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1.
Am J Physiol Gastrointest Liver Physiol ; 320(3): G366-G379, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33470189

RESUMO

Activation of mitogen-activated protein kinases (MAPKs) is a key factor in the pathogenesis of cancer, although the specific role of mitogen-activated protein kinase kinase (MEK1) is not well understood. Villin promoter-driven Cre expression was used to excise a floxed stop cassette from a phosphomimetically constitutively activated MEK1 (caMEK1) expression construct in the intestine of C57BL/6 mice. Zygosity status of caMEK1 afforded assessment of the dose dependence of the effect. The expected mendelian distribution of genotypes and sex was observed in 443 progenies. Between 21 and 63 days of life, caMEK1 had no effect on body weight in male mice, but reduced body weight in female mice homozygous for caMEK1. At 10 wk of age, the ileum of caMEK1-expressing mice was characterized by the finding of dysplasia and profound changes in overall architecture. Paneth cells were nearly absent in caMEK1 homozygotes. Targeted proteomic profiling via reverse phase protein array analyses with confirmatory Western blotting revealed significant changes in protein and phosphoprotein expression, including upregulation of proteins downstream of MEK1, associated with enhanced markers of proliferation, diminished apoptosis, alterations in cell-fate determination, cell-cell interactions, and tight junctions. Long-term viability of caMEK1 homozygous mice was reduced with no survival beyond 1 yr. Invasive adenocarcinoma developed in three of ten older mice [15 wk (homozygous), 26 wk (homozygous), and 35 wk (heterozygous) of age]. Expression of caMEK1 in enterocytes leads to marked derangements in the intestinal epithelium, which is associated with a predisposition to the development of invasive cancer.NEW & NOTEWORTHY The ileum of mice with constitutive expression of activated MEK1 (via phosphomimetic changes) in enterocytes is markedly abnormal with architectural distortion and cytologic atypia, which evolves into an adenoma invasive carcinoma sequence. Phosphoproteomic analysis reveals upregulation of proteins downstream of MEK1, associated with enhanced markers of proliferation, diminished apoptosis, alterations in cell-fate determination, cell-cell interactions, and tight junctions. This novel model provides new insights into intestinal homeostasis and carcinogenesis.


Assuntos
Enterócitos/metabolismo , Íleo/citologia , Neoplasias Intestinais/metabolismo , MAP Quinase Quinase 1/metabolismo , Animais , Diferenciação Celular/fisiologia , Feminino , Deleção de Genes , Predisposição Genética para Doença , Neoplasias Intestinais/genética , Longevidade , MAP Quinase Quinase 1/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos
2.
Mol Carcinog ; 59(10): 1147-1158, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32805066

RESUMO

Acquired resistance is a barrier to cetuximab efficacy in patients with head and neck squamous cell carcinoma (HNSCC). Secreted phosphoprotein 1 (SPP1) is involved in various biological processes, including immune responses, cancer progression, and prognosis in many cancers, while little is known in HNSCC. Bioinformatics methods were used to identify candidate genes and further in vivo and in vitro experiments were performed to examine and validate the function of SPP1. We found that SPP1 was upregulated and has been found to have an oncogenic role in HNSCC. We further confirmed that overexpression of SPP1 affected proliferation, migration, invasion, and survival, and inhibited apoptosis, whereas silencing of SPP1 yielded opposite results to those of SPP1 overexpression. In addition, activation of the KRAS/MEK pathway contributed to the SPP1-induced malignant progression of HNSCC and resistance to cetuximab. Furthermore, SPP1 knockdown or an MEK inhibitor overcame this cetuximab-resistance pattern. Taken together, our findings for the first time identify the role of SPP1 in tumor promotion, prognostic prediction, and potential therapeutic targeting, as well as resistance to cetuximab in HNSCC.


