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1.
Dermatol Clin ; 37(4): 409-423, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31466582

RESUMO

The incidence of metastatic melanoma continues to increase each decade. Although surgical treatment is often curative for localized stage I and stage II disease, the median survival for patients with distant metastases is less than 1 year. The last 2 decades have witnessed a breakthrough in therapeutic options with the development of immune checkpoint inhibitors, small molecule targeted therapy, and oncolytic viral therapy. This article provides an overview of the treatment options available for advanced melanoma, including chemotherapy, targeted therapy, immunotherapy, interleukin-2, and oncolytic viral agents.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Melanoma/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Antineoplásicos/uso terapêutico , Antígeno CTLA-4/antagonistas & inibidores , Humanos , Fatores Imunológicos/uso terapêutico , Interferon-alfa/uso terapêutico , Interleucina-2/análogos & derivados , Interleucina-2/uso terapêutico , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Melanoma/patologia , Melanoma/secundário , Metástase Neoplásica , Estadiamento de Neoplasias , Terapia Viral Oncolítica , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Recombinantes/uso terapêutico , Neoplasias Cutâneas/patologia
2.
Exp Mol Pathol ; 110: 104260, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31082388

RESUMO

Although the treatment of metastatic melanoma has been significantly improved by both anti-BRAF/MEK and checkpoint immunotherapies, resistance to these treatment modalities remains a substantial clinical problem. Multiple clinical studies are addressing the optimal sequencing of these agents in larger patient cohorts, but successful long-term individualized treatment will likely require the elucidation of resistance mechanisms from post-progression samples. Here, we describe a patient with BRAF-V600E-positive metastatic melanoma who was sequentially treated with BRAF/MEK inhibitors (dabrafenib/trametinib) and checkpoint inhibitor immunotherapy (nivolumab, followed by pembrolizumab). After the emergence of resistance, whole exome sequencing was performed, implicating MAP2K2 and B2M mutations in loss of response to anti-BRAF/MEK and anti-PD1 therapies, respectively.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase Quinases/antagonistas & inibidores , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Microglobulina beta-2/antagonistas & inibidores , Anticorpos Monoclonais Humanizados/administração & dosagem , Humanos , Imidazóis/administração & dosagem , MAP Quinase Quinase 2/genética , MAP Quinase Quinase Quinases/metabolismo , Masculino , Melanoma/genética , Pessoa de Meia-Idade , Mutação , Nivolumabe/administração & dosagem , Oximas/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Piridonas/administração & dosagem , Pirimidinonas/administração & dosagem , Neoplasias Cutâneas/genética , Falha de Tratamento , Microglobulina beta-2/genética
3.
Mol Carcinog ; 58(7): 1248-1259, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31100197

RESUMO

Purpurogallin is a natural compound that is extracted from nutgalls and oak bark and it possesses antioxidant, anticancer, and anti-inflammatory properties. However, the anticancer capacity of purpurogallin and its molecular target have not been investigated in esophageal squamous cell carcinoma (ESCC). Herein, we report that purpurogallin suppresses ESCC cell growth by directly targeting the mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling pathway. We found that purpurogallin inhibits anchorage-dependent and -independent ESCC growth. The results of in vitro kinase assays and cell-based assays indicated that purpurogallin also strongly attenuates the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and also directly binds to and inhibits MEK1 and MEK2 activity. Furthermore, purpurogallin contributed to S and G2 phase cell cycle arrest by reducing cyclin A2 and cyclin B1 expression and also induced apoptosis by activating poly (ADP ribose) polymerase (PARP). Notably, purpurogallin suppressed patient-derived ESCC tumor growth in an in vivo mouse model. These findings indicated that purpurogallin is a novel MEK1/2 inhibitor that could be useful for treating ESCC.


