seeds new neurons throughout life, but how homeostasis of adult neural stem cells (NSCs) is maintained is incompletely understood. Here, we demonstrate that the DNA methylation adapter ubiquitin-like, containing PHD and RING finger domains-1 (UHRF1) is expressed in, and regulates proliferation of, the active but not quiescent pool of adult neural progenitor cells. Mice with a neural stem cell-specific deficiency in UHRF1 exhibit a massive depletion of neurogenesis resulting in a collapse of formation of new neurons. In the absence of UHRF1, NSCs unexpectedly remain in the cell cycle but with a 17-fold increased cell cycle length due to a failure of replication phase entry caused by promoter demethylation and derepression of Cdkn1a, which encodes the cyclin-dependent kinase inhibitor p21. UHRF1 does not affect the proportion progenitor cells active within the cell cycle but among these cells, UHRF1 is critical for licensing replication re-entry. Therefore, this study shows that a UHRF1-Cdkn1a axis is essential for the control of stem cell self-renewal and neurogenesis in the adult brain. Stem Cells 2018;36:1736-1751.


Assuntos
Células-Tronco Adultas/metabolismo , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/genética , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases
2.
Bioconjug Chem ; 28(2): 539-548, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28045502

RESUMO

Conjugation of llama single domain antibody fragments (Variable Heavy chain domains of Heavy chain antibodies, VHHs) to diagnostic or therapeutic nanoparticles, peptides, proteins, or drugs offers many opportunities for optimized targeted cancer treatment. Currently, mostly nonspecific conjugation strategies or genetic fusions are used that may compromise VHH functionality. In this paper we present a versatile modular approach for bioorthogonal VHH modification and conjugation. First, sortase A mediated transPEGylation is used for introduction of a chemical click moiety. The resulting clickable VHHs are then used for conjugation to other groups employing the Cu+-independent strain-promoted alkyne-azide cycloadition (SPAAC) reaction. Using this approach, tail-to-tail bispecific VHHs and VHH-targeted nanoparticles are generated without affecting VHH functionality. Furthermore, this approach allows the bioconjugation of multiple moieties to VHHs for simple and convenient production of VHH-based theranostics.


Assuntos
Camelídeos Americanos/imunologia , Imunoconjugados/química , Cadeias Pesadas de Imunoglobulinas/química , Nanopartículas/química , Polietilenoglicóis/química , Anticorpos de Domínio Único/química , Alcinos/química , Aminoaciltransferases/metabolismo , Animais , Azidas/química , Proteínas de Bactérias/metabolismo , Química Click/métodos , Reação de Cicloadição/métodos , Cisteína Endopeptidases/metabolismo , Imunoconjugados/imunologia , Imunoconjugados/metabolismo , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/metabolismo , Polietilenoglicóis/metabolismo , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/metabolismo
3.
Ann Surg Oncol ; 23(9): 2745-52, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27334220

RESUMO

BACKGROUND: In synovial sarcomas alterations in the cyclin D1-CDK4/6-Rb axis have been described. Also, ß-catenin, a cyclin D1 regulator, is often overexpressed. Additionally, studies have shown that the t(X;18) translocation influences tumor behavior partly through cyclin D1 activation. We investigated how alterations in the cyclin D1-CDK4/6-Rb axis impact prognosis and studied effects of targeting this axis with the CDK4/6 inhibitor palbociclib. METHODS: Synovial sarcoma samples (n = 43) were immunohistochemically stained for ß-catenin, cyclin D1, p16, p21, p27, Rb, and phospho-Rb. Fluorescent in situ hybridization (FISH) was performed to detect CCND1 amplification or translocation. In 4 synovial sarcoma cell lines sensitivity to palbociclib was investigated using cell viability assays, and effects on the sensitive cell lines were evaluated on protein level and by cell cycle arrest. RESULTS: Expression of nuclear phospho-Rb and nuclear ß-catenin in the patient samples was associated with poor survival. FISH showed a sporadic translocation of CCND1 in a subset of tumors. An 8-fold CCND1 amplification was found in 1 cell line, but not in the patient samples investigated. Palbociclib effectively inhibited Rb-phosphorylation in 3 cell lines, resulting in an induction of a G1 arrest and proliferation block. CONCLUSIONS: In this series nuclear phospho-Rb and nuclear ß-catenin expression were negative prognostic factors. In vitro data suggest that palbociclib may be a potential treatment for a subset of synovial sarcoma patients. Whether this effect can be enhanced by combination treatment deserves further preclinical investigations.


