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1.
J Clin Pathol ; 73(2): 116-119, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31506288

RESUMO

Beyond targeted therapy for patients with BRAF-mutated melanomas and immunotherapy in patients lacking BRAF mutations, anti-MEK therapy has been proposed in patients with advanced melanomas harbouring BRAF fusions. BRAF fusions diagnosis in patients with advanced melanomas is the subject of the present study. Using BRAF fluorescent in situ hybridisation (FISH), we searched for BRAF fusions in 74 samples of 66 patients with advanced BRAF/NRAS/KIT wild-type melanomas. We identified 2/66 (3%) patients with BRAF fusions in a brain metastasis of one patient and in a lymph node metastasis and in a cutaneous metastasis for the second patient with 90%-95% of tumour nuclei containing isolated 3'-BRAF FISH signals. As a result, we conclude that BRAF FISH in patients with advanced BRAF/NRAS/KIT wild-type melanomas is a valuable and easy-to-perform test to diagnose BRAF fusions and to identify patients who could benefit of anti-MEK targeted therapy.


Assuntos
Biomarcadores Tumorais/genética , GTP Fosfo-Hidrolases/genética , Fusão Gênica , Hibridização in Situ Fluorescente , Melanoma/genética , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-kit/genética , Neoplasias Cutâneas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Feminino , Humanos , Masculino , Melanoma/tratamento farmacológico , Melanoma/enzimologia , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Terapia de Alvo Molecular , Seleção de Pacientes , Medicina de Precisão , Valor Preditivo dos Testes , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/enzimologia
2.
Nat Commun ; 10(1): 5143, 2019 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-31723142

RESUMO

Molecular determinants governing the evolution of tumor subclones toward phylogenetic branches or fixation remain unknown. Using sequencing data, we model the propagation and selection of clones expressing distinct categories of BRAF mutations to estimate their evolutionary trajectories. We show that strongly activating BRAF mutations demonstrate hard sweep dynamics, whereas mutations with less pronounced activation of the BRAF signaling pathway confer soft sweeps or are subclonal. We use clonal reconstructions to estimate the strength of "driver" selection in individual tumors. Using tumors cells and human-derived murine xenografts, we show that tumor sweep dynamics can significantly affect responses to targeted inhibitors of BRAF/MEK or DNA damaging agents. Our study uncovers patterns of distinct BRAF clonal evolutionary dynamics and nominates therapeutic strategies based on the identity of the BRAF mutation and its clonal composition.


Assuntos
Evolução Clonal/genética , Neoplasias/genética , Proteínas Proto-Oncogênicas B-raf/genética , Adenocarcinoma de Pulmão/patologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Dano ao DNA , Dosagem de Genes , Loci Gênicos , Humanos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação/genética , Fenótipo , Inibidores de Proteínas Quinases/farmacologia
3.
Nat Med ; 25(9): 1422-1427, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31406350

RESUMO

TRK fusions are found in a variety of cancer types, lead to oncogenic addiction, and strongly predict tumor-agnostic efficacy of TRK inhibition1-8. With the recent approval of the first selective TRK inhibitor, larotrectinib, for patients with any TRK-fusion-positive adult or pediatric solid tumor, to identify mechanisms of treatment failure after initial response has become of immediate therapeutic relevance. So far, the only known resistance mechanism is the acquisition of on-target TRK kinase domain mutations, which interfere with drug binding and can potentially be addressable through second-generation TRK inhibitors9-11. Here, we report off-target resistance in patients treated with TRK inhibitors and in patient-derived models, mediated by genomic alterations that converge to activate the mitogen-activated protein kinase (MAPK) pathway. MAPK pathway-directed targeted therapy, administered alone or in combination with TRK inhibition, re-established disease control. Experimental modeling further suggests that upfront dual inhibition of TRK and MEK may delay time to progression in cancer types prone to the genomic acquisition of MAPK pathway-activating alterations. Collectively, these data suggest that a subset of patients will develop off-target mechanisms of resistance to TRK inhibition with potential implications for clinical management and future clinical trial design.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Proteínas de Fusão Oncogênica/genética , Receptor trkA/genética , Adolescente , Adulto , Animais , Benzamidas/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Ácidos Nucleicos Livres/efeitos dos fármacos , Ácidos Nucleicos Livres/genética , Criança , Ensaios Clínicos como Assunto , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Xenoenxertos , Humanos , Imidazóis/administração & dosagem , Indazóis/administração & dosagem , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Terapia de Alvo Molecular , Neoplasias/genética , Neoplasias/patologia , Oximas/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Pirazóis/administração & dosagem , Piridonas/administração & dosagem , Pirimidinas/administração & dosagem , Pirimidinonas/administração & dosagem , Adulto Jovem
4.
Zhonghua Fu Chan Ke Za Zhi ; 54(8): 541-547, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31461811