Assuntos
Antineoplásicos/farmacologia , Benzocicloeptenos/farmacologia , Neoplasias Esofágicas/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclina A2/biossíntese , Ciclina B1/biossíntese , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Camundongos , Preparações de Plantas/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Nat Commun ; 10(1): 2030, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31048689

RESUMO

Acquired resistance to MEK1/2 inhibitors (MEKi) arises through amplification of BRAFV600E or KRASG13D to reinstate ERK1/2 signalling. Here we show that BRAFV600E amplification and MEKi resistance are reversible following drug withdrawal. Cells with BRAFV600E amplification are addicted to MEKi to maintain a precise level of ERK1/2 signalling that is optimal for cell proliferation and survival, and tumour growth in vivo. Robust ERK1/2 activation following MEKi withdrawal drives a p57KIP2-dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death, selecting against those cells with amplified BRAFV600E. p57KIP2 expression is required for loss of BRAFV600E amplification and reversal of MEKi resistance. Thus, BRAFV600E amplification confers a selective disadvantage during drug withdrawal, validating intermittent dosing to forestall resistance. In contrast, resistance driven by KRASG13D amplification is not reversible; rather ERK1/2 hyperactivation drives ZEB1-dependent epithelial-to-mesenchymal transition and chemoresistance, arguing strongly against the use of drug holidays in cases of KRASG13D amplification.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Amplificação de Genes/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias/genética , Inibidores de Proteínas Quinases/uso terapêutico , Suspensão de Tratamento , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo
5.
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
6.
Cancer Res ; 79(9): 2352-2366, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30819666

RESUMO

Combinatorial inhibition of MEK1/2 and CDK4/6 is currently undergoing clinical investigation in NRAS-mutant melanoma. To prospectively map the landscape of resistance to this investigational regimen, we utilized a series of gain- and loss-of-function forward genetic screens to identify modulators of resistance to clinical inhibitors of MEK1/2 and CDK4/6 alone and in combination. First, we identified NRAS-mutant melanoma cell lines that were dependent on NRAS for proliferation and sensitive to MEK1/2 and CDK4/6 combination treatment. We then used a genome-scale ORF overexpression screen and a CRISPR knockout screen to identify modulators of resistance to each inhibitor alone or in combination. These orthogonal screening approaches revealed concordant means of achieving resistance to this therapeutic modality, including tyrosine kinases, RAF, RAS, AKT, and PI3K signaling. Activated KRAS was sufficient to cause resistance to combined MEK/CDK inhibition and to replace genetic depletion of oncogenic NRAS. In summary, our comprehensive functional genetic screening approach revealed modulation of resistance to the inhibition of MEK1/2, CDK4/6, or their combination in NRAS-mutant melanoma. SIGNIFICANCE: These findings reveal that NRAS-mutant melanomas can acquire resistance to genetic ablation of NRAS or combination MEK1/2 and CDK4/6 inhibition by upregulating activity of the RTK-RAS-RAF and RTK-PI3K-AKT signaling cascade.


Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , GTP Fosfo-Hidrolases/genética , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Melanoma/tratamento farmacológico , Proteínas de Membrana/genética , Mutação , Antineoplásicos/farmacologia , Apoptose , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Humanos , Melanoma/genética , Melanoma/patologia , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas
7.
J Biol Chem ; 294(21): 8664-8673, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-30858179

RESUMO

Most cancer cells are dependent on a network of deregulated signaling pathways for survival and are insensitive, or rapidly evolve resistance, to selective inhibitors aimed at a single target. For these reasons, drugs that target more than one protein (polypharmacology) can be clinically advantageous. The discovery of useful polypharmacology remains serendipitous and is challenging to characterize and validate. In this study, we developed a non-genetic strategy for the identification of pathways that drive cancer cell proliferation and represent exploitable signaling vulnerabilities. Our approach is based on using a multitargeted kinase inhibitor, SM1-71, as a tool compound to identify combinations of targets whose simultaneous inhibition elicits a potent cytotoxic effect. As a proof of concept, we applied this approach to a KRAS-dependent non-small cell lung cancer (NSCLC) cell line, H23-KRASG12C Using a combination of phenotypic screens, signaling analyses, and kinase inhibitors, we found that dual inhibition of MEK1/2 and insulin-like growth factor 1 receptor (IGF1R)/insulin receptor (INSR) is critical for blocking proliferation in cells. Our work supports the value of multitargeted tool compounds with well-validated polypharmacology and target space as tools to discover kinase dependences in cancer. We propose that the strategy described here is complementary to existing genetics-based approaches, generalizable to other systems, and enabling for future mechanistic and translational studies of polypharmacology in the context of signaling vulnerabilities in cancers.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/enzimologia , Neoplasias Pulmonares/epidemiologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Receptor de Insulina/antagonistas & inibidores , Receptores de Somatomedina/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Antígenos CD/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Células HCT116 , Humanos , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptor de Insulina/metabolismo , Receptores de Somatomedina/metabolismo
8.
Oncogene ; 38(25): 5076-5090, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30905967

