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1.
Mol Cell ; 76(6): 872-884.e5, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31606273

RESUMO

The Ras GTPases are frequently mutated in human cancer, and, although the Raf kinases are essential effectors of Ras signaling, the tumorigenic properties of specific Ras-Raf complexes are not well characterized. Here, we examine the ability of individual Ras and Raf proteins to interact in live cells using bioluminescence resonance energy transfer (BRET) technology. We find that C-Raf binds all mutant Ras proteins with high affinity, whereas B-Raf exhibits a striking preference for mutant K-Ras. This selectivity is mediated by the acidic, N-terminal segment of B-Raf and requires the K-Ras polybasic region for high-affinity binding. In addition, we find that C-Raf is critical for mutant H-Ras-driven signaling and that events stabilizing B-Raf/C-Raf dimerization, such as Raf inhibitor treatment or certain B-Raf mutations, can allow mutant H-Ras to engage B-Raf with increased affinity to promote tumorigenesis, thus revealing a previously unappreciated role for C-Raf in potentiating B-Raf function.


Assuntos
Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Neoplasias/enzimologia , Quinases raf/metabolismo , Proteínas ras/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Mutação , Células NIH 3T3 , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/genética , Esferoides Celulares , Quinases raf/genética , Proteínas ras/genética
2.
Environ Toxicol Pharmacol ; 70: 103200, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31158732

RESUMO

OBJECTIVE: Plumbagin exerts effective anti-hepatocellular carcinoma (HCC) benefits, however, the detailed mechanisms behind these effects are not yet completely elucidated. The pharmacological targets and molecular mechanisms of plumbagin against HCC were revealed through conducting network pharmacology approach before experimentative verification. METHODS: The web-accessible databases of herbal ingredients' targets (HIT), Swiss-Target-Prediction and Super-Pred were used to predict the therapeutic targets of plumbagin, followed by combined with pathogenic targets of HCC from oncogenomic database of hepatocellular carcinoma (OncoDB.HCC) and Liverome databases to obtain the predominant targets of plumbagin-treating HCC. The database for annotation, visualization and integrated discovery (DAVID) was applied to output the gene ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment by use of all predominant targets for computerized visualization. The validated data of human and cell culture were subjected to a group of medical imaging, biochemical tests and immunostaining, respectively. RESULTS: As revealed in bioinformatic data, 19 predominant targets of plumbagin-treating HCC were obtained, and 5 top targets of TP53, MAPK1, MAP2K1, RAF1 and CCND1 were the most important biomolecules in anti-HCC effects exerted by plumbagin. Other identifiable 102 GO items were showed, including 66 biological processes, and 12 cellular components, 24 molecular functions. And 67 KEGG pathways were mainly involved in neoplastic signaling. In human data, HCC sections showed increased expressions of hepatocellular TP53, MAPK1, accompanied with positive clinical imaging results for HCC. In plumbagin-treated HepG2 cells, reduced TP53, MAPK1 protein expressions were observed, accompanied with cell arrest and apoptosis. CONCLUSION: Collectively, the pharmacological targets and mechanisms of plumbagin-treating HCC were predicted and integrated through the method of network pharmacology, followed by some investigative validations. Interestingly, these 5 predominant biomolecules may be the potential targets for screening and treating HCC.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Naftoquinonas/farmacologia , Biologia Computacional , Ciclina D1/metabolismo , Feminino , Células Hep G2 , Humanos , MAP Quinase Quinase 1/metabolismo , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteína Supressora de Tumor p53/metabolismo
3.
Cell Commun Signal ; 17(1): 31, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30971268