RESUMO

Objective: To detect phosphorylated-extracellular signal-regulated kinase (p-ERK1/2) protein expression in epithelial ovarian cancer and cell lines, and to examine the effects of mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor AZD6244 on cell proliferation, apoptosis as well as cell cycle of ovarian cancer cells. To explore the function and significance of MAPK/extracellular signal-regulated kinase (ERK) signaling pathway in the development of ovarian cancer. Methods: (1) A total of 104 cases of patients with ovarian cancer who accepted the treatment of gynecological surgery and being confirmed by pathological examination in First Affiliated Hospital, Dalian Medical University from January 2004 to December 2013 were selected. The expressions of p-ERK1/2 protein were detected by immunohistochemistry in ovarian cancer specimens, and the relationship between the expressions of p-ERK1/2 and the clinical features of patients was analyzed. (2) p-ERK1/2 and other related proteins were determined by western blot in various ovarian cancer cells, including SKOV3, OV2008, C13, A2780S, A2780CP, OVCAR4, OVCAR5, OVCAR8 and CAOV3 treated with or without MEK inhibitor. The cellular proliferation, apoptosis and cell cycle of ovarian cancer cells after treatment with MEK inhibitor were analyzed by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry, respectively. Results: (1) The immunohistochemical method showed that p-ERK1/2 between low grade serous carcinoma and clear cell carcinoma were not significantly higher expressed (P>0.05) . However, a lower level of the p-ERK1/2 expression were observed among high grade serous carcinoma, mucinous carcinoma and endometrioid carcinoma (all P<0.05) . There was no significant correlation between the protein expression of p-ERK1/2 and patients' age, pathological stage of surgery, and preoperative serum CA(125) level (P>0.05). (2) Western blot showed that the protein p-ERK1/2 was widely expressed in various ovarian cancer cell lines such as SKOV3, OV2008, C13, A2780S, A2780CP, OVCAR4, OVCAR5, OVCAR8 and CAOV3. After treatment with AZD6244 (5, 10 µmol/L), the level of p-ERK1/2 in OVCAR5 and OVCAR8 decreased significantly in dose-dependent manner. Additionally, we found a reduction of the expression level of cyclin D1, caspase-3 and appeared cleaved poly adenosine diphosphate ribose polymerase (PARP) in OVCAR5 and OVCAR8, compared with control groups. MTT assays showed that OVCAR5, OVCAR8 and A2780S were differently inhibited in the dose-dependent manner after being treated with different concentrations of AZD6244 (0, 2.5, 5, 10, 25, 50 and 100 µmol/L, all P<0.05). Further tested by flow cytometry, the results showed that AZD6244 (5, 10 µmol/L) was able to induce the apoptosis of OVCAR5, OVCAR8 and A2780S, as well as G(0)/G(1) phase arrest, both in a dose-dependent manner (P<0.05). Conclusions: As the main active and functional unit of MAPK/ERK signaling pathway, p-ERK1/2 protein is expressed in both the tissues and various ovarian cancer cell lines. AZD6244 could down-regulated the expression of p-ERK1/2 in ovarian cancer cells, accompanied by the decreased proliferation and increased cell apoptosis of ovarian cancer cells. In conclusion, MAPK/ERK signaling pathway might play a role in the development and progression of ovarian cancer, and may be provide a novel option for molecular targeted therapies of the disease.


Assuntos
Carcinoma Epitelial do Ovário , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias Ovarianas , Apoptose/efeitos dos fármacos , Carcinoma Epitelial do Ovário/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Proteínas Quinases Ativadas por Mitógeno/genética , Neoplasias Ovarianas/genética
5.
Nat Med ; 25(7): 1116-1122, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31263281

RESUMO

The treatment of lymphatic anomaly, a rare devastating disease spectrum of mostly unknown etiologies, depends on the patient manifestations1. Identifying the causal genes will allow for developing affordable therapies in keeping with precision medicine implementation2. Here we identified a recurrent gain-of-function ARAF mutation (c.640T>C:p.S214P) in a 12-year-old boy with advanced anomalous lymphatic disease unresponsive to conventional sirolimus therapy and in another, unrelated, adult patient. The mutation led to loss of a conserved phosphorylation site. Cells transduced with ARAF-S214P showed elevated ERK1/2 activity, enhanced lymphangiogenic capacity, and disassembly of actin skeleton and VE-cadherin junctions, which were rescued using the MEK inhibitor trametinib. The functional relevance of the mutation was also validated by recreating a lymphatic phenotype in a zebrafish model, with rescue of the anomalous phenotype using a MEK inhibitor. Subsequent therapy of the lead proband with a MEK inhibitor led to dramatic clinical improvement, with remodeling of the patient's lymphatic system with resolution of the lymphatic edema, marked improvement in his pulmonary function tests, cessation of supplemental oxygen requirements and near normalization of daily activities. Our results provide a representative demonstration of how knowledge of genetic classification and mechanistic understanding guides biologically based medical treatments, which in our instance was life-saving.