Assuntos
Antineoplásicos/uso terapêutico , Piperazinas/uso terapêutico , Piridinas/uso terapêutico , Sarcoma Sinovial/tratamento farmacológico , Sarcoma Sinovial/metabolismo , Adolescente , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina D1/genética , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Masculino , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Piridinas/farmacologia , Proteína do Retinoblastoma/metabolismo , Sarcoma Sinovial/genética , Taxa de Sobrevida , Adulto Jovem , beta Catenina/metabolismo
4.
Biochim Biophys Acta ; 1846(1): 66-74, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24747768

RESUMO

Diffuse gliomas comprise a group of primary brain tumors that originate from glial (precursor) cells and present as a variety of malignancy grades which have in common that they grow by diffuse infiltration. This phenotype complicates treatment enormously as it precludes curative surgery and radiotherapy. Furthermore, diffusely infiltrating glioma cells often hide behind a functional blood-brain barrier, hampering delivery of systemically administered therapeutic and diagnostic compounds to the tumor cells. The present review addresses the biological mechanisms that underlie the diffuse infiltrative phenotype, knowledge of which may improve treatment strategies for this disastrous tumor type. The invasive phenotype is specific for glioma: most other brain tumor types, both primary and metastatic, grow as delineated lesions. Differences between the genetic make-up of glioma and that of other tumor types may therefore help to unravel molecular pathways, involved in diffuse infiltrative growth. One such difference concerns mutations in the NADP(+)-dependent isocitrate dehydrogenase (IDH1 and IDH2) genes, which occur in >80% of cases of low grade glioma and secondary glioblastoma. In this review we present a novel hypothesis which links IDH1 and IDH2 mutations to glutamate metabolism, possibly explaining the specific biological behavior of diffuse glioma.


Assuntos
Neoplasias Encefálicas/patologia , Quimiotaxia , Glioma/patologia , Ácido Glutâmico/fisiologia , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Transformação Celular Neoplásica/genética , Quimiotaxia/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Glioma/tratamento farmacológico , Glioma/genética , Ácido Glutâmico/farmacologia , Glutamina/metabolismo , Humanos , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo
5.
Acta Neuropathol ; 130(1): 131-44, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25862637

RESUMO

MET has gained interest as a therapeutic target for a number of malignancies because of its involvement in tumorigenesis, invasion and metastasis. At present, a number of inhibitors, both antibodies against MET or its ligand hepatocyte growth factor, and small molecule MET tyrosine kinase inhibitors are in clinical trials. We here describe a novel variant of MET that is expressed in 6% of high-grade gliomas. Characterization of this mutation in a glioma cell line revealed that it consists of an intronic deletion, resulting in a splice event connecting an intact splice donor site in exon 6 with the next splice acceptor site being that of exon 9. The encoded protein lacks parts of the extracellular IPT domains 1 and 2, encoded by exons 7 and 8, resulting in a novel pseudo-IPT and is named MET(Δ7-8). MET(Δ7-8) is located predominantly in the cytosol and is constitutively active. The auto-activating nature of MET(Δ7-8), in combination with a lack of transmembrane localization, renders MET(Δ7-8) not targetable using antibodies, although the protein is efficiently deactivated by MET-specific tyrosine kinase inhibitors. Testing of MET-expressing tumors for the presence of this variant may be important for treatment decision making.