Assuntos
Biomarcadores Tumorais/metabolismo , Cetuximab/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias de Cabeça e Pescoço/patologia , MAP Quinase Quinase 1/metabolismo , Osteopontina/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Antineoplásicos Imunológicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , MAP Quinase Quinase 1/genética , Camundongos , Camundongos Nus , Invasividade Neoplásica , Osteopontina/genética , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Int J Hematol ; 112(4): 560-567, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32654047

RESUMO

In Langerhans cell histiocytosis (LCH), somatic gene mutations in the mitogen-activated protein kinase pathway have been identified in more than 80% of cases in Western countries, in which mutually exclusive BRAF and MAP2K1 mutations are involved. Among them, BRAF V600E mutation is the major contributor (50-60%). In 59 patients (50 children and nine adults) with LCH (not including pulmonary LCH) in Japan, we first screened for BRAF V600E in all patients followed by target sequencing for other gene mutations in 17 of BRAF V600E-negative patients. As a result, BRAF V600E mutation was detected in 27/59 (46%) patients. We also identified BRAF mutations other than V600E in five and MAP2K1 mutations in nine patients. Thus, gene mutations in BRAF or MAP2K1 were identified in 41/44 (93%) of the fully tested patients. Regarding the correlation of clinical features and genotype in pediatric patients, we found that BRAF V600E mutation status was not correlated with sex, age at diagnosis, disease extent, response to first-line therapy, relapse, or CNS-related sequelae. Interestingly, MAP2K1 exon 2 in-frame deletion was related to the risk organ involvement; however, further studies are required to clarify the impact of these gene mutations on the clinical features of patients with LCH.


Assuntos
Estudos de Associação Genética , Histiocitose de Células de Langerhans/genética , MAP Quinase Quinase 1/genética , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Japão , Sistema de Sinalização das MAP Quinases/genética , Masculino
4.
Virchows Arch ; 477(5): 749-753, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32372223

RESUMO

Langerhans cell histiocytosis (LCH) is characterized by mutations of the RAS-RAF-MAPK signaling pathway. We analyzed MAP2K1, NRAS and KIT mutation incidence in skin lesions of BRAF wild-type (wt) LCH patients. We evaluated the occurrence of MAP2K1, NRAS and KIT mutations in seven LCH and one indeterminate cell histiocytosis (ICH) patients. MAP2K1 mutation frequency was found to be 3/7 (42.9%) in LCH and also found in ICH. Similarly, the KIT mutation frequency was found to be equally prevalent (4/7, 57.1%) in LCH and also occurred in ICH. Involvement of KIT exons in LCH-ICH indicated that exon 9/11/18 were equally prevalent followed by exon 13. This exploratory analysis on BRAF-wt LCH revealed a KIT mutation rate comparable to MAP2K1. Although the detected KIT mutations are different from activating mutations found in other KIT-dependent neoplasms, our data suggest that KIT-inhibitors might have a role in treating BRAF-wt LCH patients.


Assuntos
Histiocitose de Células de Langerhans/genética , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-kit/genética , Dermatopatias/genética , Adolescente , Adulto , Idoso , Feminino , GTP Fosfo-Hidrolases/genética , Predisposição Genética para Doença , Histiocitose de Células de Langerhans/patologia , Histiocitose de Células de Langerhans/terapia , Humanos , Lactente , MAP Quinase Quinase 1/genética , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Taxa de Mutação , Fenótipo , Prognóstico , Dermatopatias/patologia , Dermatopatias/terapia , Adulto Jovem
5.
Cancer Cell ; 37(6): 834-849.e13, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32442403

RESUMO

Molecular mechanisms underlying adaptive targeted therapy resistance in pancreatic ductal adenocarcinoma (PDAC) are poorly understood. Here, we identify SETD5 as a major driver of PDAC resistance to MEK1/2 inhibition (MEKi). SETD5 is induced by MEKi resistance and its deletion restores refractory PDAC vulnerability to MEKi therapy in mouse models and patient-derived xenografts. SETD5 lacks histone methyltransferase activity but scaffolds a co-repressor complex, including HDAC3 and G9a. Gene silencing by the SETD5 complex regulates known drug resistance pathways to reprogram cellular responses to MEKi. Pharmacological co-targeting of MEK1/2, HDAC3, and G9a sustains PDAC tumor growth inhibition in vivo. Our work uncovers SETD5 as a key mediator of acquired MEKi therapy resistance in PDAC and suggests a context for advancing MEKi use in the clinic.


