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1.
Gene ; 733: 144369, 2020 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-31972311

RESUMO

MAP2K1 encodes mitogen-activated protein kinase 1 (MEK1). Mutations in MAP2K1 lead to continuous activation of MEK/ERK signaling pathway, giving rise to cardio-facio-cutaneous syndrome (CFCS). However, the molecular mechanisms of abnormal activation of MEK/ERK signaling pathway and the role of autophagy, if any, in manifesting CFCS in MAP2K mutants remain unclear. Here, we report three Chinese children with CFCS having MAP2K1 pathogenic variants, identified by exome sequencing. They presented with dysmorphic facial features, seizures, psychomotor retardation, and short stature. Additionally, the third child showed pulmonary valve stenosis, multiple skeletal deformities, and osteoporosis. Whole exome sequencing revealed two heterozygous missense mutations in exon 3 of MAP2K1 (c.383G>T; p.Gly128Val and c.389A>G; p.Tyr130Cys), as well as a novel heterozygous missense variant (c.170A>T; p.Lys57Met) in exon 2 of MAP2K1. In SH-SY5Y cells, we identified, for the first time, that MAP2K1 mutations can activate the p-ERK-dependent cell cycle progression and autophagy, and cause CFCS. Our results extended the mutational spectrum of MAP2K1, examined the role of MEK1 protein in nerve cell functions, and demonstrated, for the first time, that autophagy may mediate the altered MAP2K1 function, leading to CFCS phenotypes.


Assuntos
Autofagia , Displasia Ectodérmica/patologia , Insuficiência de Crescimento/patologia , Cardiopatias Congênitas/patologia , MAP Quinase Quinase 1/genética , Mutação , Adulto , Apoptose , Ciclo Celular , Movimento Celular , Proliferação de Células , Criança , Displasia Ectodérmica/genética , Displasia Ectodérmica/metabolismo , Facies , Insuficiência de Crescimento/genética , Insuficiência de Crescimento/metabolismo , Feminino , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/metabolismo , Humanos , Lactente , Sistema de Sinalização das MAP Quinases , Masculino , Fenótipo , Fosforilação , Células Tumorais Cultivadas
2.
Anticancer Res ; 39(11): 5867-5877, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31704811

RESUMO

BACKGROUND/AIM: The aim of this study was to examine clonal heterogeneity, to test the utility of liquid biopsy in monitoring disease progression and to evaluate the usefulness of ex vivo drug screening in a BRAF L597Q-mutated colorectal cancer (CRC) patient developing metastases during adjuvant therapy. MATERIALS AND METHODS: Next generation sequencing (NGS) and droplet digital PCR (ddPCR) were performed in samples from tumor tissues and liquid biopsies. Live cancer cells from a metastatic lesion were used in ex vivo drug sensitivity assays. RESULTS: We found evidence of continued dependence of MEK/MAPK pathway activation, but different activating mutations in primary tumor and metastases. Liquid biopsy based BRAF L597Q ddPCR testing was a sensitive personalized biomarker predicting the rise of clinically aggressive metastatic disease. Ex vivo drug sensitivity assays with BRAF L597Q mutated cells showed response to MEK/MAPK targeted therapies. CONCLUSION: The rare BRAF L597Q mutation may be associated with aggressive tumor behavior in CRC. Liquid biopsy can be used to capture clinically relevant tumor features.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/secundário , MAP Quinase Quinase 1/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Capecitabina/administração & dosagem , Evolução Clonal , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , MAP Quinase Quinase 1/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oxaliplatina/administração & dosagem , Prognóstico
3.
Cancer Treat Rev ; 81: 101907, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31715422

RESUMO

The aberrant activation of RAS-derived mitogen-activated protein kinase (MAPK) signaling pathway plays a prominent role in tumorigenesis of an array of malignancies. The reasons are usually the upstream activated mutations including mitogen-activated protein kinase kinase 1/2 (MEK1/2). As oncogenic mutations, MEK1 mutations have been observed in a variety of malignancies including melanoma, histiocytic neoplasms, colorectal cancer and lung cancer. Presently, the use of trametinib, a highly selective MEK1/2 inhibitor, was limited to BRAF mutations, according to the approvals of FDA. Therefore, we consider that this is a question worth studying that whether malignancies with MEK1 mutations are sensitive to the treatment of trametinib. This review discussed the function of MEK1 mutations, retrieved the frequency and distribution of MEK1 mutations in various malignancies, and reviewed the basic experiments and clinical case reports on trametinib in the treatment of cell lines or patients with MEK1 mutations. Most studies have demonstrated that trametinib was effective to cells or tumor patients harboring MEK1 mutations, which suggest that the MEK1 mutations might be potential indications of trametinib therapy. In addition, it was also reported that resistance was observed in the treatment of trametinib, suggesting that different MEK1 mutations may have different response to trametinib, and further studies are necessary to distinguish that which MEK1 mutations are appropriate for the treatment with trametinib and which are not.