RESUMO

BACKGROUND: p21-activated kinase 1 (PAK1) plays a fundamental role in promoting the development and progression of several cancers and is a potential therapeutic target. However, the biological function and underlying mechanism of PAK1 in esophageal squamous cell carcinoma (ESCC) remain unclear. METHODS: The expression of PAK1 was detected in both ESCC cell lines and clinical samples. Cell growth was measured by MTT, focus formation and soft agar assays. Cell migration and invasion were detected by wound healing and transwell assays. Animal models of subcutaneous tumourigenicity and tail vein metastasis were performed to determine the inhibitory effect of pharmacological inhibitor IPA-3 on tumor growth and metastasis of ESCC cells. RESULTS: We found that PAK1 was frequently overexpressed in ESCC. Ectopic expression of PAK1 promoted cellular growth, colony formation and anchorage-independent growth. Overexpressing PAK1 also enhanced migration, invasion and the expression of MMP-2 and MMP-9 in ESCC cells. In contrast, silencing PAK1 by lentiviral knockdown or a specific inhibitor IPA-3 resulted in a contrary effect. Subsequent investigations revealed that Raf1/MEK1/ERK signaling pathway was involved in PAK1-mediated effect. Enhanced expression of Raf1 attenuated the inhibitory functions of PAK1 shRNA. Whereas blocking of Raf1 by shRNA or specific inhibition of MEK1 by U0126 antagonized the oncogenetic effect of PAK1 on ESCC cells. More importantly, Pharmacological inhibition of PAK1 by IPA-3 significantly suppressed tumor growth and lung metastasis of ESCC cells in vivo. CONCLUSIONS: These data support that PAK1 is an ideal target for the development of potential therapeutic drugs for ESCC patients even with metastasis.


Assuntos
Dissulfetos/farmacologia , Neoplasias Esofágicas/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Naftóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinases Ativadas por p21/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Dissulfetos/uso terapêutico , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/secundário , Humanos , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Naftóis/uso terapêutico , Metástase Neoplásica , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , RNA Interferente Pequeno/metabolismo
4.
PLoS Genet ; 15(4): e1008108, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31017896

RESUMO

RASopathies are a family of related syndromes caused by mutations in regulators of the RAS/Extracellular Regulated Kinase 1/2 (ERK1/2) signaling cascade that often result in neurological deficits. RASopathy mutations in upstream regulatory components, such as NF1, PTPN11/SHP2, and RAS have been well-characterized, but mutation-specific differences in the pathogenesis of nervous system abnormalities remain poorly understood, especially those involving mutations downstream of RAS. Here, we assessed cellular and behavioral phenotypes in mice expressing a Raf1L613V gain-of-function mutation associated with the RASopathy, Noonan Syndrome. We report that Raf1L613V/wt mutants do not exhibit a significantly altered number of excitatory or inhibitory neurons in the cortex. However, we observed a significant increase in the number of specific glial subtypes in the forebrain. The density of GFAP+ astrocytes was significantly increased in the adult Raf1L613V/wt cortex and hippocampus relative to controls. OLIG2+ oligodendrocyte progenitor cells were also increased in number in mutant cortices, but we detected no significant change in myelination. Behavioral analyses revealed no significant changes in voluntary locomotor activity, anxiety-like behavior, or sociability. Surprisingly, Raf1L613V/wt mice performed better than controls in select aspects of the water radial-arm maze, Morris water maze, and cued fear conditioning tasks. Overall, these data show that increased astrocyte and oligodendrocyte progenitor cell (OPC) density in the cortex coincides with enhanced cognition in Raf1L613V/wt mutants and further highlight the distinct effects of RASopathy mutations on nervous system development and function.


Assuntos
Córtex Cerebral/metabolismo , Aprendizagem , Mutação , Neuroglia/metabolismo , Síndrome de Noonan/genética , Síndrome de Noonan/psicologia , Proteínas Proto-Oncogênicas c-raf/genética , Animais , Biomarcadores , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases , Aprendizagem em Labirinto , Memória , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Síndrome de Noonan/metabolismo , Oligodendroglia/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo
5.
Cancer Cell ; 35(4): 573-587.e6, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30975481

RESUMO

Five-year survival for pancreatic ductal adenocarcinoma (PDAC) patients remains below 7% due to the lack of effective treatments. Here, we report that combined ablation of EGFR and c-RAF expression results in complete regression of a significant percentage of PDAC tumors driven by Kras/Trp53 mutations in genetically engineered mice. Moreover, systemic elimination of these targets induces toxicities that are well tolerated. Response to this targeted therapy correlates with transcriptional profiles that resemble those observed in human PDACs. Finally, inhibition of EGFR and c-RAF expression effectively blocked tumor progression in nine independent patient-derived xenografts carrying KRAS and TP53 mutations. These results open the door to the development of targeted therapies for PDAC patients.


Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/farmacologia , Gefitinibe/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Carcinoma Ductal Pancreático/enzimologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Phytomedicine ; 61: 152857, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31035040

RESUMO

PURPOSE: This study aimed to examine the effects of the Chinese yam extract and adenosine on lipopolysaccharide (LPS)-induced cardiac anomalies and the underlying mechanisms involved. METHODS: Chinese yam extract [1630 mg/kg, intragastric (i.g.), 2 times/day] and adenosine (50 mg/kg, i.g., 2 times/day) were administered for 3 days, followed by the induction of sepsis by injecting LPS intraperitoneally [10 mg/kg, 2 h prior, intraperitoneally (i.p.)]. Also, estrogen receptor (ER)-unspecific antagonist Faslodex (ICI182,780, 0.5 mg/kg, i.p.) was administered 30 min before the treatments of Chinese yam extract or adenosine to evaluate whether the observed effects elicited by yam and adenosine were mediated via ERs. The heart function and the levels of pro-inflammatory cytokines, reversed mitogen-activated protein kinases (MAPKs), renin-angiotensin system (RAS), apoptosis markers, ER, and SHC/Ras/Raf1 were examined. The antagonistic effect of ICI182,780 (1 µM) and FTS (1 µM) against the Chinese yam extract (0.1 mg/ml) and adenosine (5 µM) in LPS (20 µg/ml, 24 h)-induced H9c2 cells was also investigated. RESULTS: The Chinese yam extract and adenosine improved heart function, downregulated pro-inflammatory cytokines, reversed MAPK and RAS, transformed the apoptosis markers, and increased the expression of ER and SHC/Ras/Raf1 following LPS challenge. These effects could be blocked by ICI182,780. FTS could not block the expression of ER on the Chinese yam extract and adenosine interposed on LPS-induced H9c2 cells, demonstrating that ER might be the upstream signaling regulator of SHC/Ras/Raf1. CONCLUSION: The Chinese yam extract and adenosine ameliorated LPS-induced cardiac contractility through the inhibition of RAS and apoptosis possibly via an ER-SHC/Ras/Raf1-dependent mechanism.


Assuntos
Adenosina/farmacologia , Dioscorea/química , Medicamentos de Ervas Chinesas/farmacologia , Cardiopatias/tratamento farmacológico , Coração/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Citocinas/metabolismo , Feminino , Fulvestranto/farmacologia , Cardiopatias/induzido quimicamente , Cardiopatias/fisiopatologia , Lipopolissacarídeos/toxicidade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas c-raf/metabolismo , Ratos Wistar , Receptores Estrogênicos/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Proteínas ras/metabolismo
7.
BMC Cancer ; 19(1): 266, 2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30909892

RESUMO

BACKGROUND: Recent advances in the treatment of melanoma that involve immunotherapy and B-Raf inhibition have revolutionised cancer care for this disease. However, an un-met clinical need remains in B-Raf inhibitor resistant patients where first-generation B-Raf inhibitors provide only short-term disease control. In these cases, B-Raf inhibition leads to paradoxical activation of the C-Raf - MEK - ERK signalling pathway, followed by metastasis. PDE8A has been shown to directly interact with and modulate the cAMP microdomain in the vicinity of C-Raf. This interaction promotes C-Raf activation by attenuating the PKA-mediated inhibitory phosphorylation of the kinase. METHODS: We have used a novel cell-penetrating peptide agent (PPL-008) that inhibits the PDE8A - C-Raf complex in a human malignant MM415 melanoma cell line and MM415 melanoma xenograft mouse model to investigate ERK MAP kinase signalling. RESULTS: We have demonstrated that the PDE8A - C-Raf complex disruptor PPL-008 increased inhibitory C-Raf-S259 phosphorylation and significantly reduced phospho-ERK signalling. We have also discovered that the ability of PPL-008 to dampen ERK signalling can be used to counter B-Raf inhibitor-driven paradoxical activation of phospho-ERK in MM415 cells treated with PLX4032 (Vemurafenib). PPL-008 treatment also significantly retarded the growth of these cells. When applied to a MM415 melanoma xenograft mouse model, PPL-008C penetrated tumour tissue and significantly reduced phospho-ERK signalling in that domain. CONCLUSION: Our data suggests that the PDE8A-C-Raf complex is a promising therapeutic treatment for B-Raf inhibitor resistant melanoma.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Peptídeos Penetradores de Células/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-raf/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacologia , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/metabolismo , Camundongos , Ligação Proteica/efeitos dos fármacos , Vemurafenib/administração & dosagem , Vemurafenib/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
8.
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
9.
Int J Mol Sci ; 20(4)2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30795516