Assuntos
Cromatina/genética , Resistencia a Medicamentos Antineoplásicos , Metiltransferases/metabolismo , Terapia de Alvo Molecular , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Apoptose , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Proliferação de Células , Feminino , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Histona Desacetilases/química , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , 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 , Metiltransferases/antagonistas & inibidores , Metiltransferases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Piridonas/farmacologia , Pirimidinonas/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(5): 567-569, 2020 May 10.
Artigo em Chinês | MEDLINE | ID: mdl-32335888

RESUMO

OBJECTIVE: To explore the genotype-phenotype correlation of Cardio-facio-cutaneous syndrome (CFCS) caused by MAP2K1 gene variants. METHODS: Genomic DNA was extracted from peripheral blood sample from a child patient and his parents. Whole exome sequencing (WES) was carried out for the patient. Suspected variant was verified by Sanger sequencing. RESULTS: The patient was a 1-year-8-month old Chinese male who manifested short stature, psychomotor retardation, relative macrocephaly, distinctive facial features, and congenital heart disease. WES test revealed a heterozygous missense c.389A>G (p.Tyr130Cys) variant in the MAP2K1 gene. Sanger sequencing has confirmed the variant as de novo. According to ACMG/AMP guidelines, the variant was classified as pathogenic. CONCLUSION: Compared with previously reported CFCS cases due to MAP2K1 variants. The patient showed obvious behavioral problems, good appetite and tricuspid regurgitation, which may to be novel features for CFCS.


Assuntos
Displasia Ectodérmica , Facies , Insuficiência de Crescimento , Variação Genética , Cardiopatias Congênitas , MAP Quinase Quinase 1 , China , Displasia Ectodérmica/genética , Insuficiência de Crescimento/genética , Estudos de Associação Genética , Cardiopatias Congênitas/genética , Heterozigoto , Humanos , Lactente , MAP Quinase Quinase 1/genética , Masculino , Mutação , Sequenciamento Completo do Exoma
7.
Nat Cell Biol ; 22(4): 389-400, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32231305

RESUMO

In mouse embryonic stem cells (mESCs), chemical blockade of Gsk3α/ß and Mek1/2 (2i) instructs a self-renewing ground state whose endogenous inducers are unknown. Here we show that the axon guidance cue Netrin-1 promotes naive pluripotency by triggering profound signalling, transcriptomic and epigenetic changes in mESCs. Furthermore, we demonstrate that Netrin-1 can substitute for blockade of Gsk3α/ß and Mek1/2 to sustain self-renewal of mESCs in combination with leukaemia inhibitory factor and regulates the formation of the mouse pluripotent blastocyst. Mechanistically, we reveal how Netrin-1 and the balance of its receptors Neo1 and Unc5B co-regulate Wnt and MAPK pathways in both mouse and human ESCs. Netrin-1 induces Fak kinase to inactivate Gsk3α/ß and stabilize ß-catenin while increasing the phosphatase activity of a Ppp2r2c-containing Pp2a complex to reduce Erk1/2 activity. Collectively, this work identifies Netrin-1 as a regulator of pluripotency and reveals that it mediates different effects in mESCs depending on its receptor dosage, opening perspectives for balancing self-renewal and lineage commitment.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Receptores de Netrina/genética , Netrina-1/genética , Receptores de Superfície Celular/genética , Via de Sinalização Wnt/genética , Animais , Linhagem Celular , Embrião de Mamíferos , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , 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 , Masculino , Camundongos , Camundongos Knockout , Camundongos SCID , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Netrina/metabolismo , Netrina-1/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Receptores de Superfície Celular/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
8.
Sci Rep ; 10(1): 4428, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32157142

RESUMO

Extracranial arteriovenous malformation (AVM) is most commonly caused by MAP2K1 mutations in the endothelial cell. The purpose of this study was to determine if local tissue overgrowth associated with AVM is caused by direct or indirect effects of the MAP2K1 mutation (i.e., cell-autonomous or cell-non autonomous). Because cartilage does not have blood vessels, we studied ear AVMs to determine if overgrown cartilage contained AVM-causing mutations. Cartilage was separated from its surrounding tissue and isolated by laser capture microdissection. Droplet digital PCR (ddPCR) was used to identify MAP2K1 mutations. MAP2K1 (p.K57N) variants were present in the tissue adjacent to the cartilage [mutant allele frequency (MAF) 6-8%], and were enriched in endothelial cells (MAF 51%) compared to non-endothelial cells (MAF 0%). MAP2K1 mutations were not identified in the overgrown cartilage, and thus local cartilage overgrowth likely results from the effects of adjacent mutant blood vessels (i.e., cell-non autonomous).


