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1.
J Cell Mol Med ; 26(14): 3902-3912, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35689379

RESUMO

Penttinen syndrome is a rare progeroid disorder caused by mutations in platelet-derived growth factor (PDGF) receptor beta (encoded by the PDGFRB proto-oncogene) and characterized by a prematurely aged appearance with lipoatrophy, skin lesions, thin hair and acro-osteolysis. Activating mutations in PDGFRB have been associated with other human diseases, including Kosaki overgrowth syndrome, infantile myofibromatosis, fusiform aneurysms, acute lymphoblastic leukaemia and myeloproliferative neoplasms associated with eosinophilia. The goal of the present study was to characterize the PDGFRB p.Val665Ala variant associated with Penttinen syndrome at the molecular level. This substitution is located in a conserved loop of the receptor tyrosine kinase domain. We observed that the mutant receptor was expressed at a lower level but showed constitutive activity. In the absence of ligand, the mutant activated STAT1 and elicited an interferon-like transcriptional response. Phosphorylation of STAT3, STAT5, AKT and phospholipase Cγ was weak or undetectable. It was devoid of oncogenic activity in two cell proliferation assays, contrasting with classical PDGF receptor oncogenic mutants. STAT1 activation was not sensitive to ruxolitinib and did not rely on interferon-JAK2 signalling. Another tyrosine kinase inhibitor, imatinib, blocked signalling by the p.Val665Ala variant at a higher concentration compared with the wild-type receptor. Importantly, this concentration remained in the therapeutic range. Dasatinib, nilotinib and ponatinib also inhibited the mutant receptor. In conclusion, the p.Val665Ala variant confers unique features to PDGF receptor ß compared with other characterized gain-of-function mutants, which may in part explain the particular set of symptoms associated with Penttinen syndrome.


Assuntos
Acro-Osteólise , Miofibromatose , Receptor beta de Fator de Crescimento Derivado de Plaquetas , Fator de Transcrição STAT1 , Acro-Osteólise/genética , Idoso , Humanos , Interferons/metabolismo , Deformidades Congênitas dos Membros/genética , Miofibromatose/genética , Miofibromatose/metabolismo , Progéria/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Transcrição STAT1/metabolismo
2.
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
3.
Genet Med ; 20(1): 142-150, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28726812

RESUMO

PurposeHeterozygous germ-line activating mutations in PDGFRB cause Kosaki and Penttinen syndromes and myofibromatosis. We describe a 10-year-old child with a germ-line PDGFRB p.N666H mutation who responded to the tyrosine kinase inhibitor imatinib by inhibition of PDGFRB.MethodsThe impact of p.N666H on PDGFRB function and sensitivity to imatinib was studied in cell culture.ResultsCells expressing the p.N666H mutation showed constitutive PDGFRB tyrosine phosphorylation. PDGF-independent proliferation was abolished by imatinib at 1 µM concentration. Patient fibroblasts showed constitutive receptor tyrosine phosphorylation that was also abrogated by imatinib with reduced proliferation of treated cells.This led to patient treatment with imatinib at 400 mg daily (340 mg/m2) for a year with objective improvement of debilitating hand and foot contractures, reduced facial coarseness, and significant improvement in quality of life. New small subcutaneous nodules developed, but remained stable. Transient leukopenia, neutropenia, and fatigue resolved without intervention; however, mildly decreased growth velocity resulted in reducing imatinib dose to 200 mg daily (170 mg/m2). The patient continues treatment with ongoing clinical response.ConclusionTo our knowledge, this is one of the first personalized treatments of a congenital disorder caused by a germ-line PDGF receptor mutation with a PDGFRB inhibitor.


Assuntos
Alelos , Substituição de Aminoácidos , Mutação com Ganho de Função , Mutação em Linhagem Germinativa , Mesilato de Imatinib/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Testes Genéticos , Humanos , Mesilato de Imatinib/farmacologia , Lactente , Imageamento por Ressonância Magnética , Masculino , Megalencefalia/diagnóstico , Megalencefalia/genética , Megalencefalia/cirurgia , Miofibromatose/congênito , Miofibromatose/diagnóstico , Miofibromatose/tratamento farmacológico , Miofibromatose/genética , Farmacogenética , Inibidores de Proteínas Quinases/farmacologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Resultado do Tratamento
4.
J Cell Mol Med ; 19(1): 239-48, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25292412

RESUMO

Platelet-derived growth factors (PDGF) bind to two related receptor tyrosine kinases, which are encoded by the PDGFRA and PDGFRB genes. Recently, heterozygous PDGFRB mutations have been described in patients diagnosed with idiopathic basal ganglia calcification (IBGC or Fahr disease), a rare inherited neurological disorder. The goal of the present study was to determine whether these mutations had a positive or negative impact on the PDGFRB activity. We first showed that the E1071V mutant behaved like wild-type PDGFRB and may represent a polymorphism unrelated to IBGC. In contrast, the L658P mutant had no kinase activity and failed to activate any of the pathways normally stimulated by PDGF. The R987W mutant activated Akt and MAP kinases but did not induce the phosphorylation of signal transducer and activator of transcription 3 (STAT3) after PDGF stimulation. Phosphorylation of phospholipase Cγ was also decreased. Finally, we showed that the R987W mutant was more rapidly degraded upon PDGF binding compared to wild-type PDGFRB. In conclusion, PDGFRB mutations associated with IBGC impair the receptor signalling. PDGFRB loss of function in IBGC is consistent with recently described inactivating mutations in the PDGF-B ligand. These results raise concerns about the long-term safety of PDGF receptor inhibition by drugs such as imatinib.