RESUMO

c-Raf is a central component of the extracellular signal-regulated kinase (ERK) pathway which is implicated in the development of many cancer types. RanBPM (Ran-Binding Protein M) was previously shown to inhibit c-Raf expression, but how this is achieved remains unclear. RanBPM is part of a recently identified E3 ubiquitin ligase complex, the CTLH (C-terminal to LisH) complex. Here, we show that the CTLH complex regulates c-Raf expression through a control of its degradation. Several domains of RanBPM were found necessary to regulate c-Raf levels, but only the C-terminal CRA (CT11-RanBPM) domain showed direct interaction with c-Raf. c-Raf ubiquitination and degradation is promoted by the CTLH complex. Furthermore, A-Raf and B-Raf protein levels are also regulated by the CTLH complex, indicating a common regulation of Raf family members. Finally, depletion of CTLH subunits RMND5A (required for meiotic nuclear division 5A) and RanBPM resulted in enhanced proliferation and loss of RanBPM promoted tumour growth in a mouse model. This study uncovers a new mode of control of c-Raf expression through regulation of its degradation by the CTLH complex. These findings also uncover a novel target of the CTLH complex, and suggest that the CTLH complex has activities that suppress cell transformation and tumour formation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Nucleares/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Animais , Células Cultivadas , Proteínas do Citoesqueleto/química , Estabilidade Enzimática , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Nucleares/química , Proteínas Proto-Oncogênicas c-raf/química , Ubiquitinação
10.
Front Immunol ; 10: 26, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30761125

RESUMO

Schistosomiasis is a major helminthic disease in which damage to the affected organs is orchestrated by a pathogenic host CD4 T helper (Th) cell-mediated immune response against parasite eggs. In the case of the species Schistosoma mansoni, the resulting granulomatous inflammation and fibrosis takes place in the liver and intestines. The magnitude of disease varies greatly from individual to individual but in a minority of patients, there is severe disease and death. S. mansoni infection in a murine model similarly results in marked strain variation of immunopathology. In the most commonly examined mouse strain, C57BL/6 (BL/6), there is relatively mild hepatic pathology arising in a Th2-dominated cytokine environment. In contrast, CBA mice develop decisively more severe lesions largely driven by proinflammatory IL-17-producing Th17 cells. Dendritic cells (DCs) from CBA mice differ sharply with those from BL/6 mice in that they vastly over-express the C-type lectin receptor (CLR) CD209a (SIGNR5), a homolog of human DC-SIGN, which senses glycans such as those produced by schistosome eggs. Silencing of CD209a, and recent studies with CD209a KO CBA mice have shown that this receptor is crucial to induce the pathogenic Th17 cell response; indeed, CD209a KO mice display markedly reduced immunopathology akin to that seen in BL/6 mice. Mechanistically, CD209a synergizes with the related CLRs Dectin-2 and Mincle to stimulate increased DC production of IL-1ß and IL-23, necessary for pathogenic Th17 cell development. These findings denote key molecular underpinnings of disease variability based on selection and function of contrasting Th cell subsets.