Assuntos
Antineoplásicos/uso terapêutico , MAP Quinase Quinase 1/genética , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Piridonas/uso terapêutico , Pirimidinonas/uso terapêutico , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Animais , Antineoplásicos/farmacologia , Grupo com Ancestrais do Continente Asiático/genética , Histiocitose de Células de Langerhans/tratamento farmacológico , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , Camundongos , Células NIH 3T3 , Neoplasias/tratamento farmacológico , Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologia , Piridonas/farmacologia , Pirimidinonas/farmacologia
4.
Nat Genet ; 51(9): 1389-1398, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31477929

RESUMO

Integrating single-cell trajectory analysis with pooled genetic screening could reveal the genetic architecture that guides cellular decisions in development and disease. We applied this paradigm to probe the genetic circuitry that controls epithelial-to-mesenchymal transition (EMT). We used single-cell RNA sequencing to profile epithelial cells undergoing a spontaneous spatially determined EMT in the presence or absence of transforming growth factor-ß. Pseudospatial trajectory analysis identified continuous waves of gene regulation as opposed to discrete 'partial' stages of EMT. KRAS was connected to the exit from the epithelial state and the acquisition of a fully mesenchymal phenotype. A pooled single-cell CRISPR-Cas9 screen identified EMT-associated receptors and transcription factors, including regulators of KRAS, whose loss impeded progress along the EMT. Inhibiting the KRAS effector MEK and its upstream activators EGFR and MET demonstrates that interruption of key signaling events reveals regulatory 'checkpoints' in the EMT continuum that mimic discrete stages, and reconciles opposing views of the program that controls EMT.


Assuntos
Transição Epitelial-Mesenquimal/genética , Redes Reguladoras de Genes , Testes Genéticos , Glândulas Mamárias Humanas/metabolismo , Análise de Célula Única/métodos , Sistemas CRISPR-Cas , Células Cultivadas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Glândulas Mamárias Humanas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/farmacologia , Transcriptoma , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
5.
Arch Pediatr ; 26(5): 301-307, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31281037

RESUMO

Langerhans cell histiocytosis (LCH) is characterized by inflammatory lesions containing abundant CD1a+ CD207+ histiocytes that lead to the destruction of affected tissues. This disease has a remarkable pleiotropic clinical presentation and most commonly affects young children. Although the current mortality rate is very low for childhood LCH patients (<2%), reactivation frequently occurs after a long period of disease control and the rates of permanent complications and sequelae remain high. Advances in genomic sequencing technologies in this past decade have highlighted somatic molecular alterations responsible for the disease in around 80% of childhood LCH cases. More than half of these cases harbored the BRAFV600E mutation, and most other mutations also concerned proteins involved in the MAPKinase pathway. In addition to improving what is known about the LCH pathology, this molecular knowledge provides opportunities to optimize patient management. The BRAFV600E mutation is associated with more severe presentations of the disease, a high reactivation rate, and a high permanent complication rate; this mutation therefore paves the way for future stratified management approaches. These therapies may be based on the patient's molecular status as well as other clinical characteristics of the disease that are independently associated with undesired events. Moreover, as observed in patients with solid tumors, the BRAFV600E allele can be detected in the circulating cell-free DNA of patients with severe BRAFV600E-mutated LCH. Quantification of the plasmatic BRAFV600E load for this group of patients can precisely monitor response to therapy. Finally, targeted therapies, such as BRAF inhibitors, are new therapeutic options especially designed for refractory multisystemic LCH involving risk organs. However, the long-term efficacy, long-term tolerance, optimal protocol scheme, and appropriate modalities of administration for these innovative therapies for children still need to be defined, a huge challenge.


Assuntos
Histiocitose de Células de Langerhans/tratamento farmacológico , Histiocitose de Células de Langerhans/genética , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/uso terapêutico , Criança , Marcadores Genéticos , Histiocitose de Células de Langerhans/diagnóstico , Histiocitose de Células de Langerhans/patologia , Humanos , Imidazóis/uso terapêutico , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , Mutação , Oximas/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Vemurafenib/uso terapêutico
6.
Cells ; 8(7)2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31323891