Assuntos
Glioma/genética , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Deleção de Sequência , Anilidas/farmacologia , Animais , Anticorpos/metabolismo , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Feminino , Glioma/tratamento farmacológico , Glioma/metabolismo , Glioma/patologia , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Masculino , Camundongos , Gradação de Tumores , Transplante de Neoplasias , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Conformação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Piridinas/farmacologia , RNA Mensageiro/metabolismo , Sarcoma/genética , Sarcoma/metabolismo , Sarcoma/patologia
6.
Acta Neuropathol ; 127(6): 897-909, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24777482

RESUMO

Diffuse intrinsic pontine glioma (DIPG), with a median survival of only 9 months, is the leading cause of pediatric brain cancer mortality. Dearth of tumor tissue for research has limited progress in this disease until recently. New experimental models for DIPG research are now emerging. To develop preclinical models of DIPG, two different methods were adopted: cells obtained at autopsy (1) were directly xenografted orthotopically into the pons of immunodeficient mice without an intervening cell culture step or (2) were first cultured in vitro and, upon successful expansion, injected in vivo. Both strategies resulted in pontine tumors histopathologically similar to the original human DIPG tumors. However, following the direct transplantation method all tumors proved to be composed of murine and not of human cells. This is in contrast to the indirect method that included initial in vitro culture and resulted in xenografts comprising human cells. Of note, direct injection of cells obtained postmortem from the pons and frontal lobe of human brains not affected by cancer did not give rise to neoplasms. The murine pontine tumors exhibited an immunophenotype similar to human DIPG, but were also positive for microglia/macrophage markers, such as CD45, CD68 and CD11b. Serial orthotopic injection of these murine cells results in lethal tumors in recipient mice. Direct injection of human DIPG cells in vivo can give rise to malignant murine tumors. This represents an important caveat for xenotransplantation models of DIPG. In contrast, an initial in vitro culture step can allow establishment of human orthotopic xenografts. The mechanism underlying this phenomenon observed with direct xenotransplantation remains an open question.


Assuntos
Neoplasias do Tronco Encefálico , Modelos Animais de Doenças , Glioma , Transplante de Neoplasias/métodos , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Neoplasias do Tronco Encefálico/genética , Neoplasias do Tronco Encefálico/patologia , Neoplasias do Tronco Encefálico/fisiopatologia , Antígeno CD11b/metabolismo , Técnicas de Cultura de Células , Criança , Feminino , Lobo Frontal/patologia , Lobo Frontal/fisiopatologia , Lobo Frontal/transplante , Glioma/genética , Glioma/patologia , Glioma/fisiopatologia , Humanos , Lactente , Antígenos Comuns de Leucócito/metabolismo , Masculino , Camundongos Nus , Camundongos SCID , Camundongos Transgênicos , Ponte/patologia , Ponte/fisiopatologia , Ponte/transplante , Adulto Jovem
7.
Nat Commun ; 14(1): 5024, 2023 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-37596278

RESUMO

A perimetastatic capsule is a strong positive prognostic factor in liver metastases, but its origin remains unclear. Here, we systematically quantify the capsule's extent and cellular composition in 263 patients with colorectal cancer liver metastases to investigate its clinical significance and origin. We show that survival improves proportionally with increasing encapsulation and decreasing tumor-hepatocyte contact. Immunostaining reveals the gradual zonation of the capsule, transitioning from benign-like NGFRhigh stroma at the liver edge to FAPhigh stroma towards the tumor. Encapsulation correlates with decreased tumor viability and preoperative chemotherapy. In mice, chemotherapy and tumor cell ablation induce capsule formation. Our results suggest that encapsulation develops where tumor invasion into the liver plates stalls, representing a reparative process rather than tumor-induced desmoplasia. We propose a model of metastases growth, where the efficient tumor colonization of the liver parenchyma and a reparative liver injury reaction are opposing determinants of metastasis aggressiveness.


Assuntos
Neoplasias Hepáticas , Animais , Camundongos , Hepatócitos , Agressão , Relevância Clínica
8.
J Pathol ; 223(5): 626-34, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21341272