Assuntos
Interações Hospedeiro-Parasita/imunologia , Lectinas Tipo C/metabolismo , Esquistossomose/imunologia , Esquistossomose/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Animais , Biomarcadores , Moléculas de Adesão Celular/metabolismo , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Ligação Proteica , Proteínas Proto-Oncogênicas c-raf/metabolismo , Receptores de Superfície Celular/metabolismo , Schistosoma mansoni/imunologia , Esquistossomose/diagnóstico , Esquistossomose/parasitologia , Índice de Gravidade de Doença , Células Th2/imunologia , Células Th2/metabolismo
11.
FASEB J ; 33(1): 1138-1150, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30106602

RESUMO

Raf1/c-Raf is a well-characterized serine/threonine-protein kinase that links Ras family members with the MAPK/ERK signaling cascade. We have identified a novel splice isoform of human Raf1 that causes protein truncation and loss of the C-terminal kinase domain (Raf1-tr). We found that Raf1-tr has increased nuclear localization compared with full-length Raf1, and this finding was secondary to reduced binding of Raf1-tr to the cytoplasmic chaperone FK506 binding protein 5. We show that Raf1-tr has increased binding to DNA-dependent protein kinase (DNA-PK), which inhibits DNA-PK function and causes amplification of irradiation- and bleomycin-induced DNA damage. We found that the human colorectal cancer cell line, HCT-116, displayed reduced expression of Raf1-tr, and reintroduction of Raf1-tr sensitized the cells to bleomycin-induced apoptosis. Furthermore, we identified differential Raf1-tr expression in breast cancer cell lines and showed that breast cancer cells with increased Raf1-tr expression become sensitized to bleomycin-induced apoptosis. Collectively, these results demonstrate a novel Raf1 isoform in humans that has a unique noncanonical role in regulating the double-stranded DNA damage response pathway through modulation of DNA-PK function.-Nixon, B. R., Sebag, S. C., Glennon, M. S., Hall, E. J., Kounlavong, E. S., Freeman, M. L., Becker, J. R. Nuclear localized Raf1 isoform alters DNA-dependent protein kinase activity and the DNA damage response.


Assuntos
Núcleo Celular/metabolismo , Dano ao DNA , Proteína Quinase Ativada por DNA/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Processamento Alternativo , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Bleomicina/farmacologia , Linhagem Celular Tumoral , DNA/efeitos dos fármacos , DNA/efeitos da radiação , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Transdução de Sinais , Proteínas de Ligação a Tacrolimo/metabolismo , Proteínas ras/metabolismo
12.
Oral Dis ; 25(2): 481-487, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30294831

RESUMO

OBJECTIVE: This study aimed to assess the frequency of KRAS mutation and its association with the presence of the MAPK/ERK signaling pathway proteins in adenomatoid odontogenic tumors. STUDY DESIGN: Paraffin-embedded tissue samples from nine cases of adenomatoid odontogenic tumor were used. Genomic DNA was extracted from each sample; in one case, genetic mutations in 50 cancer-associated genes were examined by next-generation sequencing. Hotspot mutations in the RAS family were analyzed by Luminex assay using the remaining eight cases. Subsequently, immunohistochemistry for KRAS, CRAF, BRAF, EGFR, ERK, MEK, and BRAFV600E was performed. RESULTS: A KRAS G12D missense mutation was detected in the DNA sequence of the tumor cells, but it was not detected in the stromal tissue. KRAS G12V and KRAS G12R mutations were detected in two and four cases, respectively. For immunohistochemistry, all the cases were EGFR, KRAS, BRAF, CRAF positive, one case was ERK negative,and one case was MEK and ERK negative, all the other remaining cases were MEK and ERK positive. CONCLUSION: KRAS mutation at codon 12 and the presence of MAPK/ERK pathway proteins were detected suggesting their association with tumorigenesis of adenomatoid odontogenic tumors.