RESUMO

In up to 30% of non-small cell lung cancer (NSCLC) patients, the oncogenic driver of tumor growth is a constitutively activated epidermal growth factor receptor (EGFR). Although these patients gain great benefit from treatment with EGFR tyrosine kinase inhibitors, the development of resistance is inevitable. To model the emergence of drug resistance, an EGFR-driven, patient-derived xenograft (PDX) NSCLC model was treated continuously with Gefitinib in vivo. Over a period of more than three months, three separate clones developed and were subsequently analyzed: Whole exome sequencing and reverse phase protein arrays (RPPAs) were performed to identify the mechanism of resistance. In total, 13 genes were identified, which were mutated in all three resistant lines. Amongst them the mutations in NOMO2, ARHGEF5 and SMTNL2 were predicted as deleterious. The 53 mutated genes specific for at least two of the resistant lines were mainly involved in cell cycle activities or the Fanconi anemia pathway. On a protein level, total EGFR, total Axl, phospho-NFκB, and phospho-Stat1 were upregulated. Stat1, Stat3, MEK1/2, and NFκB displayed enhanced activation in the resistant clones determined by the phosphorylated vs. total protein ratio. In summary, we developed an NSCLC PDX line modelling possible escape mechanism under EGFR treatment. We identified three genes that have not been described before to be involved in an acquired EGFR resistance. Further functional studies are needed to decipher the underlying pathway regulation.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos , Gefitinibe/farmacologia , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/farmacologia , Animais , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Feminino , Gefitinibe/uso terapêutico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/genética , MAP Quinase Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Mutação , NF-kappa B/genética , NF-kappa B/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Regulação para Cima
7.
Braz J Psychiatry ; 41(6): 485-493, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31116258

RESUMO

OBJECTIVE: Cocaine use disorders (CUDs) represent a major public health problem in many countries. To better understand the interaction between the environmental modulations and phenotype, the aim of the present study was to investigate the DNA methylation pattern of CUD patients, who had concomitant cocaine and crack dependence, and healthy controls. METHODS: We studied DNA methylation profiles in the peripheral blood of 23 CUD patients and 24 healthy control subjects using the Illumina Infinium HumanMethylation450 BeadChip arrays. RESULTS: Comparison between CUD patients and controls revealed 186 differentially methylated positions (DMPs; adjusted p-value [adjP] < 10-5) related to 152 genes, with a subset of CpGs confirmed by pyrosequencing. DNA methylation patterns discriminated CUD patients and control groups. A gene network approach showed that the EHMT1, EHMT2, MAPK1, MAPK3, MAP2K1, and HDAC5 genes, which are involved in transcription and chromatin regulation cellular signaling pathways, were also associated with cocaine dependence. CONCLUSION: The investigation of DNA methylation patterns may contribute to a better understanding of the biological mechanisms involved in CUD.


Assuntos
Transtornos Relacionados ao Uso de Cocaína/sangue , Transtornos Relacionados ao Uso de Cocaína/genética , Cocaína Crack , Metilação de DNA , Estudo de Associação Genômica Ampla/métodos , Adulto , Estudos de Casos e Controles , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Antígenos de Histocompatibilidade/genética , Histona Desacetilases/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Modelos Lineares , MAP Quinase Quinase 1/genética , Masculino , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Estatísticas não Paramétricas , Adulto Jovem
8.
Nat Commun ; 10(1): 1897, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015486

RESUMO

The cellular decision regarding whether to undergo proliferation or death is made at the restriction (R)-point, which is disrupted in nearly all tumors. The identity of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in cell biology. We found that early after mitogenic stimulation, RUNX3 binds to its target loci, where it opens chromatin structure by sequential recruitment of Trithorax group proteins and cell-cycle regulators to drive cells to the R-point. Soon after, RUNX3 closes these loci by recruiting Polycomb repressor complexes, causing the cell to pass through the R-point toward S phase. If the RAS signal is constitutively activated, RUNX3 inhibits cell cycle progression by maintaining R-point-associated genes in an open structure. Our results identify RUNX3 as a pioneer factor for the R-point and reveal the molecular mechanisms by which appropriate chromatin modifiers are selectively recruited to target loci for appropriate R-point decisions.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Cromatina/química , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Animais , Butadienos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Subunidade alfa 3 de Fator de Ligação ao Core/antagonistas & inibidores , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Imidazóis/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Nitrilos/farmacologia , Piperazinas/farmacologia , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo
9.
Oncol Rep ; 41(6): 3565-3574, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31002349