RESUMO

Currently available compounds that interfere with VEGF-A signalling effectively inhibit angiogenesis in gliomas, but influence diffuse infiltrative growth to a much lesser extent. Development of a functional tumour vascular bed not only involves VEGF-A but also requires platelet-derived growth factor receptor-ß (PDGFRß), which induces maturation of tumour blood vessels. Therefore, we tested whether combined inhibition of VEGFR and PDGFRß increases therapeutic benefit in the orthotopic glioma xenograft models E98 and E473, both displaying the diffuse infiltrative growth that is characteristically observed in most human gliomas. We used bevacizumab and vandetanib as VEGF(R) inhibitors, and sunitinib to additionally target PDGFRß. We show that combination therapy of sunitinib and vandetanib does not improve therapeutic efficacy compared to treatment with sunitinib, vandetanib or bevacizumab alone. Furthermore, all compounds induced reduction of vessel leakage in compact E98 tumour areas, resulting in decreased detectability of these mostly infiltrative xenografts in Gd-DTPA-enhanced MRI scans. These data show that inhibition of VEGF signalling cannot be optimized by additional PDGFR inhibition and support the concept that diffuse infiltrative areas in gliomas are resistant to anti-angiogenic therapy.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Inibidores da Angiogênese/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Bevacizumab , Barreira Hematoencefálica/efeitos dos fármacos , Neoplasias Encefálicas/irrigação sanguínea , Glioma/irrigação sanguínea , Humanos , Indóis/administração & dosagem , Indóis/farmacologia , Indóis/uso terapêutico , Imageamento por Ressonância Magnética/métodos , Camundongos , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Piperidinas/administração & dosagem , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Pirróis/administração & dosagem , Pirróis/farmacologia , Pirróis/uso terapêutico , Quinazolinas/administração & dosagem , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Sunitinibe , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Nucleic Acids Res ; 37(2): 322-35, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19042971

RESUMO

The tumor suppressor p53 contributes to the cellular fate after genotoxic insults, mainly through the regulation of target genes, thereby allowing e.g. repair mechanisms resulting in cell survival or inducing apoptosis. Unresolved so far is the issue, which exact mechanisms lead to one or the other cellular outcome. Here, we describe the interferon regulatory factor-2-binding protein-2 (IRF2BP2) as a new direct target gene of p53, influencing the p53-mediated cellular decision. We show that upregulation of IRF2BP2 after treatment with actinomycin D (Act.D) is dependent on functional p53 in different cell lines. This occurs in parallel with the down-regulation of the interacting partner of IRF2BP2, the interferon regulatory factor-2 (IRF2), which is known to positively influence cell growth. Analyzing the molecular functions of IRF2BP2, it appears to be able to impede on the p53-mediated transactivation of the p21- and the Bax-gene. We show here that overexpressed IRF2BP2 has an impact on the cellular stress response after Act.D treatment and that it diminishes the induction of apoptosis after doxorubicin treatment. Furthermore, the knockdown of IRF2BP2 leads to an upregulation of p21 and faster induction of apoptosis after doxorubicin as well as Act.D treatment.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas Nucleares/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Antibióticos Antineoplásicos/farmacologia , Apoptose , Sítios de Ligação , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Proteínas de Ligação a DNA , Dactinomicina/farmacologia , Dactinomicina/toxicidade , Doxorrubicina/farmacologia , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Fatores de Transcrição
10.
Acta Neuropathol ; 119(2): 157-75, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19936768

RESUMO

Gliomas are a diverse group of brain tumors of glial origin. Most are characterized by diffuse infiltrative growth in the surrounding brain. In combination with their refractive nature to chemotherapy this makes it almost impossible to cure patients using combinations of conventional therapeutic strategies. The drastically increased knowledge about the molecular underpinnings of gliomas during the last decade has elicited high expectations for a more rational and effective therapy for these tumors. Most studies on the molecular pathways involved in glioma biology thus far had a strong focus on growth factor receptor protein tyrosine kinase (PTK) and phosphatidylinositol phosphatase signaling pathways. Except for the tumor suppressor PTEN, much less attention has been paid to the PTK counterparts, the protein tyrosine phosphatase (PTP) superfamily, in gliomas. PTPs are instrumental in the reversible phosphorylation of tyrosine residues and have emerged as important regulators of signaling pathways that are linked to various developmental and disease-related processes. Here, we provide an overview of the current knowledge on PTP involvement in gliomagenesis. So far, the data point to the potential implication of receptor-type (RPTPdelta, DEP1, RPTPmicro, RPTPzeta) and intracellular (PTP1B, TCPTP, SHP2, PTPN13) classical PTPs, dual-specific PTPs (MKP-1, VHP, PRL-3, KAP, PTEN) and the CDC25B and CDC25C PTPs in glioma biology. Like PTKs, these PTPs may represent promising targets for the development of novel diagnostic and therapeutic strategies in the treatment of high-grade gliomas.