Assuntos
Anormalidades Linfáticas/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Mutação , Proteínas Proto-Oncogênicas A-raf/genética , Piridonas/uso terapêutico , Pirimidinonas/uso terapêutico , Adulto , Animais , Criança , Feminino , Células HEK293 , Humanos , Anormalidades Linfáticas/tratamento farmacológico , Masculino , Sequenciamento Completo do Exoma , Peixe-Zebra
6.
Biomed Res Int ; 2019: 5962014, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31355271

RESUMO

Background: Oxidative stress due to reactive oxygen species plays a central role in pathophysiology of neurodegenerative diseases. Inhibition of mitogen-activated protein kinase (MAPK) cascades attenuates the oxidative induced cell stress and behaves as potential neuroprotection agent. Materials and Methods: In this study, we evaluate hydrogen peroxide induced neural cell stress and determine how different MAPK inhibitors restore the cell damage. Results: The results indicated that oxidative stress induced by neural cell damage commonly exists, and MAPK inhibitors partially and selectively attenuated the cell damage by reducing ROS production and cell apoptosis. The cultured neurons are more susceptible to hydrogen peroxide than subculture cells. Conclusion: We conclude that the essential role of different MAPK inhibitors is to attenuate the hydrogen peroxide induced neuronal cell damage. Those data broaden the implication between individual neural cells and different MAPK inhibitors and give clues for oxidative stress induced neural diseases.


Assuntos
Inibidores Enzimáticos/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Antracenos/farmacologia , Butadienos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Imidazóis/farmacologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/patologia , Nitrilos/farmacologia , Células PC12 , Cultura Primária de Células , Piridinas/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo
7.
BMC Cancer ; 19(1): 502, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138163

RESUMO

BACKGROUND: Every biological experiment requires a choice of throughput balanced against physiological relevance. Most primary drug screens neglect critical parameters such as microenvironmental conditions, cell-cell heterogeneity, and specific readouts of cell fate for the sake of throughput. METHODS: Here we describe a methodology to quantify proliferation and viability of single cells in 3D culture conditions by leveraging automated microscopy and image analysis to facilitate reliable and high-throughput measurements. We detail experimental conditions that can be adjusted to increase either throughput or robustness of the assay, and we provide a stand alone image analysis program for users who wish to implement this 3D drug screening assay in high throughput. RESULTS: We demonstrate this approach by evaluating a combination of RAF and MEK inhibitors on melanoma cells, showing that cells cultured in 3D collagen-based matrices are more sensitive than cells grown in 2D culture, and that cell proliferation is much more sensitive than cell viability. We also find that cells grown in 3D cultured spheroids exhibit equivalent sensitivity to single cells grown in 3D collagen, suggesting that for the case of melanoma, a 3D single cell model may be equally effective for drug identification as 3D spheroids models. The single cell resolution of this approach enables stratification of heterogeneous populations of cells into differentially responsive subtypes upon drug treatment, which we demonstrate by determining the effect of RAK/MEK inhibition on melanoma cells co-cultured with fibroblasts. Furthermore, we show that spheroids grown from single cells exhibit dramatic heterogeneity to drug response, suggesting that heritable drug resistance can arise stochastically in single cells but be retained by subsequent generations. CONCLUSION: In summary, image-based analysis renders cell fate detection robust, sensitive, and high-throughput, enabling cell fate evaluation of single cells in more complex microenvironmental conditions.


Assuntos
Fibroblastos/citologia , Processamento de Imagem Assistida por Computador/métodos , Melanoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Esferoides Celulares/citologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Fibroblastos/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Melanoma/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Análise de Célula Única , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Microambiente Tumoral , Quinases raf/antagonistas & inibidores
8.
Nat Commun ; 10(1): 2197, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097693