RESUMO

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


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Terapia de Alvo Molecular , Compostos de Anilina/administração & dosagem , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Células HCT116 , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/genética , MAP Quinase Quinase 2/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Terapia de Alvo Molecular/métodos , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Transdução de Sinais/genética , Sulfonamidas/administração & dosagem , Células Tumorais Cultivadas
9.
J Exp Clin Cancer Res ; 38(1): 85, 2019 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-30777101

RESUMO

BACKGROUND: Despite the increasing progress in targeted and immune based-directed therapies for other solid organ malignancies, currently there is no targeted therapy available for TNBCs. A number of mechanisms have been reported both in pre-clinical and clinical settings that involve inherent, acquired and adaptive resistance to small molecule inhibitors. Here, we demonstrated a novel resistance mechanism in TNBC cells mediated by PDGFRß in response to JAK2 inhibition. METHODS: Multiple in vitro (subG1, western blotting, immunofluorescence, RT-PCR, Immunoprecipitation), in vivo and publically available datasets were used. RESULTS: We showed that TNBC cells exposed to MEK1/2-JAK2 inhibitors exhibit resistant colonies in anchorage-independent growth assays. Moreover, cells treated with various small molecule inhibitors including JAK2 promote PDGFRß upregulation. Using publically available databases, we showed that patients expressing high PDGFRß or its ligand PDGFB exhibit poor relapse-free survival upon chemotherapeutic treatment. Mechanistically we found that JAK2 expression controls steady state levels of PDGFRß. Thus, co-blockade of PDGFRß with JAK2 and MEK1/2 inhibitors completely eradicated resistant colonies in vitro. We found that triple-combined treatment had a significant impact on CD44+/CD24- stem-cell-like cells. Likewise, we found a significant tumor growth inhibition in vivo through intratumoral CD8+ T cells infiltration in a manner that is reversed by anti-CD8 antibody treatment. CONCLUSION: These findings reveal a novel regulatory role of JAK2-mediated PDGFRß proteolysis and provide an example of a PDGFRß-mediated resistance mechanism upon specific target inhibition in TNBC.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Janus Quinase 2/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Feminino , Humanos , Inibidores de Janus Quinases/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/metabolismo
10.
Biomed Pharmacother ; 109: 2548-2560, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30551515

RESUMO

Melanoma is one of the most aggressive and treatment-resistant tumors that responsible for majority of skin-cancer related deaths. Here we propose a combination of MEK inhibitor binimetinib with metformin as a promising therapy against human melanoma cells in vitro, including BRAF -mutated A375, Mel Z, and Mel IL cells, and NRAS-mutated Mel MTP and Mel Me cells. Additionally, we obtained two close to clinical practice models of melanoma progression. The first one was vemurafenib-resistant Mel IL/R melanoma cells with acquired resistance to BRAF inhibition-targeted therapy, and the second one was tumor spheroids, which are 3D in vitro model of small-size solid tumors in vivo. The cytotoxicity of binimetinib and metformin was synergistic in both 2D and 3D melanoma culture and mediated through apoptotic pathway. The combination reduced the number of melanoma-formed colonies, inhibited cell invasion and migration, and led to G0/G1 cell cycle arrest through cyclin D/CDK4/CDK6 pathway. The mechanism of metformin and binimetinib synergy in melanoma cells was associated with increased activation of p-AMPKα and decreased p-ERK, but not with alterations in p-mTOR. In summary, the combination of metformin and binimetinib resulted in stronger anti-proliferative effects on melanoma cells compared to binimetinib alone, and therefore could be promising for clinical applications.