Assuntos
Neoplasias Encefálicas/enzimologia , Glioma/enzimologia , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais/fisiologia , Animais , Humanos
11.
Nat Cell Biol ; 22(1): 97-107, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31907411

RESUMO

Diffuse brain infiltration by glioma cells causes detrimental disease progression, but its multicellular coordination is poorly understood. We show here that glioma cells infiltrate the brain collectively as multicellular networks. Contacts between moving glioma cells are adaptive epithelial-like or filamentous junctions stabilized by N-cadherin, ß-catenin and p120-catenin, which undergo kinetic turnover, transmit intercellular calcium transients and mediate directional persistence. Downregulation of p120-catenin compromises cell-cell interaction and communication, disrupts collective networks, and both the cadherin and RhoA binding domains of p120-catenin are required for network formation and migration. Deregulating p120-catenin further prevents diffuse glioma cell infiltration of the mouse brain with marginalized microlesions as the outcome. Transcriptomics analysis has identified p120-catenin as an upstream regulator of neurogenesis and cell cycle pathways and a predictor of poor clinical outcome in glioma patients. Collective glioma networks infiltrating the brain thus depend on adherens junctions dynamics, the targeting of which may offer an unanticipated strategy to halt glioma progression.


Assuntos
Junções Aderentes/metabolismo , Cateninas/metabolismo , Adesão Celular/fisiologia , Glioma/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Caderinas/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/fisiologia , Glioma/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , delta Catenina
12.
Cell Rep Med ; 1(7): 100101, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33103128

RESUMO

Tumor-educated platelets (TEPs) are potential biomarkers for cancer diagnostics. We employ TEP-derived RNA panels, determined by swarm intelligence, to detect and monitor glioblastoma. We assessed specificity by comparing the spliced RNA profile of TEPs from glioblastoma patients with multiple sclerosis and brain metastasis patients (validation series, n = 157; accuracy, 80%; AUC, 0.81 [95% CI, 0.74-0.89; p < 0.001]). Second, analysis of patients with glioblastoma versus asymptomatic healthy controls in an independent validation series (n = 347) provided a detection accuracy of 95% and AUC of 0.97 (95% CI, 0.95-0.99; p < 0.001). Finally, we developed the digitalSWARM algorithm to improve monitoring of glioblastoma progression and demonstrate that the TEP tumor scores of individual glioblastoma patients represent tumor behavior and could be used to distinguish false positive progression from true progression (validation series, n = 20; accuracy, 85%; AUC, 0.86 [95% CI, 0.70-1.00; p < 0.012]). In conclusion, TEPs have potential as a minimally invasive biosource for blood-based diagnostics and monitoring of glioblastoma patients.


Assuntos
Plaquetas/metabolismo , Neoplasias Encefálicas/diagnóstico , Glioblastoma/diagnóstico , Monitorização Fisiológica/métodos , Esclerose Múltipla/diagnóstico , RNA Neoplásico/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Plaquetas/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/cirurgia , Estudos de Casos e Controles , Progressão da Doença , Glioblastoma/genética , Glioblastoma/mortalidade , Glioblastoma/cirurgia , Humanos , Pessoa de Meia-Idade , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Metástase Neoplásica , Splicing de RNA , RNA Neoplásico/metabolismo , Curva ROC , Análise de Sobrevida , Microambiente Tumoral/genética
13.
Mol Cancer Res ; 15(11): 1587-1597, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28751462