RESUMO

In the western world, there is an increasing trend of occurrence in testicular cancer. Treatment of malignant testicular cancer is primarily combined surgery with various chemical drugs. Propofol has been frequently used as an anesthetic and sedative induction agent, which could modulate different γ­aminobutyric acid receptors in the central nervous system. Studies demonstrated that propofol activates endoplasmic reticulum stress to induce apoptosis in lung cancer. However, it remains elusive whether propofol regulates caspase and/or mitogen­activated protein kinase (MAPK) pathways to induce apoptosis in Leydig tumor cells. In the present study, MA­10 mouse Leydig tumor cells were treated with propofol, and possible signal pathways associated with apoptosis were investigated. Results demonstrated that increasing dosage of propofol (300­600 µM) for 24 h significantly decreased cell viability in MA­10 cells (P<0.05). In flow cytometry analysis, the amount of sub­G1 phase cell numbers in MA­10 cells was significantly increased by propofol (P<0.05). Additionally, Annexin V/propidium iodide double staining further confirmed that propofol could induce MA­10 cell apoptosis. Furthermore, cleaved caspase­8, ­9 and ­3, and/or poly(ADP­ribose) polymerase were significantly activated following treatment of propofol in MA­10 cells. In addition, c­Jun N­terminal kinase, extracellular signal­regulated kinase 1/2, and p38 were significantly activated by propofol in MA­10 cells (P<0.05), indicating that propofol may induce apoptosis through the MAPK pathway. Additionally, propofol diminished the phosphorylation of Akt to activate apoptosis in MA­10 cells. In conclusion, propofol may induce MA­10 cell apoptosis by activating caspase and MAPK pathways, and inhibiting the Akt pathway in MA­10 cells, demonstrating that propofol may be a potential anticancer agent against Leydig cell cancer.


Assuntos
Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Propofol/farmacologia , Neoplasias Testiculares/tratamento farmacológico , Animais , Caspases/genética , Proliferação de Células , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Tumor de Células de Leydig , MAP Quinase Quinase 1/genética , Masculino , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/efeitos dos fármacos , Neoplasias Testiculares/genética , Neoplasias Testiculares/patologia
10.
Ann Hematol ; 98(7): 1617-1626, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30923995

RESUMO

Langerhans cell histiocytosis (LCH) is a clonal histiocytic disorder with recurrent mutations of BRAF and MAP2K1, but data on the impact of genetic features on progression and long-term sequelae are sparse. Cases of pediatric LCH with long-term follow-up from our institution were analyzed for mutations in BRAFV600 and MAP2K1 exons 2 and 3 by immunostaining with mutation-specific VE1 antibody, as well as allele-specific PCR and sequencing, respectively. Clinical and follow-up data were obtained from our files and a questionnaire sent to all former patients. Sixteen of 37 (43%) evaluable cases showed BRAFV600E, one case a BRAFV600D and eleven (30%) a MAP2K1 mutation. Nine cases were unmutated for both genes. All cases with risk organ involvement showed either BRAFV600 or MAP2K1 mutation. Patients with BRAFV600 mutation excluding Hashimoto-Pritzker cases had a significantly higher risk for relapses (p = 0.02). Long-term sequelae were present in 19/46 (41%) patients (median follow-up 12.5 years, range 1.0 to 30.8) with a trend for higher rates in mutated cases (mutated = 9/17, 53% versus non-BRAFV600/MAP2K1 mutated = 2/7, 29%). In addition, 8/9 cases with skin involvement including all Hashimoto-Pritzker cases (n = 3) were positive for BRAFV600E. Infants below 2 years more frequently had BRAFV600 mutations (p = 0.013). Despite favorable prognosis, pediatric LCH shows a high frequency of relapses and long-term medical sequelae.


Assuntos
Histiocitose de Células de Langerhans/genética , MAP Quinase Quinase 1/genética , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Adolescente , Criança , Pré-Escolar , Feminino , Seguimentos , Histiocitose de Células de Langerhans/epidemiologia , Histiocitose de Células de Langerhans/patologia , Histiocitose de Células de Langerhans/terapia , Humanos , Lactente , Masculino , Estudos Retrospectivos , Fatores de Risco , Dermatopatias/epidemiologia , Dermatopatias/genética , Dermatopatias/patologia , Dermatopatias/terapia
11.
Molecules ; 24(5)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30832224

RESUMO

Background: Polyphyllin VII (PP7), a steroidal saponin from Paris polyphylla, has been found to exert strong anticancer activity. Little is known about the anti-inflammatory property of PP7. In this study, the anti-inflammatory activity and its underlying mechanisms of PP7 were evaluated in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and in multiple animal models. Methods: The content of nitric oxide (NO) was determined by spectrophotometry. The levels of prostaglandin E2 (PGE2) and cytokines were measured by enzyme-linked immunosorbent assay (ELISA) assay. The mRNA expression of pro-inflammatory genes was determined by qPCR. The total and phosphorylated protein levels were examined by Western blotting. The in vivo anti-inflammatory activities were evaluated by using mouse and zebrafish models. Results: PP7 reduced the production of NO and PGE2 and the protein and mRNA expressions of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) and enzymes (inducible NO synthase [iNOS], cyclooxygenase-2 [COX-2], and Matrix metalloproteinase-9 [MMP-9]) in LPS-induced RAW264.7 cells by suppressing the NF-κB and MAPKs pathways. Notably, PP7 markedly inhibited xylene-induced ear edema and cotton pellet-induced granuloma formation in mice and suppressed LPS and CuSO4-induced inflammation and toxicity in zebrafish embryos. Conclusion: This study demonstrates that PP7 exerts strong anti-inflammatory activities in multiple in vitro and in vivo models and suggests that PP7 is a potential novel therapeutic agent for inflammatory diseases.