Assuntos
Benzimidazóis/administração & dosagem , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Melanoma/enzimologia , Metformina/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Hipoglicemiantes/administração & dosagem , Melanoma/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem
11.
Artigo em Inglês | MEDLINE | ID: mdl-30348669

RESUMO

Cystic echinococcosis is a zoonosis caused by the larval stage of Echinococcus granulosus sensu lato There is an urgent need to develop new drugs for the treatment of this disease. In this study, we identified two new members of mitogen-activated protein kinase (MAPK) cascades, MKK3/6 and MEK1/2 homologs (termed EgMKK1 and EgMKK2, respectively), from E. granulosus sensu stricto Both EgMKK1 and EgMKK2 were expressed at the larval stages. As shown by yeast two-hybrid and coimmunoprecipitation analyses, EgMKK1 interacted with the previously identified Egp38 protein but not with EgERK. EgMKK2, on the other hand, interacted with EgERK. In addition, EgMKK1 and EgMKK2 displayed kinase activity toward the substrate myelin basic protein. When sorafenib tosylate, PD184352, or U0126-ethanol (EtOH) was added to the medium for in vitro culture of E. granulosus protoscoleces (PSCs) or cysts, an inhibitory and cytolytic effect was observed via suppressed phosphorylation of EgMKKs and EgERK. Nonviability of PSCs treated with sorafenib tosylate or U0126-EtOH, and not with PD184352, was confirmed through bioassays, i.e., inoculation of treated and untreated protoscoleces into mice. In vivo treatment of E. granulosus sensu stricto-infected mice with sorafenib tosylate or U0126-EtOH for 4 weeks demonstrated a reduction in parasite weight, but the results did not show a significant difference. In conclusion, the MAPK cascades were identified as new targets for drug development, and E. granulosus was efficiently inhibited by their inhibitors in vitro The translation of these findings into in vivo efficacy requires further adjustment of treatment regimens using sorafenib tosylate or, possibly, other kinase inhibitors.


Assuntos
Benzamidas/farmacologia , Butadienos/farmacologia , Equinococose/tratamento farmacológico , Echinococcus granulosus/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Nitrilos/farmacologia , Sorafenibe/farmacologia , Animais , Equinococose/parasitologia , Equinococose/patologia , Echinococcus granulosus/metabolismo , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 3/antagonistas & inibidores , MAP Quinase Quinase 6/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação/efeitos dos fármacos
12.
Cell Death Dis ; 9(10): 976, 2018 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-30250119

RESUMO

Inhibition of epidermal growth factor receptor (EGFR) signaling by small molecule kinase inhibitors and monoclonal antibodies has proven effective in the treatment of multiple cancers. In contrast, metastatic breast cancers (BC) derived from EGFR-expressing mammary tumors are inherently resistant to EGFR-targeted therapies. Mechanisms that contribute to this inherent resistance remain poorly defined. Here, we show that in contrast to primary tumors, ligand-mediated activation of EGFR in metastatic BC is dominated by STAT1 signaling. This change in downstream signaling leads to apoptosis and growth inhibition in response to epidermal growth factor (EGF) in metastatic BC cells. Mechanistically, these changes in downstream signaling result from an increase in the internalized pool of EGFR in metastatic cells, increasing physical access to the nuclear pool of STAT1. Along these lines, an EGFR mutant that is defective in endocytosis is unable to elicit STAT1 phosphorylation and apoptosis. Additionally, inhibition of endosomal signaling using an EGFR inhibitor linked to a nuclear localization signal specifically prevents EGF-induced STAT1 phosphorylation and cell death, without affecting EGFR:ERK1/2 signaling. Pharmacologic blockade of ERK1/2 signaling through the use of the allosteric MEK1/2 inhibitor, trametinib, dramatically biases downstream EGFR signaling toward a STAT1-dominated event, resulting in enhanced EGF-induced apoptosis in metastatic BC cells. Importantly, combined administration of trametinib and EGF also facilitated an apoptotic switch in EGFR-transformed primary tumor cells, but not normal mammary epithelial cells. These studies reveal a fundamental distinction for EGFR function in metastatic BC. Furthermore, the data demonstrate that pharmacological biasing of EGFR signaling toward STAT1 activation is capable of revealing the apoptotic function of this critical pathway.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ligantes , Neoplasias/patologia , Animais , Linhagem Celular Tumoral , Endocitose/fisiologia , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Gefitinibe/farmacologia , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridonas/farmacologia , Pirimidinonas/farmacologia , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
Stem Cell Reports ; 11(1): 58-69, 2018 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-29779897

