RESUMO
Tyrosine kinase inhibitors of epidermal growth factor receptor (EGFR-TKIs) are currently used therapy for non-small cell lung cancer (NSCLC) patients; however, drug resistance during cancer treatment is a critical problem. Survivin is an anti-apoptosis protein, which promotes cell proliferation and tumor growth that highly expressed in various human cancers. Here, we show a novel synthetic compound derived from gefitinib, do-decyl-4-(4-(3-(4-(3-chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)propyl) piper-azin-1-yl)-4-oxobutanoate, which is named as SP101 that inhibits survivin expression and tumor growth in both the EGFR-wild type and -T790M of NSCLC. SP101 blocked EGFR kinase activity and induced apoptosis in the A549 (EGFR-wild type) and H1975 (EGFR-T790M) lung cancer cells. SP101 reduced survivin proteins and increased active caspase 3 for inducing apoptosis. Ectopic expression of survivin by a survivin-expressed vector attenuated the SP101-induced cell death in lung cancer cells. Moreover, SP101 inhibited the gefitinib-resistant tumor growth in the xenograft human H1975 lung tumors of nude mice. SP101 substantially reduced survivin proteins but conversely elicited active caspase 3 proteins in tumor tissues. Besides, SP101 exerted anticancer abilities in the gefitinib resistant cancer cells separated from pleural effusion of a clinical lung cancer patient. Consistently, SP101 decreased the survivin proteins and the patient-derived xenografted lung tumor growth in nude mice. Anti-tumor ability of SP101 was also confirmed in the murine lung cancer model harboring EGFR T790M-L858R. Together, SP101 is a new EGFR inhibitor with inhibiting survivin that can be developed for treating EGFR wild-type and EGFR-mutational gefitinib-resistance in human lung cancers.
Assuntos
Gefitinibe/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Piperazinas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Quinazolinas/farmacologia , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Survivina/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Piperazinas/uso terapêutico , Quinazolinas/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mammalian target of rapamycin (mTOR) plays a range of crucial roles in cell survival, growth, proliferation, metabolism, and morphology. However, mTOR forms two distinct complexes, mTOR complex 1 and mTOR complex 2 (mTORC1 and mTORC2), via association with a series of different components; this allows the complexes to execute their wide range of functions. This study explores further the composition of the mTORC2 complex. Utilizing Rictor knock-out cells, immunoprecipitation and mass spectrometry, a novel Rictor associated protein, heterogeneous nuclear ribonucleoprotein M (hnRNP M), was identified. The association between hnRNP M and Rictor was verified using recombinant and endogenous protein and the binding site was found to be within aa 1~532 of hnRNP M. The presence of hnRNP M significantly affects phosphorylation of SGK1 S422, but not of Akt S473, PKCα S657 and PKCζ T560. Furthermore, hnRNP M also plays a critical role in muscle differentiation because knock-down of either hnRNP M or Rictor in C2C12 myoblasts reduced differentiation. This decrease is able to be rescued by overexpression SGK S422D in hnRNP M knockdown C2C12 myoblasts. Taken together, we have identified a novel Rictor/mTOR binding molecule, hnRNP M, that allows mTORC2 signaling to phosphorylate SGK1 thus regulating muscle differentiation.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Mioblastos/citologia , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Animais , Sítios de Ligação , Diferenciação Celular , Linhagem Celular , Técnicas de Inativação de Genes , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Humanos , Proteínas Imediatamente Precoces/química , Proteínas Imediatamente Precoces/metabolismo , Camundongos , Mioblastos/metabolismo , Fosforilação , Ligação Proteica , Proteína Quinase C-alfa/química , Proteína Quinase C-alfa/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de SinaisRESUMO
The presence of amyloid-ß (Aß) plaque and tau protein hyperphosphorylation in brain tissue is the pathological hallmark of Alzheimer's disease (AD). At least some Aß neurotoxicity is caused by the presence of excess glutamate that has been induced by Aß accumulation. Memantine is currently the only NMDA receptor inhibitor approved for treating moderate-to-severe AD patients. We utilized primary cortical neurons and DiBAC4(3), a slow-response voltage sensitive fluorescence dye, to create a novel system for screening herbal medicines that allows the identification of pure compounds able to ameliorate Aß-induced abnormal depolarization. The intensity of DiBAC4(3) fluorescence was increased when primary neurons were stimulated by Aß; furthermore, pre-treatment with memantine abolished this change. Using this system, we identified six crude extracts made from herbal medicines that effectively alleviated this Aß-induced abnormal depolarization. Among these herbal medicines, one pure compound, baicalein, which was known to be present in Scutellaria baricalensis and is known to improve memory using an AD mouse model, was identified by our assay. However, the compound's molecular mechanism remained unknown. We found that baicalein, in addition to inhibiting Aß-induced depolarization, possibly functions as an antagonist of AMPA and NMDA receptors. Taken together, we have established a system/platform to identify herbal medicines that ameliorate Aß-induced depolarization of neurons. Equally important, baicalein is a candidate drug with great potential for the treatment of AD patients.
