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2.
Nat Commun ; 13(1): 3246, 2022 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-35688802

RESUMO

We conducted a phase IIa, multi-centre, open label, single arm study (RADICAL; NCT01791985) of AZD4547 (a potent and selective inhibitor of Fibroblast Growth Factor Receptor (FGFR)-1, 2 and 3 receptor tyrosine kinases) administered with anastrozole or letrozole in estrogen receptor positive metastatic breast cancer patients who had become resistant to aromatase inhibitors. After a safety run-in study to assess safety and tolerability, we recruited 52 patients. The primary endpoint was change in tumour size at 12 weeks, and secondary endpoints were to assess response at 6 weeks, 20 weeks and every 8 weeks thereafter and tolerability of the combined treatment. Two partial responses (PR) and 19 stable disease (SD) patients were observed at the 12-week time point. At 28 weeks, according to centrally reviewed Response Evaluation Criteria in Solid Tumours (RECIST) criteria, five PR and 8 SD patients were observed in 50 assessable cases. Overall, objective response rate (5 PR) was of 10%, meeting the pre-specified endpoint. Fourteen patients discontinued due to adverse events. Eleven patients had retinal pigment epithelial detachments which was asymptomatic and reversible in all but one patient. Exploratory ribonucleic acid sequencing (RNA-Seq) analysis was done on patients' samples: 6 differentially-expressed-genes could distinguish those who benefited from the addition of AZD4547.


Assuntos
Benzamidas , Neoplasias da Mama , Piperazinas , Pirazóis , Antineoplásicos/efeitos adversos , Benzamidas/efeitos adversos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Feminino , Humanos , Piperazinas/efeitos adversos , Pirazóis/efeitos adversos , Resultado do Tratamento
4.
Cell Death Dis ; 6: e1671, 2015 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-25741596

RESUMO

Death-associated protein kinase (DAPK) 2 is a serine/threonine kinase that belongs to the DAPK family. Although it shows significant structural differences from DAPK1, the founding member of this protein family, DAPK2 is also thought to be a putative tumour suppressor. Like DAPK1, it has been implicated in programmed cell death, the regulation of autophagy and diverse developmental processes. In contrast to DAPK1, however, few mechanistic studies have been carried out on DAPK2 and the majority of these have made use of tagged DAPK2, which almost invariably leads to overexpression of the protein. As a consequence, physiological roles of this kinase are still poorly understood. Using two genetically distinct cancer cell lines as models, we have identified a new role for DAPK2 in the regulation of mitochondrial integrity. RNA interference-mediated depletion of DAPK2 leads to fundamental metabolic changes, including significantly decreased rate of oxidative phosphorylation in combination with overall destabilised mitochondrial membrane potential. This phenotype is further corroborated by an increase in the production of mitochondrial superoxide anions and increased oxidative stress. This then leads to the activation of classical stress-activated kinases such as ERK, JNK and p38, which is observed on DAPK2 genetic ablation. Interestingly, the generation of oxidative stress is further enhanced on overexpression of a kinase-dead DAPK2 mutant indicating that it is the kinase domain of DAPK2 that is important to maintain mitochondrial integrity and, by inference, for cellular metabolism.


Assuntos
Proteínas Quinases Associadas com Morte Celular/metabolismo , Mitocôndrias/metabolismo , Linhagem Celular Tumoral , Proteínas Quinases Associadas com Morte Celular/genética , Citometria de Fluxo , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo
5.
Oncogene ; 30(32): 3513-21, 2011 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-21423205

RESUMO

We performed a kinome-wide siRNA screen and identified 70 kinases altering cell migration in A549 lung cancer cells. In particular, ribosomal S6 kinase 1 (RSK1) silencing increased, whereas RSK2 and RSK4 downregulation inhibited cell motility. In a secondary collagen-based three-dimensional invasion screen, 38 of our hits cross-validated, including RSK1 and RSK4. In two further lung cancer cell lines, RSK1 but not RSK4 silencing showed identical modulation of cell motility. We therefore selected RSK1 for further investigation. Bioinformatic analysis followed by co-immunoprecipitation-based validation revealed that the actin regulators VASP and Mena interact with RSK1. Moreover, RSK1 phosphorylated VASP on T278, a site regulating its binding to actin. In addition, silencing of RSK1 enhanced the metastatic potential of these cells in vivo using a zebrafish model. Finally, we investigated the relevance of this finding in human lung cancer samples. In isogenically matched tissue, RSK1 was reduced in metastatic versus primary lung cancer lesions. Moreover, patients with RSK1-negative lung tumours showed increased number of metastases. Our results suggest that the findings of our high-throughput in vitro screen can reliably identify relevant clinical targets and as a proof of principle, RSK1 may provide a biomarker for metastasis in lung cancer patients.