RESUMO

In colorectal cancer (CRC), aberrant Wnt signalling is essential for tumorigenesis and maintenance of cancer stem cells. However, how other oncogenic pathways converge on Wnt signalling to modulate stem cell homeostasis in CRC currently remains poorly understood. Using large-scale compound screens in CRC, we identify MEK1/2 inhibitors as potent activators of Wnt/ß-catenin signalling. Targeting MEK increases Wnt activity in different CRC cell lines and murine intestine in vivo. Truncating mutations of APC generated by CRISPR/Cas9 strongly synergize with MEK inhibitors in enhancing Wnt responses in isogenic CRC models. Mechanistically, we demonstrate that MEK inhibition induces a rapid downregulation of AXIN1. Using patient-derived CRC organoids, we show that MEK inhibition leads to increased Wnt activity, elevated LGR5 levels and enrichment of gene signatures associated with stemness and cancer relapse. Our study demonstrates that clinically used MEK inhibitors inadvertently induce stem cell plasticity, revealing an unknown side effect of RAS pathway inhibition.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Antineoplásicos/uso terapêutico , Biópsia , Sistemas CRISPR-Cas/genética , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Plasticidade Celular/efeitos dos fármacos , Neoplasias Colorretais/patologia , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Intestinos/citologia , Intestinos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteômica , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/metabolismo
9.
PLoS Pathog ; 15(4): e1007728, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30998777

RESUMO

Plant viruses have evolved multiple strategies to overcome host defense to establish an infection. Here, we identified two components of a host mitogen-activated protein kinase (MAPK) cascade, MKK2 and MPK4, as bona fide targets of the ßC1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNV). ßC1 interacts with the kinase domain of MKK2 and inhibits its activity. In vivo, ßC1 suppresses flagellin-induced MAPK activation and downstream responses by targeting MKK2. Furthermore, ßC1 also interacts with MPK4 and inhibits its kinase activity. TYLCCNV infection induces the activation of the MAPK cascade, mutation in MKK2 or MPK4 renders the plant more susceptible to TYLCCNV, and can complement the lack of ßC1. This work shows for the first time that a plant virus both activates and suppresses a MAPK cascade, and the discovery of the ability of ßC1 to selectively interfere with the host MAPK activation illustrates a novel virulence function and counter-host defense mechanism of geminiviruses.


Assuntos
Arabidopsis/imunologia , Geminiviridae/imunologia , Interações Hospedeiro-Patógeno/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Tabaco/imunologia , Proteínas Virais/metabolismo , Arabidopsis/metabolismo , Arabidopsis/virologia , Proteínas de Arabidopsis/antagonistas & inibidores , Geminiviridae/metabolismo , Geminiviridae/patogenicidade , Fosforilação , Tabaco/metabolismo , Tabaco/virologia
10.
Expert Opin Drug Saf ; 18(5): 381-392, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30977681

RESUMO

INTRODUCTION: Selective inhibition of the MAPK pathway with BRAF and MEK inhibitors has emerged as a key component of the treatment of BRAF-mutant unresectable/locally advanced metastatic melanoma. AREAS COVERED: Current data are presented on the efficacy and safety of BRAFi + MEKi combination therapy (dabrafenib/trametinib, vemurafenib/cobimetinib, and encorafenib/binimetinib) from phase I, II, and III trials in the unresectable/locally advanced metastatic setting, as well as neoadjuvant and adjuvant applications. The theoretical basis, pre-clinical findings, clinical trial results and current ongoing clinical studies of combined BRAF/MEK inhibition with immunotherapy, also known as 'triplet therapy,' are also explored. EXPERT OPINION: Combination therapy with BRAF and MEK inhibitors dramatically improves response rates, progression-free survival and overall survival in patients with BRAF-mutant metastatic melanoma compared to historical treatments such as chemotherapy. Some serious adverse effects, including cutaneous squamous cell carcinoma, are attenuated with combination therapy, while less severe and reversible effects including pyrexia, left ventricular dysfunction, and ocular events can be more common with combination therapy. Existing data are insufficient to recommend triplet therapy, or a particular treatment sequence, with respect to BRAF and MEK inhibitors and immune therapies, though results from multiple ongoing trials are anticipated.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Intervalo Livre de Doença , Humanos , Imunoterapia/métodos , Melanoma/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Taxa de Sobrevida
11.
PLoS One ; 14(4): e0215398, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30978262