Assuntos
Anti-Inflamatórios/farmacologia , Inflamação/tratamento farmacológico , Óxido Nítrico/genética , Saponinas/farmacologia , Animais , Anti-Inflamatórios/química , Ciclo-Oxigenase 2/genética , Dinoprostona/genética , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/induzido quimicamente , Inflamação/patologia , Interleucina-1beta/genética , Interleucina-6/genética , Lipopolissacarídeos/toxicidade , MAP Quinase Quinase 1/genética , Camundongos , NF-kappa B/genética , Óxido Nítrico Sintase Tipo II/genética , Células RAW 264.7/efeitos dos fármacos , Saponinas/química , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética
12.
Breast Cancer Res Treat ; 175(2): 339-351, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30826934

RESUMO

PURPOSE: Triple-negative breast cancer (TNBC) lacks the receptor targets estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, and thus, it does not respond to receptor-targeted treatments. TNBC has higher recurrence, metastasis, and mortality rates than other subtypes of breast cancer. Mounting data suggest that the MAPK (also known as RAS-RAF-MEK-ERK) pathway is an important therapeutic target in TNBC. METHODS: To evaluate anti-tumor and anti-metastasis efficacy of E6201, we used cell proliferation assay, soft agar assay, cell cycle assay, Annexin V staining assay, immunoblotting analysis, immunohistochemistry, migration assay, invasion assay, mammary fat pad xenograft, and experimental and spontaneous metastasis xenograft models. We also evaluated the anti-tumor efficacy of E6201 plus CDK4/6 inhibitor, mTOR inhibitor, or ATR inhibitor. RESULTS: E6201 inhibited TNBC cell colony formation, migration, and invasion in a dose-dependent manner. E6201 induced G1 cell cycle arrest and apoptosis. E6201 inhibited TNBC xenograft growth and inhibited TNBC lung metastasis and improved mouse survival in experimental metastasis and spontaneous metastasis assays. Immunohistochemical staining demonstrated that E6201 decreased the metastatic burden in the lung and decreased phosphorylated ERK expression in a dose-dependent manner. Combination of E6201 with CDK4/6 inhibitor or mTOR inhibitor enhanced E6201's in vitro anti-tumor efficacy. CONCLUSION: These results indicate that E6201 exhibits anti-tumor efficacy against TNBC in vitro and anti-metastasis efficacy against TNBC in vivo. These results provide a rationale for further clinical development of E6201 as a MAPK-pathway-targeted therapy for TNBC.


Assuntos
Proliferação de Células/efeitos dos fármacos , Lactonas/farmacologia , MAP Quinase Quinase 1/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Feminino , Xenoenxertos , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , Camundongos , Metástase Neoplásica , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
13.
Molecules ; 24(6)2019 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-30909606

RESUMO

We describe herein the preparation of certain 2-substituted 3-arylquinoline derivatives and the evaluation of their anti-inflammatory effects in LPS-activated murine J774A.1 macrophage cells. Among these newly synthesized 2-substituted 3-arylquinoline derivatives, 2-(4-methoxy- benzoyl)-3-(3,4,5-trimethoxyphenyl)quinoline (18a) and 2-(4-fluorobenzoyl)-3-(3,4,5-trimethoxy- phenyl)quinoline (18b) are two of the most active compounds which can inhibit the production of NO at non-cytotoxic concentrations. Our results have also indicated that compounds 18a and 18b significantly decrease the secretion of pro-inflammatory cytokines (TNF-á and IL-6), inhibit the expression of iNOS, suppress the phosphorylation of MAPKs, and attenuate the activity of NF-êB by LPS-activated macrophages. Through molecular docking analysis, we found that 18b could fit into the middle of the TNF-á dimer and form hydrophobic interactions with Leu55, Leu57 chain A and B, Tyr59, Val123 chain B and D, Ile 155. These results suggest that both 18a and 18b are potential lead compounds in inhibiting LPS-induced inflammatory responses. Further structural optimization to discover novel anti-inflammatory agents is ongoing.