RESUMO

The receptor tyrosine kinase (RTK) MET represents a promising tumor target in a subset of glioblastomas. Most RTK inhibitors available in the clinic today, including those inhibiting MET, affect multiple targets simultaneously. Previously, it was demonstrated that treatment with cabozantinib (MET/VEGFR2/RET inhibitor) prolonged survival of mice carrying orthotopic patient-derived xenografts (PDX) of the MET-addicted glioblastoma model E98, yet did not prevent development of recurrent and cabozantinib-resistant tumors. To exclude VEGFR2 inhibition-inflicted blood-brain barrier normalization and diminished tumor distribution of the drug, we have now investigated the effects of the novel MET-selective inhibitor Compound A in the orthotopic E98 xenograft model. In vitro, Compound A proved a highly potent inhibitor of proliferation of MET-addicted cell lines. In line with its target selectivity, Compound A did not restore the leaky blood-brain barrier and was more effective than cabozantinib in inhibiting MET phosphorylation in vivo Compound A treatment significantly prolonged survival of mice carrying E98 tumor xenografts, but did not prevent eventual progression. Contrasting in vitro results, the Compound A-treated xenografts displayed high levels of AKT phosphorylation despite the absence of phosphorylated MET. Profiling by RNA sequencing showed that in vivo transcriptomes differed significantly from those in control xenografts.Implications: Collectively, these findings demonstrate the plasticity of paracrine growth factor receptor signaling in vivo and urge for prudency with in vitro drug-testing strategies to validate monotherapies. Mol Cancer Res; 15(11); 1587-97. ©2017 AACR.


Assuntos
Aminopiridinas/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Perfilação da Expressão Gênica/métodos , Glioma/tratamento farmacológico , Pirazóis/administração & dosagem , Análise de Sequência de RNA/métodos , Bibliotecas de Moléculas Pequenas/administração & dosagem , Aminopiridinas/farmacologia , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/genética , Células HT29 , Humanos , Camundongos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Acta Neuropathol Commun ; 4(1): 96, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27586084

RESUMO

The infiltrative behavior of diffuse gliomas severely reduces therapeutic potential of surgical resection and radiotherapy, and urges for the identification of new drug-targets affecting glioma growth and migration. To address the potential role of protein tyrosine phosphatases (PTPs), we performed mRNA expression profiling for 91 of the 109 known human PTP genes on a series of clinical diffuse glioma samples of different grades and compared our findings with in silico knowledge from REMBRANDT and TCGA databases. Overall PTP family expression levels appeared independent of characteristic genetic aberrations associated with lower grade or high grade gliomas. Notably, seven PTP genes (DUSP26, MTMR4, PTEN, PTPRM, PTPRN2, PTPRT and PTPRZ1) were differentially expressed between grade II-III gliomas and (grade IV) glioblastomas. For DUSP26, PTEN, PTPRM and PTPRT, lower expression levels correlated with poor prognosis, and overexpression of DUSP26 or PTPRT in E98 glioblastoma cells reduced tumorigenicity. Our study represents the first in-depth analysis of PTP family expression in diffuse glioma subtypes and warrants further investigations into PTP-dependent signaling events as new entry points for improved therapy.


Assuntos
Neoplasias Encefálicas/enzimologia , Glioma/enzimologia , Proteínas Tirosina Fosfatases/metabolismo , Neoplasias Encefálicas/patologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Progressão da Doença , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/patologia , Células HEK293 , Humanos , Imuno-Histoquímica , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Estimativa de Kaplan-Meier , Gradação de Tumores , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Sci Rep ; 6: 30486, 2016 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-27460417

RESUMO

The majority of low-grade and secondary high-grade gliomas carry heterozygous hotspot mutations in cytosolic isocitrate dehydrogenase 1 (IDH1) or the mitochondrial variant IDH2. These mutations mostly involve Arg132 in IDH1, and Arg172 or Arg140 in IDH2. Whereas IDHs convert isocitrate to alpha-ketoglutarate (α-KG) with simultaneous reduction of NADP(+) to NADPH, these IDH mutants reduce α-KG to D-2-hydroxyglutarate (D-2-HG) while oxidizing NADPH. D-2-HG is a proposed oncometabolite, acting via competitive inhibition of α-KG-dependent enzymes that are involved in metabolism and epigenetic regulation. However, much less is known about the implications of the metabolic stress, imposed by decreased α-KG and NADPH production, for tumor biology. We here present a novel heterozygous IDH1 mutation, IDH1(R314C), which was identified by targeted next generation sequencing of a high grade glioma from which a mouse xenograft model and a cell line were generated. IDH1(R314C) lacks isocitrate-to-α-KG conversion activity due to reduced affinity for NADP(+), and differs from the IDH1(R132) mutants in that it does not produce D-2-HG. Because IDH1(R314C) is defective in producing α-KG and NADPH, without concomitant production of the D-2-HG, it represents a valuable tool to study the effects of IDH1-dysfunction on cellular metabolism in the absence of this oncometabolite.


