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1.
Hum Mol Genet ; 26(10): 1801-1810, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28334876

RESUMO

Infantile myofibromatosis is one of the most prevalent soft tissue tumors of infancy and childhood. Multifocal nodules with visceral lesions are associated with a poor prognosis. A few familial cases have been linked to mutations in various genes including PDGFRB. In this study, we sequenced PDGFRB, which encodes a receptor tyrosine kinase, in 16 cases of myofibromatosis or solitary myofibroma. Mutations in the coding sequence of PDGFRB were identified in 6 out of 8 patients with the sporadic multicentric form of the disease and in 1 out of 8 patients with isolated myofibroma. Two patients had the same mutation in multiple separated lesions. By contrast, a third patient had three different PDGFRB mutations in the three nodules analyzed. Mutations were located in the transmembrane, juxtamembrane and kinase domains of the receptor. We showed that these mutations activated receptor signaling in the absence of ligand and transformed fibroblasts. In one case, a weakly-activating germline variant was associated with a stronger somatic mutation, suggesting a two-hit model for familial myofibromatosis. Furthermore, the mutant receptors were sensitive to the tyrosine kinase inhibitor imatinib, except D850V, which was inhibited by dasatinib and ponatinib, suggesting a targeted therapy for severe myofibromatosis. In conclusion, we identified gain-of-function PDGFRB mutations in the majority of multifocal infantile myofibromatosis cases, shedding light on the mechanism of disease development, which is reminiscent of multifocal venous malformations induced by TIE2 mutations. Our results provide a genetic test to facilitate diagnosis, and preclinical data for development of molecular therapies.


Assuntos
Mutação , Miofibromatose/congênito , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Miofibromatose/genética , Miofibromatose/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor TIE-2/genética
2.
Mol Oncol ; 8(3): 728-40, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24618081

RESUMO

Activated forms of the platelet derived growth factor receptor alpha (PDGFRα) have been described in various tumors, including FIP1L1-PDGFRα in patients with myeloproliferative diseases associated with hypereosinophilia and the PDGFRα(D842V) mutant in gastrointestinal stromal tumors and inflammatory fibroid polyps. To gain a better insight into the signal transduction mechanisms of PDGFRα oncogenes, we mutated twelve potentially phosphorylated tyrosine residues of FIP1L1-PDGFRα and identified three mutations that affected cell proliferation. In particular, mutation of tyrosine 720 in FIP1L1-PDGFRα or PDGFRα(D842V) inhibited cell growth and blocked ERK signaling in Ba/F3 cells. This mutation also decreased myeloproliferation in transplanted mice and the proliferation of human CD34(+) hematopoietic progenitors transduced with FIP1L1-PDGFRα. We showed that the non-receptor protein tyrosine phosphatase SHP2 bound directly to tyrosine 720 of FIP1L1-PDGFRα. SHP2 knock-down decreased proliferation of Ba/F3 cells transformed with FIP1L1-PDGFRα and PDGFRα(D842V) and affected ERK signaling, but not STAT5 phosphorylation. Remarkably, SHP2 was not essential for cell proliferation and ERK phosphorylation induced by the wild-type PDGF receptor in response to ligand stimulation, suggesting a shift in the function of SHP2 downstream of oncogenic receptors. In conclusion, our results indicate that SHP2 is required for cell transformation and ERK activation by mutant PDGF receptors.


Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Células Cultivadas , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Mutação , Fosforilação , Ligação Proteica , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais
3.
Diabetes ; 62(5): 1612-22, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23382449

RESUMO

We evaluated the role of ATP-sensitive K⁺ (K(ATP)) channels, somatostatin, and Zn²âº in the control of glucagon secretion from mouse islets. Switching from 1 to 7 mmol/L glucose inhibited glucagon release. Diazoxide did not reverse the glucagonostatic effect of glucose. Tolbutamide decreased glucagon secretion at 1 mmol/L glucose (G1) but stimulated it at 7 mmol/L glucose (G7). The reduced glucagon secretion produced by high concentrations of tolbutamide or diazoxide, or disruption of K(ATP) channels (Sur1(-/-) mice) at G1 could be inhibited further by G7. Removal of the somatostatin paracrine influence (Sst(-/-) mice or pretreatement with pertussis toxin) strongly increased glucagon release, did not prevent the glucagonostatic effect of G7, and unmasked a marked glucagonotropic effect of tolbutamide. Glucose inhibited glucagon release in the absence of functional K(ATP) channels and somatostatin signaling. Knockout of the Zn²âº transporter ZnT8 (ZnT8(-/-) mice) did not prevent the glucagonostatic effect of glucose. In conclusion, glucose can inhibit glucagon release independently of Zn²âº, K(ATP) channels, and somatostatin. Closure of K(ATP) channels controls glucagon secretion by two mechanisms, a direct stimulation of α-cells and an indirect inhibition via somatostatin released from δ-cells. The net effect on glucagon release results from a balance between both effects.