Assuntos
Anti-Inflamatórios/química , Inflamação/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Quinolinas/química , Aminoácidos/química , Animais , Anti-Inflamatórios/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Inflamação/induzido quimicamente , Inflamação/patologia , Lipopolissacarídeos/toxicidade , MAP Quinase Quinase 1/química , MAP Quinase Quinase 1/genética , Macrófagos/patologia , Camundongos , Simulação de Acoplamento Molecular , Óxido Nítrico/metabolismo , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genética
14.
Lipids Health Dis ; 18(1): 71, 2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30909920

RESUMO

BACKGROUND: Obesity increases the risk of developing diabetes mellitus. Clinical studies suggest that risk factors like palmitic acid (PA) and lipopolysaccharide (LPS) exist simultaneously in diabetes with obesity. Combination of PA and LPS even at low concentration can induce strong inflammatory reaction. Monocyte chemoattractant protein-1 (MCP-1) is an important inflammatory chemokine related to insulin resistance and type II diabetes. Our previous study using PCR array revealed that LPS and PA synergistically induce MCP-1 mRNA expression in macrophage cells RAW264.7, while the protein expression of MCP-1 in this case was not investigated. Moreover, the underling mechanism in the synergistic effect of MCP-1 expression or production induced by treatment of LPS and PA combination remains unclear. METHODS: Protein secretion of MCP-1 was measured by the enzyme-linked immunosorbent assay (ELISA) and mRNA levels of MCP-1 and Toll-like receptor 4 (TLR4) were measured by real-time PCR. Statistical analysis was conducted using SPSS software. RESULTS: LPS could increase MCP-1 transcription as well as secretion in RAW264.7, and PA amplified this effect obviously. Meanwhile, combination of LPS with PA increased TLR4 mRNA expression while LPS alone or PA alone could not, TLR4 knockdown inhibited MCP-1 transcription/secretion induced by LPS plus PA. Moreover, not NF-κB inhibitor but inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways, including c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPK were found to block MCP-1 generation stimulated by LPS plus PA. CONCLUSION: LPS and PA synergistically induced MCP-1 secretion in RAW264.7 macrophage cells, in which MCP-1 transcription mediated by MAPK/TLR4 signaling pathways was involved. Combined treatment of PA and LPS in RAW264.7 cells mimics the situation of diabetes with obesity that has higher level of PA and LPS, MAPK/TLR4/ MCP-1 might be potential therapeutic targets for diabetes with obesity.


Assuntos
Quimiocina CCL2/genética , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase 1/genética , Ácido Palmítico/farmacologia , Receptor 4 Toll-Like/genética , Animais , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/genética , Diabetes Mellitus/patologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Obesidade/tratamento farmacológico , Obesidade/genética , Obesidade/patologia , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo
15.
Food Chem Toxicol ; 126: 303-312, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30840849

RESUMO

With the introduction of arsenic trioxide and all-trans retinoic acid, the prognosis of acute promyelocytic leukemia has greatly improved. However, all-trans retinoic acid resistance is still unresolved in acute promyelocytic leukemia relapsed patients. In this study, the clinical achievable concentration of 7-hydroxystaurosporine synergized with all-trans retinoic acid to induce terminal differentiation in all-trans retinoic acid resistant acute promyelocytic leukemia cell lines. Though 7-hydroxystaurosporine is a PKC inhibitor, PKC might not be involved in the combination-induced differentiation since other PKC selective inhibitors, Gö 6976 and rottlerin failed to cooperate with all-trans retinoic acid to trigger differentiation. The combination significantly enhanced the protein level of CCAAT/enhancer binding protein ß and/or PU.1 as well as activated MEK/ERK. U0126 (MEK specific inhibitor) not only suppressed the combination-induced differentiation but also restored the protein level of CCAAT/enhancer binding protein ß and/or PU.1. However, RAF-1 inhibitor had no inhibitory effect on MEK activation and the combination-induced differentiation. Therefore, the combination overcame differentiation block via RAF-1 independent MEK/ERK modulation of the protein level of CCAAT/enhancer binding protein ß and/or PU.1. These findings may provide a preclinical rationale for the potential role of this combination in the treatment of all-trans retinoic acid resistant acute promyelocytic leukemia patients.


Assuntos
Antineoplásicos/farmacologia , Leucemia Promielocítica Aguda/enzimologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Estaurosporina/análogos & derivados , Tretinoína/administração & dosagem , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática/efeitos dos fármacos , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/genética , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Proto-Oncogênicas c-raf/genética , Estaurosporina/farmacologia
16.
Biochem Biophys Res Commun ; 512(1): 125-130, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30876692

RESUMO

Oxidative stress injury is one of the main mechanisms of ischemia-reperfusion (I/R) injury. The extracellular signal-regulated kinase (ERK1/2) pathway plays an important role in cardioprotective during acute myocardial infarction. In this study, we used constitutively active MEK1 gene (CaMEK) transfection strategy to investigate whether CaMEK provides a protective effect against apoptosis and autophagy induced by Hydrogen peroxide (H2O2) in neonatal rat cardiac ventricular cardiomyocytes (NCMs) and the underlying mechanisms. As a result, CaMEK attenuated H2O2-induced apoptosis and cytotoxicity in NCMs, evidenced by decreased apoptotic cells and the ratio of Bax/Bcl-2, increased the mitochondrial membrane potential (Δψm) and cell vitality and reduced the level of lactate dehydrogenase (LDH). Further studies revealed that CaMEK attenuated H2O2-induced autophagy, evidenced by the decreased LC3-Ⅱ/LC3-Ⅰratio and SQSTM1/p62 (p62) degradation. Furthermore, we demonstrated that CaMEK phosphorylated the ERK1/2 pathway-related proteins, ERK1/2, p70S6K and GSK3ß, in NCMs with H2O2 stimulation. In contrast, these effects could be reversed by co-treatment with the ERK1/2 inhibitor, PD98059. These results suggest that CaMEK plays an important role in protecting cardiomyocytes against H2O2-induced injury and autophagy in NCMs via ERK1/2 pathway. Therefore, transfection of CaMEK may provide a hopeful therapeutic strategy for I/R.