Assuntos
Malformações Arteriovenosas/complicações , Doenças das Cartilagens/etiologia , Doenças das Cartilagens/patologia , Células Endoteliais/patologia , MAP Quinase Quinase 1/genética , Mutação , Adolescente , Adulto , Criança , Células Endoteliais/metabolismo , Feminino , Humanos , Masculino , Prognóstico , Adulto Jovem
9.
Int J Mol Sci ; 21(4)2020 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-32070055

RESUMO

Prostate cancer is one of the leading causes of cancer mortality in men worldwide. An unusual but unique environment for studying tumor cell processes is provided by microgravity, either in space or simulated by ground-based devices like a random positioning machine (RPM). In this study, prostate adenocarcinoma-derived PC-3 cells were cultivated on an RPM for time periods of 3 and 5 days. We investigated the genes associated with the cytoskeleton, focal adhesions, extracellular matrix, growth, survival, angiogenesis, and metastasis. The gene expression of signaling factors of the vascular endothelial growth factor (VEGF), mitogen-activated protein kinase (MAPK), and PI3K/AKT/mTOR (PAM) pathways was investigated using qPCR. We performed immunofluorescence to study the cytoskeleton, histological staining to examine the morphology, and a time-resolved immunofluorometric assay to analyze the cell culture supernatants. When PC-3 cells were exposed to simulated microgravity (s-µg), some cells remained growing as adherent cells (AD), while most cells detached from the cell culture flask bottom and formed multicellular spheroids (MCS). After 3-day RPM exposure, PC-3 cells revealed significant downregulation of the VEGF, SRC1, AKT, MTOR, and COL1A1 gene expression in MCS, whereas FLT1, RAF1, MEK1, ERK1, FAK1, RICTOR, ACTB, TUBB, and TLN1 mRNAs were not significantly changed. ERK2 and TLN1 were elevated in AD, and FLK1, LAMA3, COL4A5, FN1, VCL, CDH1, and NGAL mRNAs were significantly upregulated in AD and MCS after 3 days. After a 5-day culture in s-µg, the PC-3 cells showed significant downregulations of VEGF mRNA in AD and MCS, and FN1, CDH1, and LAMA3 in AD and SCR1 in MCS. In addition, we measured significant upregulations in FLT1, AKT, ERK1, ERK2, LCN2, COL1A1, TUBB, and VCL mRNAs in AD and MCS, and increases in FLK1, FN1, and COL4A5 in MCS as well as LAMB2, CDH1, RAF1, MEK1, SRC1, and MTOR mRNAs in AD. FAK1 and RICTOR were not altered by s-µg. In parallel, the secretion rate of VEGFA and NGAL proteins decreased. Cytoskeletal alterations (F-actin) were visible, as well as a deposition of collagen in the MCS. In conclusion, RPM-exposure of PC-3 cells induced changes in their morphology, cytoskeleton, and extracellular matrix protein synthesis, as well as in their focal adhesion complex and growth behavior. The significant upregulation of genes belonging to the PAM pathway indicated their involvement in the cellular changes occurring in microgravity.


Assuntos
Proteínas da Matriz Extracelular/genética , Proteínas de Neoplasias/genética , Neoplasias da Próstata/radioterapia , Simulação de Ausência de Peso , Linhagem Celular Tumoral , Citoesqueleto/genética , Matriz Extracelular/genética , Adesões Focais/genética , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , MAP Quinase Quinase 1/genética , Masculino , Fosfatidilinositol 3-Quinases/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Serina-Treonina Quinases TOR/genética , Fator A de Crescimento do Endotélio Vascular/genética
10.
Cardiovasc Drugs Ther ; 34(1): 3-14, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32103377

RESUMO

PURPOSE: We investigated whether increased expression of activated mitogen-activated protein kinase (MAPK) kinases 1 (MEK1) restores ischemic post-conditioning (IPostC) protection in hypertrophic myocardium following ischemia/reperfusion (I/R) injury. METHODS: C57Bl/6 mice received recombinant adeno-associated virus type 9 (rAAV9)-mediated activated MEK1 gene delivery systemically, then following the induction of cardiac hypertrophy via transverse aortic constriction for 4 weeks. In a Langendorff model, hypertrophic hearts were subjected to 40 min/60 min I/R or with IPostC intervention consisting of 6 cycles of 10 s reperfusion and 10 s no-flow before a 60-min reperfusion. Hemodynamics, infarct size (IS), myocyte apoptosis and changes in expression of reperfusion injury salvage kinase (RISK) pathway were examined. RESULTS: rAAV9-MEK1 gene delivery led to a 4.3-fold and 2.7-fold increase in MEK1 mRNA and protein expression in the heart versus their control values. I/R resulted in a larger IS in hypertrophic than in non-hypertrophic hearts (52.3 ± 4.7% vs. 40.0 ± 2.5%, P < 0.05). IPostC mediated IS reduction in non-hypertrophic hearts (27.6 ± 2.6%, P < 0.05), while it had no significant effect in hypertrophic hearts (46.5 ± 3.1%, P=NS) compared with the IS in non-hypertrophic or hypertrophic hearts subjected to I/R injury only, respectively. Hemodynamic decline induced by I/R was preserved by IPostC in non-hypertrophic hearts but not in hypertrophic hearts. rAAV9-MEK1 gene delivery restored IPostC protection in hypertrophic hearts evidenced by reduced IS (32.0 ± 2.8% vs. 46.5 ± 3.1%) and cardiac cell apoptosis and largely preserved hemodynamic parameters. These protective effects were associated with significantly increased phosphorylation of ERK1/2 and ribosomal protein S6 kinases (p70S6K), but it had no influence on Akt and glycogen synthase kinase-3ß. CONCLUSION: These results demonstrated that rAAV9-mediated activated MEK1 expression restores IPostC protection in the hypertrophic heart against I/R injury through the activation of ERK pathway.