Assuntos
Glucagon/metabolismo , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Canais KATP/metabolismo , Células Secretoras de Somatostatina/efeitos dos fármacos , Tolbutamida/farmacologia , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Cruzamentos Genéticos , Diazóxido/farmacologia , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Canais KATP/agonistas , Canais KATP/antagonistas & inibidores , Moduladores de Transporte de Membrana/farmacologia , Camundongos , Camundongos Knockout , Concentração Osmolar , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Receptores de Droga/genética , Receptores de Droga/metabolismo , Somatostatina/genética , Somatostatina/metabolismo , Células Secretoras de Somatostatina/metabolismo , Receptores de Sulfonilureias , Técnicas de Cultura de Tecidos , Transportador 8 de Zinco
4.
Haematologica ; 97(7): 1064-72, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22271894

RESUMO

BACKGROUND: ETV6-PDGFRB (also called TEL-PDGFRB) and FIP1L1-PDGFRA are receptor-tyrosine kinase fusion genes that cause chronic myeloid malignancies associated with hypereosinophilia. The aim of this work was to gain insight into the mechanisms whereby fusion genes affect human hematopoietic cells and in particular the eosinophil lineage. DESIGN AND METHODS: We introduced ETV6-PDGFRB and FIP1L1-PDGFRA into human CD34(+) hematopoietic progenitor and stem cells isolated from umbilical cord blood. RESULTS: Cells transduced with these oncogenes formed hematopoietic colonies even in the absence of cytokines. Both oncogenes also stimulated the proliferation of cells in liquid culture and their differentiation into eosinophils. This model thus recapitulated key features of the myeloid neoplasms induced by ETV6-PDGFRB and FIP1L1-PDGFRA. We next showed that both fusion genes activated the transcription factors STAT1, STAT3, STAT5 and nuclear factor-κB. Phosphatidylinositol-3 kinase inhibition blocked nuclear factor-κB activation in transduced progenitor cells and patients' cells. Nuclear factor-κB was also activated in the human FIP1L1-PDGFRA-positive leukemia cell line EOL1, the proliferation of which was blocked by bortezomib and the IκB kinase inhibitor BMS-345541. A mutant IκB that prevents nuclear translocation of nuclear factor-κB inhibited cell growth and the expression of eosinophil markers, such as the interleukin-5 receptor and eosinophil peroxidase, in progenitors transduced with ETV6-PDGFRB. In addition, several potential regulators of this process, including HES6, MYC and FOXO3 were identified using expression microarrays. CONCLUSIONS: We show that human CD34(+) cells expressing PDGFR fusion oncogenes proliferate autonomously and differentiate towards the eosinophil lineage in a process that requires nuclear factor-κB. These results suggest new treatment possibilities for imatinib-resistant myeloid neoplasms associated with PDGFR mutations.


Assuntos
Eosinófilos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , NF-kappa B/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Antígenos CD34/genética , Antígenos CD34/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Eosinofilia/complicações , Eosinofilia/genética , Eosinofilia/metabolismo , Eosinofilia/patologia , Eosinófilos/citologia , Eosinófilos/efeitos dos fármacos , Sangue Fetal , Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/complicações , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , NF-kappa B/genética , Proteínas de Fusão Oncogênica/genética , Inibidores de Proteínas Quinases/farmacologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução Genética , Transgenes , Fatores de Poliadenilação e Clivagem de mRNA/genética
5.
Haematologica ; 96(10): 1406-14, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21685469

RESUMO

BACKGROUND: KANK1-PDGFRB is a fusion gene generated by the t(5;9) translocation between KANK1 and the platelet-derived growth factor receptor beta gene PDGFRB. This hybrid was identified in a myeloproliferative neoplasm featuring severe thrombocythemia, in the absence of the JAK2 V617F mutation. DESIGN AND METHODS: KANK1-PDGFRB was transduced into Ba/F3 cells and CD34(+) human progenitor cells to gain insights into the mechanisms whereby this fusion gene transforms cells. RESULTS: Although platelet-derived growth factor receptors are capable of activating JAK2, KANK1-PDGFRß did not induce JAK2 phosphorylation in hematopoietic cells and a JAK inhibitor did not affect KANK1-PDGFRß-induced cell growth. Like JAK2 V617F, KANK1-PDGFRß constitutively activated STAT5 transcription factors, but this did not require JAK kinases. In addition KANK1-PDGFRß induced the phosphorylation of phospholipase C-γ, ERK1 and ERK2, like wild-type PDGFRß and TEL-PDGFRß, another hybrid protein found in myeloid malignancies. We next tested various mutant forms of KANK1-PDGFRß in Ba/F3 cells and human CD34(+) hematopoietic progenitors. The three coiled-coil domains located in the N-terminus of KANK1 were required for KANK1-PDGFRß-induced cell growth and signaling via STAT5 and ERK. However, the coiled-coils were not essential for KANK1-PDGFRß oligomerization, which could be mediated by another new oligomerization domain. KANK1-PDGFRß formed homotrimeric complexes and heavier oligomers. CONCLUSIONS: KANK1-PDGFRB is a unique example of a thrombocythemia-associated oncogene that does not signal via JAK2. The fusion protein is activated by multiple oligomerization domains, which are required for signaling and cell growth stimulation.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Janus Quinase 2/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular Transformada , Proliferação de Células , Proteínas do Citoesqueleto , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células-Tronco Hematopoéticas/metabolismo , Humanos , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Proteínas de Fusão Oncogênica/genética , Fosforilação , Multimerização Proteica , Estrutura Terciária de Proteína , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Proteínas Supressoras de Tumor/genética
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