RESUMO

Embryonic stem cells (ESCs) display heterogeneous expression of pluripotency factors such as Nanog when cultured with serum and leukemia inhibitory factor (LIF). In contrast, dual inhibition of the signaling kinases GSK3 and MEK (2i) converts ESC cultures into a state with more uniform and high Nanog expression. However, it is so far unclear whether 2i acts through an inductive or selective mechanism. Here, we use continuous time-lapse imaging to quantify the dynamics of death, proliferation, and Nanog expression in mouse ESCs after 2i addition. We show that 2i has a dual effect: it both leads to increased cell death of Nanog low ESCs (selective effect) and induces and maintains high Nanog levels (inductive effect) in single ESCs. Genetic manipulation further showed that presence of NANOG protein is important for cell viability in 2i medium. This demonstrates complex Nanog-dependent effects of 2i treatment on ESC cultures.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , MAP Quinase Quinase 2/metabolismo , Proteína Homeobox Nanog/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Expressão Gênica , Técnicas de Inativação de Genes , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Camundongos , Proteína Homeobox Nanog/genética , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Análise de Célula Única
15.
Br J Haematol ; 182(3): 360-372, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29767411

RESUMO

The survival and proliferation of chronic lymphocytic leukaemia (CLL) cells is driven by multiple signalling pathways, including those mediated by the B cell, Toll-like and chemokine receptors. Many of these pathways converge on the same signalling molecules, including those involved in the Raf-1/MEK/Erk1/2-MAPK pathway. We investigated the effects of the MEK1/2 (also termed MAP2K1/2) inhibitor, binimetinib, against CLL cells cultured under conditions that mimic aspects of the tumour microenvironment. Binimetinib blocked CLL cell survival induced by stroma-conditioned media and phorbol myristylate (PMA). Binimetinib was also significantly more toxic towards CLL cells cultured in the presence of either anti-IgM antibody or stroma-derived factor-1α (SDF-1α) and reduced CLL cell cycle progression and proliferation. Furthermore, binimetinib significantly increased the sensitivity of CLL cells co-cultured with CD40 ligand (CD40L)-expressing fibroblasts to the BH3-mimetics ABT-737 and Venetoclax (ABT-199) via a mechanism involving down-regulation of Mcl-1 (MCL1) activity and Bim (BCL2L11) and Bcl-xL (BCL2L1) expression. Collectively, these data suggest that binimetinib may have both cytotoxic and cytostatic effects on CLL cells by blocking microenvironment-derived signals known to drive survival and proliferation. The combination of binimetinib with a BH3 mimetic may be an effective treatment strategy for CLL, particularly against the proliferative fraction of the disease within the tumour microenvironment.