Assuntos
Ameloblastoma/genética , Ameloblastoma/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Adolescente , Adulto , Criança , Pré-Escolar , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Adulto Jovem
13.
Hum Genet ; 138(1): 21-35, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30368668

RESUMO

RASopathies are a group of developmental disorders caused by mutations in genes that regulate the RAS/MAPK pathway and include Noonan syndrome (NS), Costello syndrome, cardiofaciocutaneous syndrome and other related disorders. Whole exome sequencing studies recently identified LZTR1, PPP1CB and MRAS as new causative genes in RASopathies. However, information on the phenotypes of LZTR1 mutation-positive patients and functional properties of the mutations are limited. To identify variants of LZTR1, PPP1CB, and MRAS, we performed a targeted next-generation sequencing and reexamined previously analyzed exome data in 166 patients with suspected RASopathies. We identified eight LZTR1 variants, including a de novo variant, in seven probands who were suspicious for NS and one known de novo PPP1CB variant in a patient with NS. One of the seven probands had two compound heterozygous LZTR1 variants, suggesting autosomal recessive inheritance. All probands with LZTR1 variants had cardiac defects, including hypertrophic cardiomyopathy and atrial septal defect. Five of the seven probands had short stature or intellectual disabilities. Immunoprecipitation of endogenous LZTR1 followed by western blotting showed that LZTR1 bound to the RAF1-PPP1CB complex. Cells transfected with a small interfering RNA against LZTR1 exhibited decreased levels of RAF1 phosphorylated at Ser259. These are the first results to demonstrate LZTR1 in association with the RAF1-PPP1CB complex as a component of the RAS/MAPK pathway.


Assuntos
Biomarcadores/análise , Mutação , Síndrome de Noonan/genética , Proteína Fosfatase 1/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Fatores de Transcrição/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Exoma , Feminino , Seguimentos , Humanos , Masculino , Síndrome de Noonan/metabolismo , Síndrome de Noonan/patologia , Fenótipo , Prognóstico , Ligação Proteica , Proteína Fosfatase 1/genética , Proteínas Proto-Oncogênicas c-raf/genética , Fatores de Transcrição/genética , Adulto Jovem
14.
Mol Carcinog ; 58(4): 554-564, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30520151

RESUMO

Emerging research has indicated that circular RNAs (circRNAs), a novel class of non-coding RNAs, play a vital role in human tumorigenesis and progression. Our previous results suggested that hsa_circ_0006528 (circ_0006528), a circRNA with an unknown function, mediates adriamycin resistance in human breast cancer cells. However, the role of circ_0006528 in breast cancer progression remains unknown. Here, we investigated the probable involvement of circ_0006528 in breast cancer. We analyzed a cohort of 97 patients and found that circ_0006528 expression was significantly upregulated in human breast cancer tissues compared with that in adjacent non-tumorous tissues and was significantly associated with advanced tumor-node-metastasis (TNM) stage and poor prognosis. In addition, we found that in breast cancer cells, circ_0006528 could promote DNA synthesis and cell proliferation, invasion, and migration. Downregulating circ_0006528 induced G2 phase arrest and cell apoptosis. Further mechanistic studies revealed that circ_0006528 could sponge endogenous miR-7-5p and inhibit its activity. We also identified Raf1, which activates the MAPK/ERK signaling pathway, as a target of miR-7-5p and determined that circ_0006528 promotes breast cancer growth, invasion, and migration by promoting the expression of Raf1 and activates the MAPK/ERK pathway. Thus, this study provides the first evidence of the circ_0006528/miR-7-5p/Raf1/MEK/ERK regulatory network in the development of breast cancer and suggests that circ_0006528 is a potential therapeutic target and prognostic predictor for breast cancer.


Assuntos
Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , RNA/genética , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proliferação de Células , Progressão da Doença , Feminino , Seguimentos , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Prognóstico , Proteínas Proto-Oncogênicas c-raf/genética , Transdução de Sinais , Células Tumorais Cultivadas
15.
Mol Biol Cell ; 30(4): 506-523, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30586319