Assuntos
Astrocitoma/enzimologia , Astrocitoma/genética , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/genética , Isocitrato Desidrogenase/genética , Mutação/genética , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Glutaratos/metabolismo , Células HEK293 , Heterozigoto , Humanos , Isocitratos/metabolismo , Camundongos , NADP/metabolismo , Gradação de Tumores , Multimerização Proteica
16.
Cancer Res ; 74(17): 4898-907, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25005896

RESUMO

Many patients with glioma harbor specific mutations in the isocitrate dehydrogenase gene IDH1 that associate with a relatively better prognosis. IDH1-mutated tumors produce the oncometabolite 2-hydroxyglutarate. Because IDH1 also regulates several pathways leading to lipid synthesis, we hypothesized that IDH1-mutant tumors have an altered phospholipid metabolite profile that would impinge on tumor pathobiology. To investigate this hypothesis, we performed (31)P-MRS imaging in mouse xenograft models of four human gliomas, one of which harbored the IDH1-R132H mutation. (31)P-MR spectra from the IDH1-mutant tumor displayed a pattern distinct from that of the three IDH1 wild-type tumors, characterized by decreased levels of phosphoethanolamine and increased levels of glycerophosphocholine. This spectral profile was confirmed by ex vivo analysis of tumor extracts, and it was also observed in human surgical biopsies of IDH1-mutated tumors by (31)P high-resolution magic angle spinning spectroscopy. The specificity of this profile for the IDH1-R132H mutation was established by in vitro (31)P-NMR of extracts of cells overexpressing IDH1 or IDH1-R132H. Overall, our results provide evidence that the IDH1-R132H mutation alters phospholipid metabolism in gliomas involving phosphoethanolamine and glycerophosphocholine. These new noninvasive biomarkers can assist in the identification of the mutation and in research toward novel treatments that target aberrant metabolism in IDH1-mutant glioma.


Assuntos
Glioma/genética , Glioma/metabolismo , Isocitrato Desidrogenase/genética , Mutação/genética , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Etanolaminas/metabolismo , Feminino , Humanos , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus
17.
Oncotarget ; 5(18): 8690-702, 2014 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-25238264

RESUMO

Gliomas are primary brain tumors for which surgical resection and radiotherapy is difficult because of the diffuse infiltrative growth of the tumor into the brain parenchyma. For development of alternative, drug-based, therapies more insight in the molecular processes that steer this typical growth and morphodynamic behavior of glioma cells is needed. Protein tyrosine phosphatase PTPRZ-B is a transmembrane signaling molecule that is found to be strongly up-regulated in glioma specimens. We assessed the contribution of PTPRZ-B protein domains to tumor cell growth and migration, via lentiviral knock-down and over-expression using clinically relevant glioma xenografts and their derived cell models. PTPRZ-B knock-down resulted in reduced migration and proliferation of glioma cells in vitro and also inhibited tumor growth in vivo. Interestingly, expression of only the PTPRZ-B extracellular segment was sufficient to rescue the in vitro migratory phenotype that resulted from PTPRZ-B knock-down. In contrast, PTPRZ-B knock-down effects on proliferation could be reverted only after re-expression of PTPRZ-B variants that contained its C-terminal PDZ binding domain. Thus, distinct domains of PTPRZ-B are differentially required for migration and proliferation of glioma cells, respectively. PTPRZ-B signaling pathways therefore represent attractive therapeutic entry points to combat these tumors.