Assuntos
Flavanonas/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptores de AMPA/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Peptídeos beta-Amiloides/toxicidade , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Células HEK293 , Humanos , MAP Quinase Quinase 4/metabolismo , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/metabolismo , Neurotransmissores/farmacologia , Fragmentos de Peptídeos/toxicidade , Fitoterapia , Extratos Vegetais/farmacologia , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Simple construction and manipulation of low-molecular-weight supramolecular nanogels, based on the introduction of multiple hydrogen bonding interactions, with the desired physical properties to achieve effective and safe delivery of drugs for cancer therapy remain highly challenging. Herein, a novel supramolecular oligomer cytosine (Cy)-polypropylene glycol containing self-complementary multiple hydrogen-bonded Cy moieties is developed, which undergoes spontaneous self-assembly to form nanosized particles in an aqueous environment. Phase transitions and scattering studies confirm that the supramolecular nanogels can be readily tailored to obtain the desired phase-transition temperature and temperature-induced release of the anticancer drug doxorubicin (DOX). The resulting nanogels exhibit an extremely high load carrying capacity (up to 24.8%) and drug-entrapment stability, making the loading processes highly efficient. Importantly, in vitro cytotoxicity assays indicate that DOX-loaded nanogels possess excellent biosafety for drug delivery applications under physiological conditions. When the environmental temperature is increased to 40 °C, DOX-loaded nanogels trigger rapid DOX release and exert cytotoxic effects, significantly reducing the dose required compared to free DOX. Given its simplicity, low cost, high reliability, and efficiency, this newly developed temperature-responsive nanocarrier has highly promising potential for controlled release drug delivery systems.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Portadores de Fármacos , Nanoestruturas , Antibióticos Antineoplásicos/química , Linhagem Celular , Doxorrubicina/química , Humanos , Ligação de Hidrogênio , Microscopia Eletrônica de Transmissão , Nanoestruturas/química , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
BACKGROUND: Vorinostat, a histone deacetylase (HDAC) inhibitor, is a promising agent for cancer therapy. Combining vorinostat with cisplatin may relax the chromatin structure and facilitate the accessibility of cisplatin, thus enhancing its cytotoxicity. Studies have not yet investigated the effects of the combination of vorinostat and cisplatin on small cell lung cancer (SCLC). METHODS: We first assessed the efficacy of vorinostat with etoposide/cisplatin (EP; triple combination) and then investigated the effects of cotreatment with vorinostat and cisplatin on H209 and H146 SCLC cell lines. The anticancer effects of various combinations were determined in terms of cell viability, apoptosis, cell cycle distribution, and vorinostat-regulated proteins. We also evaluated the efficacy of vorinostat/cisplatin combination in H209 xenograft nude mice. RESULTS: Our data revealed that the triple combination engendered a significant reduction of cell viability and high apoptotic cell death. In addition, vorinostat combined with cisplatin enhanced cell growth inhibition, induced apoptosis, and promoted cell cycle arrest. We observed that the acetylation levels of histone H3 and α-tubulin were higher in combination treatments than in vorinostat treatment alone. Moreover, vorinostat reduced the expression of thymidylate synthase (TS), and TS remained inhibited after cotreament with cisplatin. Furthermore, an in vivo study revealed that the combination of vorinostat and cisplatin significantly inhibited tumor growth in xenograft nude mice (tumor growth inhibition T/C% = 20.5 %). CONCLUSIONS: Combined treatments with vorinostat promote the cytotoxicity of cisplatin and induce the expression of vorinostat-regulated acetyl proteins, eventually enhancing antitumor effects in SCLC cell lines. Triple combinations with a low dosage of cisplatin demonstrate similar therapeutic effects. Such triple combinations, if applied clinically, may reduce the undesired adverse effects of cisplatin. The effects of the combination of vorinostat and cisplatin should be evaluated further before conducting clinical trials for SCLC treatment.