RESUMO

Vascular pathophysiological changes after haemorrhagic stroke, such as phenotypic modulation of the cerebral arteries and cerebral vasospasms, are associated with delayed cerebral ischemia (DCI) and poor outcome. The only currently approved drug treatment shown to reduce the risk of DCI and improve neurologic outcome after aneurysmal subarachnoid haemorrhage (SAH) is nimodipine, a dihydropyridine L-type voltage-gated Ca2+ channel blocker. MEK1/2 mediated transcriptional upregulation of contractile receptors, including endothelin-1 (ET-1) receptors, has previously been shown to be a factor in the pathology of SAH. The aim of the study was to compare intrathecal and subcutaneous treatment regimens of nimodipine and intrathecal treatment regimens of U0126, a MEK1/2 inhibitor, in a single injection experimental rat SAH model with post 48 h endpoints consisting of wire myography of cerebral arteries, flow cytometry of cerebral arterial tissue and behavioural evaluation. Following ET-1 concentration-response curves, U0126 exposed arteries had a significantly lower ET-1max than vehicle arteries. Arteries from both the intrathecal- and subcutaneous nimodipine treated animals had significantly higher ET-1max contractions than the U0126 arteries. Furthermore, Ca2+ concentration response curves (precontracted with ET-1 and in the presence of nimodipine) showed that nimodipine treatment could result in larger nimodipine insensitive contractions compared to U0126. Flow cytometry showed decreased protein expression of the ETB receptor in U0126 treated cerebral vascular smooth muscle cells compared to vehicle. Only U0126 treatment lowered ET-1max contractions and ETB receptor levels, as well as decreased the contractions involving nimodipine-insensitive Ca2+ channels, when compared to both intrathecal and subcutaneous nimodipine treatment. This indicate that targeting gene expression might be a better strategy than blocking specific receptors or ion channels in future treatments of SAH.


Assuntos
Butadienos/farmacologia , Nitrilos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Hemorragia Subaracnóidea/tratamento farmacológico , Hemorragia Subaracnóidea/fisiopatologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Artérias Cerebrais/efeitos dos fármacos , Artérias Cerebrais/fisiopatologia , Modelos Animais de Doenças , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiopatologia , Nimodipina/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor de Endotelina B/genética , Receptor de Endotelina B/fisiologia , Hemorragia Subaracnóidea/genética , Regulação para Cima/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia
12.
Phytomedicine ; 61: 152813, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31035049

RESUMO

BACKGROUND: Colorectal cancer is one of the most common causes of cancer death worldwide. Unfortunately, chemotherapies are limited due to many complications and development of resistance and recurrence. The T-lymphokine-activated killer cell-originated protein kinase (TOPK) is highly expressed and activated in colon cancer, and plays an important role in inflammation, proliferation, and survival of cancer cells. Therefore, suppressing TOPK activity and its downstream signaling cascades is considered to be a rational therapeutic/preventive strategy against colon cancers. PURPOSE: 3-Deoxysappanchalcone (3-DSC), a component of Caesalpinia sappan L., is a natural oriental medicine. In this study, we investigated the effects of 3-DSC on colon cancer cell growth and elucidated its underlying molecular mechanism of targeting TOPK. STUDY DESIGN AND METHODS: To evaluate the effects of 3-DSC against colon cancer, we performed cell proliferation assays, propidium iodide- and annexin V-staining analyses and Western blotting. Targeting TOPK by 3-DSC was identified by a kinase-binding assay and computational docking models. RESULTS: 3-DSC inhibited the kinase activity of TOPK, but not mitogen-activated protein kinase (MEK). The direct binding of 3-DSC with TOPK was explored using a computational docking model and binding assay in vitro and ex vivo. 3-DSC inhibited colon cancer cell proliferation and anchorage-independent cell growth, and induced G2/M cell cycle arrest and apoptosis. Treatment of colon cancer cells with 3-DSC induced expression of protein that are involved in cell cycle (cyclin B1) and apoptosis (cleaved-PARP, cleaved-caspase-3, and cleaved-caspase-7), and suppressed protein expressions of extracellular signal-regulated kinase (ERK)-1/2, ribosomal S6 kinase (RSK), and c-Jun, which are regulated by the upstream kinase, TOPK. CONCLUSION: 3-DSC suppresses colon cancer cell growth by directly targeting the TOPK- mediated signaling pathway.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Chalconas/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chalconas/química , Neoplasias do Colo/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/química , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular/métodos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos
13.
Sci Transl Med ; 11(483)2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867319

RESUMO

Mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitors have failed to show clinical benefit in Kirsten rat sarcoma (KRAS) mutant lung cancer due to various resistance mechanisms. To identify differential therapeutic sensitivities between epithelial and mesenchymal lung tumors, we performed in vivo small hairpin RNA screens, proteomic profiling, and analysis of patient tumor datasets, which revealed an inverse correlation between mitogen-activated protein kinase (MAPK) signaling dependency and a zinc finger E-box binding homeobox 1 (ZEB1)-regulated epithelial-to-mesenchymal transition. Mechanistic studies determined that MAPK signaling dependency in epithelial lung cancer cells is due to the scaffold protein interleukin-17 receptor D (IL17RD), which is directly repressed by ZEB1. Lung tumors in multiple Kras mutant murine models with increased ZEB1 displayed low IL17RD expression, accompanied by MAPK-independent tumor growth and therapeutic resistance to MEK inhibition. Suppression of ZEB1 function with miR-200 expression or the histone deacetylase inhibitor mocetinostat sensitized resistant cancer cells to MEK inhibition and markedly reduced in vivo tumor growth, showing a promising combinatorial treatment strategy for KRAS mutant cancers. In human lung tumor samples, high ZEB1 and low IL17RD expression correlated with low MAPK signaling, presenting potential markers that predict patient response to MEK inhibitors.


Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Mutação/genética , Neoplasias/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptores de Interleucina-17/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Animais , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Células Epiteliais/patologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Mesoderma/patologia , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias/tratamento farmacológico , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico
14.
Nature ; 567(7749): 521-524, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30867592

RESUMO

Histiocytic neoplasms are a heterogeneous group of clonal haematopoietic disorders that are marked by diverse mutations in the mitogen-activated protein kinase (MAPK) pathway1,2. For the 50% of patients with histiocytosis who have BRAFV600 mutations3-5, RAF inhibition is highly efficacious and has markedly altered the natural history of the disease6,7. However, no standard therapy exists for the remaining 50% of patients who lack BRAFV600 mutations. Although ERK dependence has been hypothesized to be a consistent feature across histiocytic neoplasms, this remains clinically unproven and many of the kinase mutations that are found in patients who lack BRAFV600 mutations have not previously been biologically characterized. Here we show ERK dependency in histiocytoses through a proof-of-concept clinical trial of cobimetinib, an oral inhibitor of MEK1 and MEK2, in patients with histiocytoses. Patients were enrolled regardless of their tumour genotype. In parallel, MAPK alterations that were identified in treated patients were characterized for their ability to activate ERK. In the 18 patients that we treated, the overall response rate was 89% (90% confidence interval of 73-100). Responses were durable, with no acquired resistance to date. At one year, 100% of responses were ongoing and 94% of patients remained progression-free. Cobimetinib treatment was efficacious regardless of genotype, and responses were observed in patients with ARAF, BRAF, RAF1, NRAS, KRAS, MEK1 (also known as MAP2K1) and MEK2 (also known as MAP2K2) mutations. Consistent with the observed responses, the characterization of the mutations that we identified in these patients confirmed that the MAPK-pathway mutations were activating. Collectively, these data demonstrate that histiocytic neoplasms are characterized by a notable dependence on MAPK signalling-and that they are consequently responsive to MEK inhibition. These results extend the benefits of molecularly targeted therapy to the entire spectrum of patients with histiocytosis.


Assuntos
Azetidinas/uso terapêutico , Transtornos Histiocíticos Malignos/tratamento farmacológico , Transtornos Histiocíticos Malignos/enzimologia , Histiocitose/tratamento farmacológico , Histiocitose/enzimologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Piperidinas/uso terapêutico , Azetidinas/farmacologia , Transtornos Histiocíticos Malignos/genética , Transtornos Histiocíticos Malignos/patologia , Histiocitose/genética , Histiocitose/patologia , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mutação , Piperidinas/farmacologia , Intervalo Livre de Progressão , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/genética
15.
Mol Cell Proteomics ; 18(6): 1096-1109, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30890564

RESUMO

Targeted inhibition of mutated kinases using selective MAP kinase inhibitors in malignant melanoma often results in temporary improvement of clinical symptoms followed by rapid development of resistance. To gain insights in molecular processes that govern resistance, we performed SILAC-based quantitative proteomics profiling of vemurafenib-resistant and -sensitive melanoma cells. Among downregulated proteins in vemurafenib-resistant cell lines we detected multiple proteins involved in cytoskeletal organization and signaling, including the intermediate filament nestin, which was one of the most downregulated proteins. Previous studies showed that nestin is expressed in various types of solid tumors and its abundance correlates with malignant phenotype of transformed cells. However, the role of nestin in cancer cells regarding acquired resistance is still poorly understood. We performed CRISPR/Cas9 knockout of the nestin gene (NES) in vemurafenib-sensitive cells and showed that loss of nestin leads to increased cellular proliferation and colony formation upon treatment with BRAFV600E and MEK inhibitors. Moreover, nestin depletion led to increased invasiveness and metalloproteinase activity like the phenotype of melanoma cells with acquired resistance to the BRAF inhibitor. Finally, phosphoproteome analysis revealed that nestin depletion influenced signaling through integrin and PI3K/AKT/mTOR pathways and led to increased focal adhesion kinase abundance and phosphorylation. Taken together, our results reveal that nestin is associated with acquired vemurafenib resistance in melanoma cells.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Filamentos Intermediários/metabolismo , Melanoma/metabolismo , Nestina/metabolismo , Proteômica , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Filamentos Intermediários/efeitos dos fármacos , Metaloproteinases da Matriz/metabolismo , Melanoma/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ensaio Tumoral de Célula-Tronco , Vemurafenib/farmacologia
16.
Plant Sci ; 280: 416-423, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30824021