Assuntos
MAP Quinase Quinase 1/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cardiotônicos/metabolismo , Células Cultivadas , Feminino , Peróxido de Hidrogênio/toxicidade , MAP Quinase Quinase 1/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção
17.
Oncogene ; 38(25): 5076-5090, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30905967

RESUMO

Genomic alterations in cancer cells result in vulnerabilities that clinicians can exploit using molecularly targeted drugs, guided by knowledge of the tumour genotype. However, the selective activity of these drugs exerts an evolutionary pressure on cancers that can result in the outgrowth of resistant clones. Use of rational drug combinations can overcome resistance to targeted drugs, but resistance may eventually develop to combinatorial therapies. We selected MAPK- and PI3K-pathway inhibition in colorectal cancer as a model system to dissect out mechanisms of resistance. We focused on these signalling pathways because they are frequently activated in colorectal tumours, have well-characterised mutations and are clinically relevant. By treating a panel of 47 human colorectal cancer cell lines with a combination of MEK- and PI3K-inhibitors, we observe a synergistic inhibition of growth in almost all cell lines. Cells with KRAS mutations are less sensitive to PI3K inhibition, but are particularly sensitive to the combined treatment. Colorectal cancer cell lines with inherent or acquired resistance to monotherapy do not show a synergistic response to the combination treatment. Cells that acquire resistance to an MEK-PI3K inhibitor combination treatment still respond to an ERK-PI3K inhibitor regimen, but subsequently also acquire resistance to this combination treatment. Importantly, the mechanisms of resistance to MEK and PI3K inhibitors observed, MEK1/2 mutation or loss of PTEN, are similar to those detected in the clinic. ERK inhibitors may have clinical utility in overcoming resistance to MEK inhibitor regimes; however, we find a recurrent active site mutation of ERK2 that drives resistance to ERK inhibitors in mono- or combined regimens, suggesting that resistance will remain a hurdle. Importantly, we find that the addition of low concentrations of the BCL2-family inhibitor navitoclax to the MEK-PI3K inhibitor regimen improves the synergistic interaction and blocks the acquisition of resistance.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Terapia de Alvo Molecular , Compostos de Anilina/administração & dosagem , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Células HCT116 , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/genética , MAP Quinase Quinase 2/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Terapia de Alvo Molecular/métodos , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Transdução de Sinais/genética , Sulfonamidas/administração & dosagem , Células Tumorais Cultivadas
18.
Biochim Biophys Acta Proteins Proteom ; 1867(1): 62-70, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29753091

RESUMO

The kinase MEK1 is an essential component of the mitogen-activated protein kinase cascades. Somatic mutations that have been identified in the MEK1-coding gene generally enhance kinase activity. Consequently, MEK1 has attracted much interest as a target for cancer therapy to block the aberrant activity. By using Phos-tag affinity electrophoresis, we found that the introduction of mutations detected in certain sporadic cancers or in MEK-inhibitor-resistant cancer cells produced constitutively active MEK1 species containing phosphorylated Ser-218 and Ser-222 residues; it also enhanced the constitutive activity of the kinase. Phosphorylation profiling of the mutants in the presence of inhibitors of RAF/MEK demonstrated that several mutations conferred resistance to multiple inhibitors as a result of an increase in the quantity of active MEK1 species containing the two phosphorylated Ser-218 and Ser-222 residues. Phos-tag-based phosphorylation profiling of MEK1 can therefore provide clinical insights into characteristics of individual mutations in the MEK1-coding gene.