Assuntos
Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Leucemia Linfocítica Crônica de Células B/patologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Microambiente Tumoral/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Compostos de Bifenilo/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Avaliação Pré-Clínica de Medicamentos/métodos , Sinergismo Farmacológico , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Sulfonamidas/farmacologia , Células Tumorais Cultivadas
16.
Inflamm Bowel Dis ; 24(6): 1251-1265, 2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29669006

RESUMO

Background: Anti-tumor necrosis factor alpha (anti-TNFα) therapy has become the mainstay of therapy for Crohn's disease (CD). However, post-therapy, the recurrence rate is still high. The aim of this study was to dissect the molecular mechanism for recurrence of CD treated with anti-TNFα therapy and investigate novel therapeutic options that could induce complete remission. Methods: We re-analyzed publicly available mucosal gene expression data from CD patients pre- and post-infliximab therapy to extract the transcriptional differences between responders and healthy controls. We used a systematic computational approach based on identified differences to discover novel therapies and validated this prediction through in vitro and in vivo experimentation. Results: We identified a set of 3545 anti-TNFα therapy-untreatable genes (TUGs) that are significantly regulated in intestinal epithelial cells, which remain altered during remission. Pathway enrichment analysis of these genes clearly showed excessive growth state and suppressed terminal differentiation, whereas immune components were clearly resolved. Through in silico screening strategy, we observed that MEK inhibitors were predicted to revert expression of genes dysregulated in infliximab responders. In vitro transcriptome analysis demonstrated that selective MEK1/2 inhibitor significantly normalized reference genes from TUGs. In addition, in vitro functional study proved that MEK1/2 inhibitor facilitated intestinal epithelial differentiation. Finally, using murine colitis model, administration of MEK1/2 inhibitor significantly improved diarrhea and histological score. Conclusions: Our data revealed the abnormalities in anti-TNFα responders' CD colons that would be cause of recurrence of CD. Also, we provided evidence regarding MEK1/2 inhibitor as a potential treatment against CD to achieve sustainable remission.


Assuntos
Doença de Crohn/tratamento farmacológico , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adalimumab , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Células CACO-2 , Colo/patologia , Doença de Crohn/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Infliximab , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Recidiva , Indução de Remissão
17.
Eur J Drug Metab Pharmacokinet ; 43(4): 373-382, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29488172

RESUMO

Pimasertib belongs to the growing family of mitogen activated protein kinase (MEK1/2) inhibitors undergoing clinical development for various cancer indications. Since the MEK inhibition in several cell signalling transduction cascades within tumours was considered therapeutically beneficial, number of clinical investigations of pimasertib have been reported. Despite being orally bioavailable in cancer patients, pimasertib undergoes faster clearance with a short elimination half-life. In addition, due to occurrence of toxicity, the development of pimasertib appears to be stalled. Case studies are provided on the possible utilization of pimasertib in combination therapies with other approved drugs. Based on the review, it appeared that there was the need to identify the optimal dose and the dosing regimen of pimasertib to provide a balance between safety and efficacy when combined with approved therapies.


Assuntos
MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Niacinamida/análogos & derivados , Inibidores de Proteínas Quinases/uso terapêutico , Humanos , Niacinamida/administração & dosagem , Niacinamida/efeitos adversos , Niacinamida/farmacocinética , Niacinamida/uso terapêutico , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacocinética
18.
Cancer Discov ; 8(5): 568-581, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29496664

RESUMO

Targeting cyclin-dependent kinases 4/6 (CDK4/6) represents a therapeutic option in combination with BRAF inhibitor and/or MEK inhibitor (MEKi) in melanoma; however, continuous dosing elicits toxicities in patients. Using quantitative and temporal in vivo reporting, we show that continuous MEKi with intermittent CDK4/6 inhibitor (CDK4/6i) led to more complete tumor responses versus other combination schedules. Nevertheless, some tumors acquired resistance that was associated with enhanced phosphorylation of ribosomal S6 protein. These data were supported by phospho-S6 staining of melanoma biopsies from patients treated with CDK4/6i plus targeted inhibitors. Enhanced phospho-S6 in resistant tumors provided a therapeutic window for the mTORC1/2 inhibitor AZD2014. Mechanistically, upregulation or mutation of NRAS was associated with resistance in in vivo models and patient samples, respectively, and mutant NRAS was sufficient to enhance resistance. This study utilizes an in vivo reporter model to optimize schedules and supports targeting mTORC1/2 to overcome MEKi plus CDK4/6i resistance.Significance: Mutant BRAF and NRAS melanomas acquire resistance to combined MEK and CDK4/6 inhibition via upregulation of mTOR pathway signaling. This resistance mechanism provides the preclinical basis to utilize mTORC1/2 inhibitors to improve MEKi plus CDK4/6i drug regimens. Cancer Discov; 8(5); 568-81. ©2018 AACR.See related commentary by Sullivan, p. 532See related article by Romano et al., p. 556This article is highlighted in the In This Issue feature, p. 517.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Fatores de Transcrição E2F/metabolismo , Expressão Gênica , Genes Reporter , Inibidores de Proteínas Quinases/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Modelos Animais de Doenças , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Fosforilação , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/antagonistas & inibidores , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Dig Liver Dis ; 50(5): 501-506, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29428513