RESUMO

Activation of the epidermal growth factor (EGF) receptor (EGFR) at the cell surface initiates signaling through the RAS-RAF-MAPK/ERK1/2 pathway and receptor endocytosis. Whether this signaling continues from endosomes remains unclear, because RAS is predominantly located on the plasma membrane, and the localization of endogenous RAF kinases, downstream effectors of RAS, is not defined. To examine RAF localization, we labeled endogenous RAF1 with mVenus using gene editing. From 10 to 15% of RAF1-mVenus (<2000 molecules/cell), which was initially entirely cytosolic, transiently translocated to the plasma membrane after EGF stimulation. Following an early burst of translocation, the membrane-associated RAF1-mVenus was undetectable by microscopy or subcellular fractionation, and this pool was estimated to be <200 molecules per cell. In contrast, persistent EGF-dependent translocation of RAF1-mVenus to the plasma membrane was driven by the RAF inhibitor sorafenib, which increases the affinity of Ras-GTP:RAF1 interactions. RAF1-mVenus was not found in EGFR-containing endosomes under any conditions. Computational modeling of RAF1 dynamics revealed that RAF1 membrane abundance is controlled most prominently by association and dissociation rates from RAS-GTP and by RAS-GTP concentration. The model further suggested that the relatively protracted activation of the RAF-MEK1/2-ERK1/2 module, in comparison with RAF1 membrane localization, may involve multiple rounds of cytosolic RAF1 rebinding to active RAS at the membrane.


Assuntos
Fator de Crescimento Epidérmico/farmacologia , Proteínas Proto-Oncogênicas c-raf/metabolismo , Coloração e Rotulagem , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Fluorescência , Células HeLa , Humanos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Sorafenibe/farmacologia , Frações Subcelulares/metabolismo , Fatores de Tempo
16.
Chem Commun (Camb) ; 55(1): 111-114, 2018 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-30515494

RESUMO

We report the first supramolecular stabilizers of the interaction between 14-3-3ζ and two of its effectors, Tau and C-Raf, which are involved in neurodegenerative diseases and proliferative signal transduction, respectively. These supramolecular ligands open up an opportunity to modulate functions of 14-3-3 with these effectors.


Assuntos
Proteínas 14-3-3/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas tau/metabolismo , Proteínas 14-3-3/química , Sítios de Ligação , Dimerização , Humanos , Ligantes , Simulação de Dinâmica Molecular , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-raf/química , Transdução de Sinais , Proteínas tau/química
17.
Proc Natl Acad Sci U S A ; 115(50): E11661-E11670, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30478051

RESUMO

Long noncoding RNAs (lncRNAs) function through a diverse array of mechanisms that are not presently fully understood. Here, we sought to find lncRNAs differentially regulated in cancer cells resistant to either TNF-related apoptosis-inducing ligand (TRAIL) or the Mcl-1 inhibitor UMI-77, agents that act through the extrinsic and intrinsic apoptotic pathways, respectively. This work identified a commonly up-regulated lncRNA, ovarian adenocarcinoma-amplified lncRNA (OVAAL), that conferred apoptotic resistance in multiple cancer types. Analysis of clinical samples revealed OVAAL expression was significantly increased in colorectal cancers and melanoma in comparison to the corresponding normal tissues. Functional investigations showed that OVAAL depletion significantly inhibited cancer cell proliferation and retarded tumor xenograft growth. Mechanically, OVAAL physically interacted with serine/threonine-protein kinase 3 (STK3), which, in turn, enhanced the binding between STK3 and Raf-1. The ternary complex OVAAL/STK3/Raf-1 enhanced the activation of the RAF protooncogene serine/threonine-protein kinase (RAF)/mitogen-activated protein kinase kinase 1 (MEK)/ERK signaling cascade, thus promoting c-Myc-mediated cell proliferation and Mcl-1-mediated cell survival. On the other hand, depletion of OVAAL triggered cellular senescence through polypyrimidine tract-binding protein 1 (PTBP1)-mediated p27 expression, which was regulated by competitive binding between OVAAL and p27 mRNA to PTBP1. Additionally, c-Myc was demonstrated to drive OVAAL transcription, indicating a positive feedback loop between c-Myc and OVAAL in controlling tumor growth. Taken together, these results reveal that OVAAL contributes to the survival of cancer cells through dual mechanisms controlling RAF/MEK/ERK signaling and p27-mediated cell senescence.