Assuntos
Neoplasias Pulmonares/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Animais , Sítios de Ligação , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microscopia de Fluorescência , Metástase Neoplásica , Transplante de Neoplasias , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Treonina/genética , Treonina/metabolismo , Transplante Heterólogo , Peixe-Zebra/embriologia
6.
Oncogene ; 25(6): 877-87, 2006 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-16170339

RESUMO

The impact of the 3-hydroxy-3methylglutaryl CoA reductase inhibitor simvastatin on human small-cell lung cancer (SCLC) cell growth and survival was investigated. Simvastatin profoundly impaired basal and growth factor-stimulated SCLC cell growth in vitro and induced apoptosis. SCLC cells treated with simvastatin were sensitized to the effects of the chemotherapeutic agent etoposide. Moreover, SCLC tumour growth in vivo was inhibited by simvastatin. These responses correlated with the inhibition of stem cell factor (SCF)-stimulated activation of extracellular signal-regulated kinase (Erk), protein kinase B (PKB) and ribosomal S6 kinase by simvastatin. Constitutive activation of the Erk pathway was sufficient to rescue SCLC cell from the effects of simvastatin. The drug did not directly affect activation of c-Kit or its localization to lipid rafts, but in addition to its ability to block Ras membrane localization, it selectively downregulated H-Ras protein levels at the post-translational level. Downregulation of either H- or K-Ras by RNA interference (RNAi) did not impair Erk activation by growth factors, whereas an RNAi specific for N-Ras inhibited activation of Erk, PKB and SCLC cell growth. Together our data demonstrate that inhibiting Ras signalling with simvastatin potently disrupts growth and survival in human SCLC cells.


Assuntos
Carcinoma de Células Pequenas/tratamento farmacológico , Substâncias de Crescimento/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Sinvastatina/farmacologia , Proteínas ras/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Sequência de Bases , Carcinoma de Células Pequenas/metabolismo , Carcinoma de Células Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Etoposídeo/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , MAP Quinase Quinase Quinases/efeitos dos fármacos , MAP Quinase Quinase Quinases/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-kit/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Regulação para Cima , Proteínas ras/efeitos dos fármacos , Proteínas ras/genética
7.
Oncogene ; 20(52): 7658-67, 2001 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-11753643

RESUMO

Here, we show that fibroblast growth factor-2 (FGF-2) induces proliferation of H-510 and H-69 small cell lung cancer (SCLC) cells. However, the optimal response to FGF-2 was obtained at 10-fold lower concentrations in H-510 cells. This correlated with the selective activation of the mitogen-activated protein kinase kinase (MEK) pathway in H-510, but not H-69 cells. Moreover, inhibition of MEK with PD098059 blocked FGF-2-induced proliferation in H-510 cells only. Similarly, ribosomal protein S6 kinase 2 (S6K2), a recently identified homologue of S6K1 was activated by FGF-2 in H-510, but not H-69 cells. This activation was independent of phosphatidylinositol-3 kinase, but was sensitive to inhibition of the MEK pathway. These data suggest that S6K2 is a novel downstream target of MEK. The potency of FGF-2 in H-510 cells might reflect this additional MEK/S6K2 signalling. In contrast to S6K2, S6K1 was activated in both SCLC cell lines. Inhibition of the mammalian target of rapamycin with 10 ng/ml rapamycin blocked S6K1 activation and proliferation of both lines. However, even at 100 ng/ml, rapamycin only partially inhibited S6K2. Strikingly, this correlated with inhibition of MEK signalling. Our data indicate that S6K1, and possibly S6K2, are involved in FGF-2-induced SCLC cell growth, a notion supported by the overexpression and higher baseline activity of both isoforms in SCLC lines, as compared to normal human type-II pneumocytes.