Assuntos
Neoplasias Encefálicas/enzimologia , Movimento Celular , Proliferação de Células , Glioma/enzimologia , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioma/genética , Glioma/patologia , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fenótipo , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/genética , Transdução de Sinais , Transfecção
18.
PLoS One ; 8(3): e58262, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23484006

RESUMO

Anti-angiogenic treatment of glioblastoma with Vascular Endothelial Growth Factor (VEGF)- or VEGF Receptor 2 (VEGFR2) inhibitors normalizes tumor vessels, resulting in a profound radiologic response and improved quality of life. This approach however does not halt tumor progression by diffuse infiltration, as this phenotype is less angiogenesis dependent. Combined inhibition of angiogenesis and diffuse infiltrative growth would therefore be a more effective treatment approach in these tumors. The HGF/c-MET axis is important in both angiogenesis and cell migration in several tumor types including glioma. We therefore analyzed the effects of the c-MET- and VEGFR2 tyrosine kinase inhibitor cabozantinib (XL184, Exelixis) on c-MET positive orthotopic E98 glioblastoma xenografts, which routinely present with angiogenesis-dependent areas of tumor growth, as well as diffuse infiltrative growth. In in vitro cultures of E98 cells, cabozantinib effectively inhibited c-MET phosphorylation, concomitant with inhibitory effects on AKT and ERK1/2 phosphorylation, and cell proliferation and migration. VEGFR2 activation in endothelial cells was also effectively inhibited in vitro. Treatment of BALB/c nu/nu mice carrying orthotopic E98 xenografts resulted in a significant increase in overall survival. Cabozantinib effectively inhibited angiogenesis, resulting in increased hypoxia in angiogenesis-dependent tumor areas, and induced vessel normalization. Yet, tumors ultimately escaped cabozantinib therapy by diffuse infiltrative outgrowth via vessel co-option. Of importance, in contrast to the results from in vitro experiments, in vivo blockade of c-MET activation was incomplete, possibly due to multiple factors including restoration of the blood-brain barrier resulting from cabozantinib-induced VEGFR2 inhibition. In conclusion, cabozantinib is a promising therapy for c-MET positive glioma, but improving delivery of the drug to the tumor and/or the surrounding tissue may be needed for full activity.


Assuntos
Anilidas/farmacologia , Glioblastoma/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Piridinas/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Sistemas de Liberação de Medicamentos , Glioblastoma/tratamento farmacológico , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Fisiológica/fisiologia , Fosforilação/efeitos dos fármacos , Transplante Heterólogo , Células Tumorais Cultivadas
19.
Acta Neuropathol Commun ; 1: 18, 2013 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-24252742

RESUMO

BACKGROUND: Point mutations in genes encoding NADP+-dependent isocitrate dehydrogenases (especially IDH1) are common in lower grade diffuse gliomas and secondary glioblastomas and occur early during tumor development. The contribution of these mutations to gliomagenesis is not completely understood and research is hampered by the lack of relevant tumor models. We previously described the development of the patient-derived high-grade oligodendroglioma xenograft model E478 that carries the commonly occurring IDH1-R132H mutation. We here report on the analyses of E478 xenografts at the genetic, histologic and metabolic level. RESULTS: LC-MS and in situ mass spectrometric imaging by LESA-nano ESI-FTICR revealed high levels of the proposed oncometabolite D-2-hydroxyglutarate (D-2HG), the product of enzymatic conversion of α-ketoglutarate (α-KG) by IDH1-R132H, in the tumor but not in surrounding brain parenchyma. α-KG levels and total NADP+-dependent IDH activity were similar in IDH1-mutant and -wildtype xenografts, demonstrating that IDH1-mutated cancer cells maintain α-KG levels. Interestingly, IDH1-mutant tumor cells in vivo present with high densities of mitochondria and increased levels of mitochondrial activity as compared to IDH1-wildtype xenografts. It is not yet clear whether this altered mitochondrial activity is a driver or a consequence of tumorigenesis. CONCLUSIONS: The oligodendroglioma model presented here is a valuable model for further functional elucidation of the effects of IDH1 mutations on tumor metabolism and may aid in the rational development of novel therapeutic strategies for the large subgroup of gliomas carrying IDH1 mutations.


Assuntos
Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Mitocôndrias/fisiologia , Oligodendroglioma/genética , Oligodendroglioma/fisiopatologia , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/fisiopatologia , Células Cultivadas , Feminino , Glutaratos/metabolismo , Humanos , Ácidos Cetoglutáricos/metabolismo , Camundongos Endogâmicos BALB C , Mutação de Sentido Incorreto , Transplante de Neoplasias , Oligodendroglioma/patologia
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