RESUMO

Cadmium (Cd) toxicity induces oxidative burst and leads to programmed cell death (PCD) in plant cells. The role of salicylic acid-induced protein kinase (SIPK) signaling pathway in Cd-induced oxidative stress was investigated in suspension-cultured tobacco (Nicotiana tabacum L. cv. Barley 21). The cells were pretreated with 40 µM PD98059 (inhibitor of MAPKK) and then exposed to 50 µM Cd for 24 h. The percentages of cell viability, apoptosis, necrosis, and the content of reactive oxygen species (ROS) were monitored by flow cytometry. Expression of PCD related gene (Hsr203J) and the contents of certain signaling molecules were measured as well. The results showed that Cd increased the expression of SIPK, Hsr203J, and CAT genes, the activities of catalase and caspase-3-like enzymes. Addition of PD98059 inhibitor reduced the expression of Hsr203J and CAT genes, decreased CAT activity, but increased ROS and SA contents, and caspase-3-like activity and apoptosis rate. The highest apoptosis level was accompanied by the highest level of Hsr203J gene expression. From the results it can be suggested that upon treatment of tobacco cells with Cd, internal SA content increased and induced the SIPK signaling pathway, thereby inhibited the antioxidant system and led to PCD.


Assuntos
Antioxidantes/metabolismo , Cádmio/toxicidade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Tabaco/fisiologia , Apoptose , Catalase/genética , Catalase/metabolismo , Esterases/efeitos dos fármacos , Esterases/genética , Flavonoides/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Estresse Oxidativo , Proteínas de Plantas/efeitos dos fármacos , Proteínas de Plantas/genética , Inibidores de Proteínas Quinases/farmacologia , Ácido Salicílico/metabolismo , Tabaco/efeitos dos fármacos , Tabaco/genética
17.
Skin Therapy Lett ; 24(1): 8-11, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30817881

RESUMO

Today, a number of treatment options are now available for metastatic melanoma. Within the last decade, the development of novel immunotherapies for cancer has significantly altered the course of the disease in patients with melanoma. With more patients receiving these potentially life-saving treatments, not only have we learned more about the interplay between the immune system and melanoma, but more importantly, which treatment options are most appropriate given the clinical picture.


Assuntos
Imunoterapia/métodos , Melanoma/tratamento farmacológico , Antígeno CTLA-4/antagonistas & inibidores , Humanos , Melanoma/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Metástase Neoplásica , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores
18.
Nat Med ; 25(4): 620-627, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30833748

RESUMO

Pancreatic ductal adenocarcinoma (PDA) was responsible for ~ 44,000 deaths in the United States in 2018 and is the epitome of a recalcitrant cancer driven by a pharmacologically intractable oncoprotein, KRAS1-4. Downstream of KRAS, the RAF→MEK→ERK signaling pathway plays a central role in pancreatic carcinogenesis5. However, paradoxically, inhibition of this pathway has provided no clinical benefit to patients with PDA6. Here we show that inhibition of KRAS→RAF→MEK→ERK signaling elicits autophagy, a process of cellular recycling that protects PDA cells from the cytotoxic effects of KRAS pathway inhibition. Mechanistically, inhibition of MEK1/2 leads to activation of the LKB1→AMPK→ULK1 signaling axis, a key regulator of autophagy. Furthermore, combined inhibition of MEK1/2 plus autophagy displays synergistic anti-proliferative effects against PDA cell lines in vitro and promotes regression of xenografted patient-derived PDA tumors in mice. The observed effect of combination trametinib plus chloroquine was not restricted to PDA as other tumors, including patient-derived xenografts (PDX) of NRAS-mutated melanoma and BRAF-mutated colorectal cancer displayed similar responses. Finally, treatment of a patient with PDA with the combination of trametinib plus hydroxychloroquine resulted in a partial, but nonetheless striking disease response. These data suggest that this combination therapy may represent a novel strategy to target RAS-driven cancers.