Assuntos
Carcinoma de Células Pequenas/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Neoplasias Pulmonares/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Divisão Celular/efeitos dos fármacos , Ativação Enzimática , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mitógenos/metabolismo , Mitógenos/farmacologia , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases , Proteínas Quinases/metabolismo , Proteínas Quinases/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Quinases S6 Ribossômicas/fisiologia , Serina-Treonina Quinases TOR , Células Tumorais Cultivadas , Regulação para Cima
8.
Anticancer Drug Des ; 16(6): 261-70, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12375879

RESUMO

Nordihydroguaiaretic acid (NDGA) 1 is a constituent of the creosote bush Larrea divaricata and is well known to be a selective inhibitor of lipoxygenases. NDGA can also inhibit the platelet derived growth factor receptor and the protein kinase C intracellular signalling family, which both play an important role in proliferation and survival of cancers. Moreover, NDGA induces apoptosis in tumour xenografts. Although it is likely to have several targets of action, NDGA is well tolerated in animals. These encouraging results have prompted interest in the compound for clinical study. However, high concentrations of NDGA are required for efficacy and more potent analogues are required. We have synthesized five analogues of NDGA with different lengths of carbon bridge between the two catechol moieties in order to establish the spacing required for optimum anticancer effect and to compare their activities with NDGA. In order to ascertain if the catechol moieties are essential for anticancer activity, we prepared five analogues of NDGA containing only one hydroxyl group on each aromatic ring. NDGA 1, its racemic form 2, the catechol derivatives 5, 6 with five or six carbon atom bridges and the phenol analogues 8-11 with bridges of three to six carbon atoms all showed similar activity, with IC50 values of approximately 3-5 microM against the H-69 small cell lung cancer cell line. Analogues with shorter (3) or longer bridges (7, 12) were much less active. The most potent analogue was the biscatechol with a four-carbon bridge 4 which was > 10 times more active than NDGA and therefore represents a new lead compound in this area. Surprisingly, the tetramethyl ether 14 of this compound was slightly more active than NDGA, but the trihydroxy analogue 13 was less active than NDGA. The conformationally restricted analogue 15 was also less active than NDGA. In summary, simplification of the structure of NDGA by removal of the methyl groups has produced a new lead compound 4, which is >10 times more potent than NDGA as a proliferative inhibitor of H-69 small cell lung cancer cells.


Assuntos
Antineoplásicos/síntese química , Divisão Celular/efeitos dos fármacos , Inibidores de Lipoxigenase/síntese química , Masoprocol/síntese química , Células Tumorais Cultivadas/efeitos dos fármacos , Antineoplásicos/farmacologia , Carcinoma de Células Pequenas/tratamento farmacológico , Humanos , Concentração Inibidora 50 , Inibidores de Lipoxigenase/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Masoprocol/análogos & derivados , Masoprocol/farmacologia , Oxirredução , Relação Estrutura-Atividade
10.
Hum Gene Ther ; 10(14): 2373-9, 1999 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-10515457

RESUMO

To optimize gene delivery for the treatment of malignant mesothelioma, expression of the beta-galactosidase marker gene was examined in a murine model of intraperitoneal malignant mesothelioma. The beta-galactosidase gene was delivered to the peritoneal cavity of tumor-bearing mice by various plasmid-liposome complexes or by replication-incompetent retrovirus, used alone or complexed to liposomes. In tumor samples from immunodeficient nude mice, moderate levels of gene expression were achieved by liposome-complexed plasmids. Retroviral gene delivery was more effective, and was increased nearly 10-fold by complexing the retrovirus to liposomes. In contrast, in tumor samples from immunocompetent CBA mice treated with the same vectors, no marker gene expression was detected. In immunodeficient mice, tumor growth was not affected by beta-galactosidase gene transfer. However, immunocompetent mice showed a significant decrease in tumor size and increase in survival time after beta-galactosidase delivery. Induction of cytotoxic T cells capable of lysing beta-Gal-transfected tumor cells suggests that tumor cells transduced with the bacterial beta-galactosidase gene may be eliminated in immunocompetent hosts. Our findings also indicate that plasmid-liposome complexes, which achieve a low level of gene expression, and retrovirus-liposome complexes, which result in nearly 100 times higher levels of gene expression in tumor cells in vivo, are similarly effective in inducing an antitumor immune response.


Assuntos
Terapia Genética , Mesotelioma/terapia , Neoplasias Peritoneais/terapia , Animais , Citotoxicidade Imunológica , Expressão Gênica/genética , Expressão Gênica/imunologia , Técnicas de Transferência de Genes , Genes Bacterianos , Vetores Genéticos/genética , Lipossomos , Mesotelioma/imunologia , Mesotelioma/patologia , Camundongos , Camundongos Endogâmicos CBA , Camundongos Nus , Transplante de Neoplasias , Neoplasias Peritoneais/imunologia , Neoplasias Peritoneais/patologia , Plasmídeos/genética , Retroviridae/genética , Linfócitos T Citotóxicos/patologia , Células Tumorais Cultivadas , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
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