Assuntos
Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Hipertrofia Ventricular Esquerda/terapia , Pós-Condicionamento Isquêmico , MAP Quinase Quinase 1/biossíntese , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/enzimologia , Animais , Apoptose , Modelos Animais de Doenças , Indução Enzimática , Hipertrofia Ventricular Esquerda/enzimologia , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/patologia , Preparação de Coração Isolado , MAP Quinase Quinase 1/genética , Masculino , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Fosforilação , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo
11.
Blood ; 135(16): 1311-1318, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32107533

RESUMO

Erdheim-Chester disease (ECD) is characterized by the infiltration of tissues by foamy CD68+CD1a- histiocytes, with 1500 known cases since 1930. Mutations activating the MAPK pathway are found in more than 80% of patients with ECD, mainly the BRAFV600E activating mutation in 57% to 70% of cases, followed by MAP2K1 in close to 20%. The discovery of BRAF mutations and of other MAP kinase pathway alterations, as well as the co-occurrence of ECD with LCH in 15% of patients with ECD, led to the 2016 revision of the classification of histiocytoses in which LCH and ECD belong to the "L" group. Both conditions are considered inflammatory myeloid neoplasms. Ten percent of ECD cases are associated with myeloproliferative neoplasms and/or myelodysplastic syndromes. Some of the most striking signs of ECD are the long bone involvement (80%-95%), as well as the hairy kidney appearance on computed tomography scan (63%), the coated aorta (40%), and the right atrium pseudo-tumoral infiltration (36%). Central nervous system involvement is a strong prognostic factor and independent predictor of death. Interferon-α seems to be the best initial treatment of ECD. Since 2012, more than 200 patients worldwide with multisystem or refractory ECD have benefitted from highly effective therapy with BRAF and MEK inhibitors. Targeted therapies have an overall, robust, and reproducible efficacy in ECD, with no acquired resistance to date, but their use may be best reserved for the most severe manifestations of the disease, as they may be associated with serious adverse effects and as-yet-unknown long-term consequences.


Assuntos
Doença de Erdheim-Chester/complicações , Doença de Erdheim-Chester/diagnóstico , Animais , Doença de Erdheim-Chester/genética , Doença de Erdheim-Chester/terapia , Humanos , Leucemia Mieloide/complicações , MAP Quinase Quinase 1/genética , Mutação , Síndromes Mielodisplásicas/complicações , Transtornos Mieloproliferativos/complicações , Prognóstico , Proteínas Proto-Oncogênicas B-raf/genética
12.
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
13.
J Biol Chem ; 295(3): 800-807, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31836663

RESUMO

Sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the surface of human hepatocytes and functions as an entry receptor of hepatitis B virus (HBV). Recently, we have reported that epidermal growth factor receptor (EGFR) is involved in NTCP-mediated viral internalization during the cell entry process. Here, we analyzed which function of EGFR is essential for mediating HBV internalization. In contrast to the reported crucial function of EGFR-downstream signaling for the entry of hepatitis C virus (HCV), blockade of EGFR-downstream signaling proteins, including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and signal transducer and activator of transcription (STAT), had no or only minor effects on HBV infection. Instead, deficiency of EGFR endocytosis resulting from either a deleterious mutation in EGFR or genetic knockdown of endocytosis adaptor molecules abrogated internalization of HBV via NTCP and prevented viral infection. EGFR activation triggered a time-dependent relocalization of HBV preS1 to the early and late endosomes and to lysosomes in concert with EGFR transport. Suppression of EGFR ubiquitination by site-directed mutagenesis or by knocking down two EGFR-sorting molecules, signal-transducing adaptor molecule (STAM) and lysosomal protein transmembrane 4ß (LAPTM4B), suggested that EGFR transport to the late endosome is critical for efficient HBV infection. Cumulatively, these results support the idea that the EGFR endocytosis/sorting machinery drives the translocation of NTCP-bound HBV from the cell surface to the endosomal network, which eventually enables productive viral infection.