Assuntos
MAP Quinase Quinase 1/genética , Neoplasias/genética , Linhagem Celular , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Humanos , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases , Mutagênese Sítio-Dirigida , Mutação , Neoplasias/enzimologia , Fosfoproteínas/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia
19.
J Cell Physiol ; 234(3): 2822-2836, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30066962

RESUMO

Resveratrol, a natural polyphenol compound, has been shown to possess anticancer activity. However, how resveratrol inhibits cancer cell adhesion has not been fully elucidated. Here, we show that resveratrol suppressed the basal or type I insulin-like growth factor (IGF)-1-stimulated adhesion of cancer cells (Rh1, Rh30, HT29, and HeLa cells) by inhibiting the extracellular signal-regulated kinase 1/2 (Erk1/2) pathway. Inhibition of Erk1/2 with U0126, knockdown of Erk1/2, or overexpression of dominant-negative mitogen-activated protein kinase kinase 1 (MKK1) strengthened resveratrol's inhibition of the basal or IGF-1-stimulated of Erk1/2 phosphorylation and cell adhesion, whereas ectopic expression of constitutively active MKK1 attenuated the inhibitory effects of resveratrol. Further research revealed that both protein phosphatase 2A (PP2A) and phosphatase and tensin homolog (PTEN)-Akt were implicated in resveratrol-inactivated Erk1/2-dependent cell adhesion. Inhibition of PP2A with okadaic acid or overexpression of dominant-negative PP2A rendered resistance to resveratrol's suppression of the basal or IGF-1-stimulated phospho-Erk1/2 and cell adhesion, whereas expression of wild-type PP2A enhanced resveratrol's inhibitory effects. Overexpression of wild-type PTEN or dominant-negative Akt or inhibition of Akt with Akt inhibitor X strengthened resveratrol's inhibition of the basal or IGF-1-stimulated Erk1/2 phosphorylation and cell adhesion. Furthermore, inhibition of mechanistic/mammalian target of rapamycin (mTOR) with rapamycin or silencing mTOR enhanced resveratrol's inhibitory effects on the basal and IGF-1-induced inhibition of PP2A-PTEN, activation of Akt-Erk1/2, and cell adhesion. The results indicate that resveratrol inhibits Erk1/2-mediated adhesion of cancer cells via activating PP2A-PTEN signaling network. Our data highlight that resveratrol has a great potential in the prevention of cancer cell adhesion.


Assuntos
Adesão Celular/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/genética , Neoplasias/tratamento farmacológico , Resveratrol/farmacologia , Butadienos/farmacologia , Adesão Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HT29 , Células HeLa , Humanos , MAP Quinase Quinase 1/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/genética , Neoplasias/patologia , Nitrilos/farmacologia , Ácido Okadáico/farmacologia , Proteína Oncogênica v-akt/genética , PTEN Fosfo-Hidrolase/genética , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/genética , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética
20.
Br J Dermatol ; 180(1): 172-180, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30141192

RESUMO

BACKGROUND: Data on dermatological manifestations of cardiofaciocutaneous syndrome (CFCS) remain heterogeneous and almost without expert dermatological classification. OBJECTIVES: To describe the dermatological manifestations of CFCS; to compare them with the literature findings; to assess those discriminating CFCS from other RASopathies, including Noonan syndrome (NS) and Costello syndrome (CS); and to test for dermatological phenotype-genotype correlations. METHODS: We performed a 4-year, large, prospective, multicentric, collaborative dermatological and genetic study. RESULTS: Forty-five patients were enrolled. Hair abnormalities were ubiquitous, including scarcity or absence of eyebrows and wavy or curly hair in 73% and 69% of patients, respectively. Keratosis pilaris (KP), ulerythema ophryogenes (UO), palmoplantar hyperkeratosis (PPHK) and multiple melanocytic naevi (MMN; over 50 naevi) were noted in 82%, 44%, 27% and 29% of patients, respectively. Scarcity or absence of eyebrows, association of UO and PPHK, diffuse KP and MMN best differentiated CFCS from NS and CS. Oral acitretin may be highly beneficial for therapeutic management of PPHK, whereas treatment of UO by topical sirolimus 1% failed. No significant dermatological phenotype-genotype correlation was determined. CONCLUSIONS: A thorough knowledge of CFCS skin manifestations would help in making a positive diagnosis and differentiating CFCS from CS and NS.


Assuntos
Displasia Ectodérmica/diagnóstico , Insuficiência de Crescimento/diagnóstico , Cardiopatias Congênitas/diagnóstico , Acitretina/administração & dosagem , Administração Cutânea , Administração Oral , Adolescente , Criança , Pré-Escolar , Síndrome de Costello/diagnóstico , Diagnóstico Diferencial , Displasia Ectodérmica/tratamento farmacológico , Displasia Ectodérmica/genética , Facies , Insuficiência de Crescimento/tratamento farmacológico , Insuficiência de Crescimento/genética , Feminino , França , Estudos de Associação Genética , Cardiopatias Congênitas/tratamento farmacológico , Cardiopatias Congênitas/genética , Humanos , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 2/genética , Masculino , Mutação , Síndrome de Noonan/diagnóstico , Estudos Prospectivos , Proteínas Proto-Oncogênicas B-raf/genética , Sirolimo/administração & dosagem , Resultado do Tratamento , Adulto Jovem
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