Assuntos
Doenças dos Gânglios da Base/genética , Calcinose/genética , Mutação/genética , Doenças Neurodegenerativas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais/genética , Substituição de Aminoácidos , Linhagem Celular Tumoral , Células HEK293 , Humanos , Proteínas Mutantes/metabolismo , Fosfolipase C gama/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-sis/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos
5.
JAMA Dermatol ; 155(8): 946-950, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31017643

RESUMO

IMPORTANCE: Myofibroma is the most frequent fibrous tumor in children. Multicentric myofibroma (referred to as infantile myofibromatosis) is a life-threatening disease. OBJECTIVE: To determine the frequency, spectrum, and clinical implications of mutations in the PDGFRB receptor tyrosine kinase found in sporadic myofibroma and myofibromatosis. DESIGN, SETTING, AND PARTICIPANTS: In this retrospective study of 69 patients with sporadic myofibroma or myofibromatosis, 85 tumor samples were obtained and analyzed by targeted deep sequencing of PDGFRB. Mutations were confirmed by an alternative method of sequencing and were experimentally characterized to confirm gain of function and sensitivity to the tyrosine kinase inhibitor imatinib. MAIN OUTCOMES AND MEASURES: Frequency of gain-of-function PDGFRB mutations in sporadic myofibroma and myofibromatosis. Sensitivity to imatinib, as assessed experimentally. RESULTS: Of the 69 patients with tumor samples (mean [SD] age, 7.8 [12.7] years), 60 were children (87%; 29 girls [48%]) and 9 were adults (13%; 4 women [44%]). Gain-of-function PDGFRB mutations were found in samples from 25 children, with no mutation found in samples from adults. Mutations were particularly associated with severe multicentric disease (13 of 19 myofibromatosis cases [68%]). Although patients had no familial history, 3 of 25 mutations (12%) were likely to be germline, suggesting de novo heritable alterations. All of the PDGFRB mutations were associated with ligand-independent receptor activation, and all but one were sensitive to imatinib at clinically relevant concentrations. CONCLUSIONS AND RELEVANCE: Gain-of-function mutations of PDGFRB in myofibromas may affect only children and be more frequent in the multicentric form of disease, albeit present in solitary pediatric myofibromas. These alterations may be sensitive to tyrosine kinase inhibitors. The PDGFRB sequencing appears to have a high value for diagnosis, prognosis, and therapy of soft-tissue tumors in children.

6.
Hum Pathol ; 70: 98-104, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29079174

RESUMO

Ovarian cancer is the fifth most common cancer in women worldwide and has the highest mortality amongst gynecological cancers. miRNAs are a class of non-coding RNAs, approximately 22 nt long, that negatively regulate gene expression and have roles in cell growth, differentiation, metabolism, apoptosis and tumorigenesis. Dysregulated miRNA-223 expression has been implicated in a wide range of cancer subtypes. SMARCD1 is an integral protein component of the SWI/SNF complex, which remodels chromatin, and which has important roles in transcriptional control, DNA replication, recombination and repair. In this study, we examined whether the expression levels of miR-223 and SMARCD1 are altered in ovarian serous neoplasia and whether miR-223 functionally regulates the gene and protein expression of SMARCD1 in vivo, as has been predicted by in silico methods. Benign, atypical proliferative serous tumors (borderline) and malignant serous tumors (n = 144) were laser-capture microdissected, and relative expression levels of miR-223 and SMARCD1 were quantified by RT-PCR. Ovarian cancer cell line OC316 was reverse transfected with a miR-223 mimic, and relative expression levels of miR-223 and SMARCD1 were quantified by reverse-transcription polymerase chain reaction; protein expression of SMARCD1 was evaluated by Western blot. miR-223 expression was up-regulated in high-grade ovarian serous carcinoma samples (median RQ = 4.8881, P = .0045), whilst SMARCD1 was down-regulated (median RQ = 0.5107, P = .0492). In OC316 cells transfected with a miR-223 mimic, SMARCD1 gene expression was down-regulated 3-fold (P = .001), and SMARCD1 protein expression was down-regulated 2-fold (P = .002). These results suggest a regulatory role for miR-223 in ovarian serous neoplasia, linking it with SMARCD1.


Assuntos
Proliferação de Células , MicroRNAs/metabolismo , Neoplasias Císticas, Mucinosas e Serosas/metabolismo , Neoplasias Ovarianas/metabolismo , Fatores de Transcrição/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias Císticas, Mucinosas e Serosas/etiologia , Neoplasias Císticas, Mucinosas e Serosas/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Transdução de Sinais , Fatores de Transcrição/genética
7.
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
8.
Oncotarget ; 8(13): 20523-20524, 2017 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-28423556

Assuntos
Mutação , Transgenes
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