Assuntos
Endocitose/genética , Endossomos/genética , Receptores ErbB/genética , Hepatite B/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/química , Receptores ErbB/química , Células Hep G2 , Hepacivirus/química , Hepacivirus/genética , Hepacivirus/patogenicidade , Hepatite B/metabolismo , Hepatite B/virologia , Vírus da Hepatite B/química , Vírus da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , MAP Quinase Quinase 1/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio , Fosfatidilinositol 3-Quinases/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Fatores de Transcrição STAT/genética , Simportadores , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Internalização do Vírus
14.
Int J Biochem Cell Biol ; 118: 105645, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31733402

RESUMO

OBJECTIVE: The pathogenesis of coronary artery calcification (CAC) in coronary heart disease (CHD) is mediated by exosomes derived from vascular smooth muscle cells (VSMCs). However, little is known about their underlying mechanism. In this study, we aimed to investigate the differentially expressed miRNAs in VSMCs undergoing induced calcification. METHODS: A cellular calcification model was established using the mouse VSMC line MOVAS-1. Calcium deposition was evaluated by Alizarin Red staining. Exosome sizes were determined by Nanoparticle Tracking Analysis (NTA), and exosome morphology was examined by transmission electron microscopy (TEM). The expression of exosome and calcification biomarkers was analyzed by quantitative real-time PCR (qPCR) and western blotting. Differential miRNA profiles were determined by deep sequencing and bioinformatics. Protein levels in VSMCs experiencing interference by a miR-324-3p inhibitor were detected by western blotting. RESULTS: The MOVAS-1 calcification model was confirmed by Alizarin Red staining and expressional alteration of α-SMA, BMP-2, OPN, and MGP. Exosomes from the calcification model showed expression of exosomal biomarkers and regular exosome diameters, which caused significant calcification in MOVAS-1 cells. In total, 987 and 92 miRNAs were significantly upregulated and downregulated in exosomes from the cellular calcification model as compared with those from MOVAS-1 cells, respectively. Target genes of differential miRNAs were involved in various biological processes such as development, metabolism, and cellular component organization and biogenesis as well as multiple signaling pathways such as protein kinase B (AKT) signaling. The most differentially expressed miRNAs were validated by qPCR, which showed that mmu-let-7e-5p was downregulated and mmu-miR-324-3p was upregulated in exosomes from the MOVAS-1 cellular calcification model. The expression of IGF1R was increased, and the expressions of PIK3CA and MAP2K1 were reduced in MOVAS-1 transfected with a miR-324-3p inhibitor. CONCLUSION: microRNA profiles were significantly altered in exosomes from VSMCs undergoing calcification.


Assuntos
MicroRNAs/genética , Receptor IGF Tipo 1/genética , Calcificação Vascular/genética , Animais , Cálcio/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/genética , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Exossomos/genética , Exossomos/metabolismo , Regulação da Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MAP Quinase Quinase 1/genética , Camundongos , MicroRNAs/antagonistas & inibidores , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Transdução de Sinais/genética , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia
15.
Mol Med Rep ; 21(1): 420-428, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31746389