Assuntos
Senescência Celular/genética , Senescência Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Sistema de Sinalização das MAP Quinases , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Xenoenxertos , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Nus , Estabilidade Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo
18.
Cell Cycle ; 17(24): 2666-2683, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30382763

RESUMO

Multiple myeloma (MM) is a cancer that occurs in plasma cells, which fall under the category of white blood cells that are in charge of antibody production. According to previous studies, microRNA-497 (miR-497) functions as a tumor suppressor in several types of cancer, including gastric cancer and colorectal cancer. Therefore, the present study aims to investigate the effects of miR-497 on cellular function of human MM cells through the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway by targeting Raf-1. The differentially expressed genes and miRs in MM, and the relationship between the miR and gene were verified. It was found that Raf-1 was a target gene of miR-497. The data obtained from MM tissues showed increased Raf-1 level and decreased miR-497 level. MM cells were treated with mimic, inhibitor and siRNA in order to evaluate the role of miR-497, Raf-1 and MAPK/ERK in MM. The expression pattern of miR-497, Raf-1, ERK1/2, survivin, B-cell lymphoma-2 (Bcl-2) and BCL2-Associated X (Bax) as well as the extent of ERK1/2 phosphorylation were determined. Retored miR-497 and si-Raf-1 resulted in increases in the Bax expression and cell apoptosis and decreases in the expressions of Raf-1, MEK-2, survivin, Bcl-2, along with the extent of ERK1/2 phosphorylation. In addition, the biological function evaluations of MM cells revealed that miR-497 mimic or si-Raf-1 led to suppression in cell proliferation, invasion and migration. In conclusion, our results have demonstrated that miR-497 targets Raf-1 in order to inhibit the progression of MM by blocking the MAPK/ERK signaling pathway.


Assuntos
MicroRNAs/metabolismo , Mieloma Múltiplo/patologia , Proteínas Proto-Oncogênicas c-raf/metabolismo , Animais , Antagomirs/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mieloma Múltiplo/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo
19.
Biochem Soc Trans ; 46(6): 1393-1406, 2018 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-30381334

RESUMO

RAF (rapidly accelerated fibrosarcoma) Ser/Thr kinases (ARAF, BRAF, and CRAF) link the RAS (rat sarcoma) protein family with the MAPK (mitogen-activated protein kinase) pathway and control cell growth, differentiation, development, aging, and tumorigenesis. Their activity is specifically modulated by protein-protein interactions, post-translational modifications, and conformational changes in specific spatiotemporal patterns via various upstream regulators, including the kinases, phosphatase, GTPases, and scaffold and modulator proteins. Dephosphorylation of Ser-259 (CRAF numbering) and dissociation of 14-3-3 release the RAF regulatory domains RAS-binding domain and cysteine-rich domain for interaction with RAS-GTP and membrane lipids. This, in turn, results in RAF phosphorylation at Ser-621 and 14-3-3 reassociation, followed by its dimerization and ultimately substrate binding and phosphorylation. This review focuses on structural understanding of how distinct binding partners trigger a cascade of molecular events that induces RAF kinase activation.


Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas A-raf/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Animais , Humanos , Proteínas Quinases Ativadas por Mitógeno/genética , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas A-raf/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/genética
20.
Bull Exp Biol Med ; 166(2): 257-259, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30488209

RESUMO

We analyzed association of the levels of VEGFA, RAF1, and mTOR gene expression in the tissue of clear-cell renal cell carcinoma (ccRCC) with tumor metastasizing. Significant association with metastases was found only for VEGFA gene: OR=6.641, 95%CI=2.111-20.696. The risk of metastasis associated with reduced expression of VEGFA gene - 2.467, 95%CI=1.238-4.915. An association of VEGFA gene expression with the time to the metastasis appearance was revealed (p=0.0005). Reduced expression of the VEGFA gene is associated with reduction of the time to metastasis appearance; the median of this time is shifted from 46 to 2 months. Analysis of tumor samples with reduced expression of the VEGFA gene revealed association of increased expression of RAF1 (p=0.003) and mTOR genes (p=0.038) with metastasis.


Assuntos
Carcinoma de Células Renais/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Proteínas Proto-Oncogênicas c-raf/genética , Serina-Treonina Quinases TOR/genética , Fator A de Crescimento do Endotélio Vascular/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/cirurgia , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Neoplasias Renais/cirurgia , Metástase Neoplásica , Proteínas Proto-Oncogênicas c-raf/metabolismo , Curva ROC , Risco , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismo
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