RESUMO

BACKGROUND AND OBJECTIVE: P27 is a putative tumor suppressor when located in the nucleus and AKT is an inhibitor of P27 which promotes growth of cholangiocarcinoma. We hypothesized that AKT-dependent phosphorylation at the P27 nuclear localization sequence T157 leads to nuclear export of P27, and thus loss of its tumor suppressive function. This study investigated whether loss of cell cycle regulation in cholangiocarcinoma due to subcellular localization of P27. METHODS: Human cholangiocarcinoma cells were transfected with AKT. P27 was tagged with yellow fluorescence protein. Cell cycle progression was determined by flow cytometry. Migration and invasion of was measured by transwell assay. RESULTS: Overexpression of wildtype P27 or P27-T157A in Mz-ChA-1 cells resulted in G1 arrest; expression of myr-AKT caused translocation of P27-YFP and endogenous P27 from the nucleus to the cytoplasm, leading to inhibition of P27-dependent G1 arrest; the AKT inhibitor and expression of dnAKT increased P27-YFP accumulation in the nucleus and promoted G1 arrest. In contrast, cells expressing YFP-P27-T157A or P27-YFP accumulated only in the nucleus. Co-expression of myr-AKT failed to induce P27-YFP translocation to the cytoplasm or inhibit G1 arrest. Overexpression of P27-T157A significantly increased migration and invasion. CONCLUSIONS: Cholangiocarcinoma growth is associated with nuclear export of P27 that is due to AKT-mediated phosphorylation of P27 at T157.


Assuntos
Neoplasias dos Ductos Biliares/metabolismo , Colangiocarcinoma/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Butadienos/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Morfolinas/farmacologia , Nitrilos/farmacologia , Fosforilação , Sistemas de Translocação de Proteínas/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/genética , Transfecção
20.
Genes Cells ; 23(3): 146-160, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29356242

RESUMO

Epigenetic properties of cultured embryonic stem cells (ESCs), including DNA methylation imprinting, are important because they affect the developmental potential. Here, we tested a variety of culture media, including knockout serum replacement (KSR) and fetal bovine serum (FBS) with or without inhibitors of Gsk3ß and Mek1/2 (2i) at various time points. In addition to the previously known passage-dependent global changes, unexpected dynamic DNA methylation changes occurred in both maternal and paternal differentially methylated regions: under the widely used condition of KSR with 2i, a highly hypomethylated state occurred at early passages (P1-7) as well as P10, but DNA methylation increased over further passages in most conditions, except under KSR with 2i at P25. Dramatic DNA demethylation under KSR+2i until P25 was associated with upregulated Tet1 and Parp1, and their related genes, whereas 2i regulated the expressions of DNA methyltransferase-related genes for the change in DNA methylation during the cumulative number of passages. Although DNA methylation imprinting is more labile under KSR with and without 2i, it can be more faithfully maintained under condition of cooperative FBS and 2i. Thus, our study will provide the useful information for improved epigenetic control of ESCs and iPSCs in applications in regenerative medicine.


Assuntos
Técnicas de Cultura de Células , Metilação de DNA , Epigênese Genética , Impressão Genômica , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Embrionárias Murinas/citologia , Animais , Meios de Cultura , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Inibidores de Proteínas Quinases/farmacologia
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