RESUMO

Malignant melanoma has the highest malignancy rate among all skin cancer and is characterized by an insidious onset, high invasion and poor patient prognosis. Yet, the mechanisms involved remain unclear and warrant further investigation. Based on bioinformatic analysis, phospholipase C ß2 (PLCB2) has been found to be correlated with melanoma growth. The present study was the first to demonstrate that PLCB2 is a key factor affecting melanoma proliferation and apoptosis. Here, microarray datasets from the publicly available Gene Expression Omnibus (GEO) database were employed, and gene set enrichment analysis (GSEA) was introduced to identify candidate transcription factors. PLCB2 was identified as a crucial gene in the protein­protein interaction (PPI) network. The expression of PLCB2 mRNA in various cancer lines was analyzed by reverse transcription­polymerase chain reaction (RT­PCR). In addition, the proliferation ability and apoptosis rate in human melanoma cells overexpressing or not overexpressing PLCB2 were assessed using colony formation assay, flow cytometry and the Cell Counting Kit­8 (CCK­8) assay. Cell viability and apoptosis­related factors, such as p53, Bcl­2, Bax and caspase­3 were significantly regulated. Knockdown of PLCB2 suppressed the activation of the Ras/Raf/MAPK signaling pathway. In conclusion, knockdown of PLCB2 suppressed cell viability and promoted cell apoptosis by activating the Ras/Raf/MAPK pathway. Thus, PLCB2 may utilized as a potential therapeutic target in patients with melanoma.


Assuntos
Proliferação de Células/genética , Melanoma/genética , Proteínas de Neoplasias/genética , Fosfolipase C beta/genética , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Genes ras/genética , Humanos , MAP Quinase Quinase 1/genética , Melanoma/patologia , Análise em Microsséries , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas c-raf/genética , Transdução de Sinais/genética
16.
JCI Insight ; 4(23)2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31801908

RESUMO

The MEK1/2-ERK1/2 pathway has been implicated in regulating the inflammatory response to lung injury and infection, and pharmacologic MEK1/2 inhibitor compounds are reported to reduce detrimental inflammation in multiple animal models of disease, in part through modulation of leukocyte responses. However, the specific contribution of myeloid MEK1 in regulating acute lung injury (ALI) and its resolution remain unknown. Here, the role of myeloid Mek1 was investigated in a murine model of LPS-induced ALI (LPS-ALI) by genetic deletion using the Cre-floxed system (LysMCre × Mekfl), and human alveolar macrophages from healthy volunteers and patients with acute respiratory distress syndrome (ARDS) were obtained to assess activation of the MEK1/2-ERK1/2 pathway. Myeloid Mek1 deletion results in a failure to resolve LPS-ALI, and alveolar macrophages lacking MEK1 had increased activation of MEK2 and the downstream target ERK1/2 on day 4 of LPS-ALI. The clinical significance of these findings is supported by increased activation of the MEK1/2-ERK1/2 pathway in alveolar macrophages from patients with ARDS compared with alveolar macrophages from healthy volunteers. This study reveals a critical role for myeloid MEK1 in promoting resolution of LPS-ALI and controlling the duration of macrophage proinflammatory responses.


Assuntos
Lesão Pulmonar Aguda/metabolismo , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Macrófagos Alveolares/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/patologia , Animais , Feminino , Humanos , Imunidade Inata , Inflamação/metabolismo , Lipopolissacarídeos/efeitos adversos , Pulmão/patologia , MAP Quinase Quinase 2/metabolismo , Sistema de Sinalização das MAP Quinases , Macrófagos Alveolares/imunologia , Masculino , Camundongos , Camundongos Knockout , Transcriptoma
17.
Aging (Albany NY) ; 11(23): 11040-11053, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31809267

RESUMO

The malignant bone tumors that are categorized as chondrosarcomas display a high potential for metastasis in late-stage disease. Higher-grade chondrosarcomas contain higher levels of expression of platelet-derived growth factor (PDGF) and its receptor. The phosphorylation of sphingosine by sphingosine kinase enzymes SphK1 and SphK2 generates sphingosine-1-phosphate (S1P), which inhibits human chondrosarcoma cell migration, while SphK1 overexpression suppresses lung metastasis of chondrosarcoma. We sought to determine whether S1P mediates levels of PDGF-A expression and angiogenesis in chondrosarcoma. Surprisingly, our investigations found that treatment of chondrosarcoma cells with S1P and transfecting them with SphK1 cDNA increased PDGF-A expression and induced angiogenesis of endothelial progenitor cells (EPCs). Ras, Raf, MEK, ERK and AP-1 inhibitors and their small interfering RNAs (siRNAs) inhibited S1P-induced PDGF-A expression and EPC angiogenesis. Our results indicate that S1P promotes the expression of PDGF-A in chondrosarcoma via the Ras/Raf/MEK/ERK/AP-1 signaling cascade and stimulates EPC angiogenesis.