Assuntos
Autofagia/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas ras/metabolismo , Animais , Antígeno CA-19-9/metabolismo , Linhagem Celular Tumoral , Cloroquina/farmacologia , Humanos , Camundongos SCID , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Piridonas/farmacologia , Pirimidinonas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Neuropharmacology ; 149: 133-148, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30772375

RESUMO

Leptin is produced in the adipocytes and plays a pivotal role in regulation of energy balance by controlling appetite and metabolism. Leptin receptors are widely distributed in the brain, especially in the hypothalamus, hippocampus, and neocortex. The insular cortex (IC) processes gustatory and visceral information, which functionally correlate to feeding behavior. However, it is still an open issue whether and how leptin modulates IC neural activities. Our paired whole-cell patch-clamp recordings using IC slice preparations demonstrated that unitary inhibitory postsynaptic currents (uIPSCs) but not uEPSCs were potentiated by leptin in the connections between pyramidal (PNs) and fast-spiking neurons (FSNs). The leptin-induced increase in uIPSC amplitude was accompanied by a decrease in paired-pulse ratio. Under application of inhibitors of JAK2-PI3K but not MAPK pathway, leptin did not change uIPSC amplitude. Variance-mean analysis revealed that leptin increased the release probability but not the quantal size and the number of release site. These electrophysiological findings suggest that the leptin-induced uIPSC increase is mediated by activation of JAK2-PI3K pathway in presynaptic FSNs. An in vivo optical imaging revealed that leptin application decreased excitatory propagation in IC induced by electrical stimulation of IC. These leptin-induced effects were not observed under the low energy states: low glucose concentration (2.5 mM) in vitro and one-day-fasting condition in vivo. However, leptin enhanced uIPSCs under application of low glucose with an AMPK inhibitor. These results suggest that leptin suppresses IC excitation by facilitating GABA release in FSN→PN connections, which may not occur under a hunger state.


Assuntos
Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Janus Quinase 2/antagonistas & inibidores , Leptina/farmacologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Transdução de Sinais/efeitos dos fármacos , Animais , Córtex Cerebral/efeitos dos fármacos , Cromonas/farmacologia , Excitabilidade Cortical/efeitos dos fármacos , Estimulação Elétrica , Flavonoides/farmacologia , Neurônios GABAérgicos/efeitos dos fármacos , Glucose/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Morfolinas/farmacologia , Técnicas de Patch-Clamp , Cultura Primária de Células , Proteínas Quinases , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Ratos , Ratos Transgênicos , Rotenona/análogos & derivados , Rotenona/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Wortmanina/farmacologia
20.
Int J Oncol ; 54(3): 1123-1133, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30747223

RESUMO

Mutations affecting the Wnt/ß­catenin pathway have been identified in 26­40% of hepatocellular carcinoma (HCC) cases. Aberrant activation of this pathway leads to uncontrolled cell proliferation and survival. Thus, identifying Wnt/ß­catenin pathway inhibitors may benefit a subset of patients with HCC. In the present study, the effects of sorafenib and a MEK inhibitor on tumor growth and Wnt/ß­catenin signaling in HCC models were evaluated. A ß­catenin mutant and ß­catenin wild­type HCC models were treated once daily with i) 10 mg/kg sorafenib, ii) 15 mg/kg refametinib (or 25 mg/kg selumetinib), or iii) sorafenib/refametinib. Western blotting was employed to determine changes in biomarkers relevant to Wnt/ß­catenin signaling. Apoptosis, cell proliferation and ß­catenin localization were analyzed by immunohistochemistry. Sorafenib/refametinib markedly inhibited tumor growth and cell proliferation, and caused cell death in naïve and sorafenib­resistant HCC models. Despite similar total ß­catenin levels, significant reductions in phosphorylated (p)­RanBP3 Ser58, p­ß­catenin Tyr142, active ß­catenin and ß­catenin target genes were observed in sorafenib/refametinib­treated tumors. Greater levels of ß­catenin in sorafenib/refametinib­treated tumors were accumulated at the membrane, as compared with in the control. In vitro, sorafenib/refametinib inhibited the Wnt/ß­catenin pathway and suppressed Wnt­3A­induced p­low­density lipoprotein receptor­related protein 6 Ser1490, p­RanBP3 Ser58 and p­ß­catenin Tyr142 in HCC cells. Combination of sorafenib and refametinib inhibits the growth of naïve and sorafenib resistant HCC tumors in association with active suppression of ß­catenin signaling regardless of ß­catenin mutational status. Thus, the sorafenib/MEK inhibitor combination may represent an alternative treatment for patients with HCC whose tumors develop resistance to sorafenib therapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Benzimidazóis/administração & dosagem , Difenilamina/administração & dosagem , Difenilamina/análogos & derivados , Humanos , Masculino , Camundongos , Camundongos SCID , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Sorafenibe/administração & dosagem , Sulfonamidas/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto
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