Assuntos
Condrossarcoma/metabolismo , Lisofosfolipídeos/metabolismo , Lisofosfolipídeos/farmacologia , Neovascularização Patológica/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Esfingosina/análogos & derivados , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Fator de Crescimento Derivado de Plaquetas/genética , Esfingosina/metabolismo , Esfingosina/farmacologia , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Quinases raf/genética , Quinases raf/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo
18.
Nat Commun ; 10(1): 5472, 2019 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-31784532

RESUMO

Adenocarcinoma in situ and minimally invasive adenocarcinoma are the pre-invasive forms of lung adenocarcinoma. The genomic and immune profiles of these lesions are poorly understood. Here we report exome and transcriptome sequencing of 98 lung adenocarcinoma precursor lesions and 99 invasive adenocarcinomas. We have identified EGFR, RBM10, BRAF, ERBB2, TP53, KRAS, MAP2K1 and MET as significantly mutated genes in the pre/minimally invasive group. Classes of genome alterations that increase in frequency during the progression to malignancy are revealed. These include mutations in TP53, arm-level copy number alterations, and HLA loss of heterozygosity. Immune infiltration is correlated with copy number alterations of chromosome arm 6p, suggesting a link between arm-level events and the tumor immune environment.


Assuntos
Adenocarcinoma in Situ/genética , Adenocarcinoma de Pulmão/genética , Neoplasias Pulmonares/genética , Adenocarcinoma in Situ/imunologia , Adenocarcinoma in Situ/patologia , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Variações do Número de Cópias de DNA , Receptores ErbB/genética , Feminino , Perfilação da Expressão Gênica , Antígenos HLA/genética , Antígenos HLA/imunologia , Humanos , Perda de Heterozigosidade , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , MAP Quinase Quinase 1/genética , Masculino , Pessoa de Meia-Idade , Mutação , Invasividade Neoplásica , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas de Ligação a RNA/genética , Receptor ErbB-2/genética , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos T/genética , Proteína Supressora de Tumor p53/genética , Sequenciamento Completo do Exoma
19.
Braz. J. Psychiatry (São Paulo, 1999, Impr.) ; 41(6): 485-493, Nov.-Dec. 2019. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1055347

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
Humanos , Masculino , Adulto , Adulto Jovem , Cocaína Crack , Metilação de DNA , Transtornos Relacionados ao Uso de Cocaína/genética , Transtornos Relacionados ao Uso de Cocaína/sangue , Estudo de Associação Genômica Ampla/métodos , Estudos de Casos e Controles , Modelos Lineares , Histona-Lisina N-Metiltransferase/genética , Estatísticas não Paramétricas , Proteína Quinase 1 Ativada por Mitógeno/genética , MAP Quinase Quinase 1/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Antígenos de Histocompatibilidade/genética , Histona Desacetilases/genética
20.
J Recept Signal Transduct Res ; 39(4): 368-372, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31774019

RESUMO

Although ecotropic viral integration site 2 A (EVI2A) plays key roles in several cancers, the expression and function of EVI2A in osteosarcoma (OS) have not been investigated. Hence, we explored the expression of EVI2A and its clinical significance of EVI2A of OS. Firstly, we investigated the expression of EVI2A in OS tissues. The relationship of EVI2A expression and survival time was analyzed using Kaplan-Meier plotter. Then, we used quantitative reverse transcription PCR (qRT-PCR) to confirm the expression level of EVI2A in OS cell lines. Cell proliferation, and wound-healing experiments were used to identify the biological function of EVI2A. Moreover, EVI2A-mediated MEK/ERK signaling pathway was evaluated using western blotting. Data suggested that EVI2A was highly expressed in OS tissues, and high-expression of EVI2A was associated with worse overall survival in OS patients. Moreover, the up-regulation of it was observed in OS cell lines (Saos2, and MG63). Knockdown of EVI2A suppressed cell proliferation and migration of OS. Western blotting revealed that the inactivation of MEK/ERK pathway was found in OS cells after EVI2A knockdown. Our data implicated the crucial role of EVI2A in the progression of OS, demonstrating that expression of EVI2A may offer an attractive novel prognostic signature for OS.


Assuntos
Neoplasias Ósseas/patologia , Regulação Neoplásica da Expressão Gênica , MAP Quinase Quinase 1/metabolismo , Proteínas de Membrana/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Osteossarcoma/patologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/metabolismo , Movimento Celular , Proliferação de Células , Progressão da Doença , Humanos , MAP Quinase Quinase 1/genética , Proteínas de Membrana/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Osteossarcoma/metabolismo , Prognóstico , Transdução de Sinais , Células Tumorais Cultivadas
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