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/patologia , Vorinostat , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The novel concept of modifying and enhancing the properties of existing functional micelles through self-complementary interactions has significant potential. In this study, a practical approach to living polymerization of functionalized thermoresponsive monomers enabled the incorporation of self-constituted multiple hydrogen bonded groups into micelles that have potential as supramolecular drug-delivery systems. Phase transitions and morphological studies in aqueous solution showed that the microstructure can be controlled to achieve well-defined vesicle-like micelles with respect to the strength of the hydrogen bond segment. Thus, the resulting micelles have a very low critical micellization concentration and very high loading capacity (16.1%), making the loading process extremely stable and efficient. Incorporation of the anticancer drug doxorubicin (DOX) affected the micellization process in aqueous solution and enabled fine-tuning of drug loading and precise control of drug release rate with excellent sensitivity. Release studies in vitro showed that DOX-loaded micelles exerted dose-dependent cytotoxicity against human liver carcinoma (HepG2) cells at the physiological temperature of 37°C. In addition, DOX-loaded micelles were efficiently endocytosed by the cancer cells, which may enable the micelles to serve as suitable vehicles for effective delivery of anticancer drugs to primary tumors and metastatic disease. This newly developed material may provide a potential route towards next-generation drug delivery vehicles. STATEMENT OF SIGNIFICANCE: A breakthrough innovation in water-based thermo-responsive polymers has enabled significant progress in developing smart stimuli-responsive nanocarriers by generating novel "supramolecular polymeric micelles" via self-complementary hydrogen-bonding interactions. These newly developed micelles exhibit extremely high micellar stability and drug loading capacity (up to 16%), excellent thermo-responsive behavior and precise control of drug release rate due to hydrogen-bond-induced physical cross-linking. In addition, doxorubicin-loaded micelles were efficiently endocytosed by the cancer cells, which allows them to serve as suitable vehicles for effective delivery of anticancer drugs to primary tumors and metastatic disease. Thus, this work provides a potential route for the development of next generation multifunctional nanocarriers that have improved safety and to increase the therapeutic efficacy of anticancer therapy.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Polímeros/química , Materiais Biocompatíveis/farmacologia , Células HEK293 , Células Hep G2 , Humanos , Micelas , Polímeros/síntese química , Espalhamento de Radiação , TemperaturaRESUMO
A sequential single-flask multicomponent reactions is highly effective for the synthesis of 1,2-dihydroisoquinolines through amidealkylation from intermediate N-acylisoquinolinium salts under mild conditions. N-Acylisoquinolinium ions and trichloromethyl-1-(1H-indol-3-yl)isoquinoline-2(1H)-carboxylate have demonstrated their reactivity toward aromatic and aliphatic π-nucleophiles. One of the 1,2-dihydroisoquinoline derivatives was found to be a potent inhibitor for transcription factor NF-κB by blocking IκBα degradation, p65 nuclear translocation, and NF-κB DNA binding in TNF-α-induced NIH 3T3 cells.
Assuntos
Isoquinolinas/síntese química , Isoquinolinas/farmacologia , NF-kappa B/antagonistas & inibidores , Animais , Células Cultivadas , Humanos , Isoquinolinas/química , Camundongos , Estrutura Molecular , NF-kappa B/metabolismo , Células NIH 3T3 , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Small cell lung carcinoma (SCLC) is a neuroendocrine subtype of lung cancer that affects more than 200,000 people worldwide every year with a very high mortality rate. Here, we used a mouse genetics approach to characterize the cell of origin for SCLC; in this mouse model, tumors are initiated by the deletion of the Rb and p53 tumor suppressor genes in the lung epithelium of adult mice. We found that mouse SCLCs often arise in the lung epithelium, where neuroendocrine cells are located, and that the majority of early lesions were composed of proliferating neuroendocrine cells. In addition, mice in which Rb and p53 are deleted in a variety of non-neuroendocrine lung epithelial cells did not develop SCLC. These data indicate that SCLC likely arises from neuroendocrine cells in the lung.
Assuntos
Genes do Retinoblastoma/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Células Neuroendócrinas/patologia , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia , Proteína Supressora de Tumor p53/genética , Animais , Epitélio , Camundongos , Camundongos Transgênicos , Deleção de SequênciaRESUMO
ErbB3 is a critical activator of phosphoinositide 3-kinase (PI3K) signaling in epidermal growth factor receptor (EGFR; ErbB1), ErbB2 [human epidermal growth factor receptor 2 (HER2)], and [hepatocyte growth factor receptor (MET)] addicted cancers, and reactivation of ErbB3 is a prominent method for cancers to become resistant to ErbB inhibitors. In this study, we evaluated the in vivo efficacy of a therapeutic anti-ErbB3 antibody, MM-121. We found that MM-121 effectively blocked ligand-dependent activation of ErbB3 induced by either EGFR, HER2, or MET. Assessment of several cancer cell lines revealed that MM-121 reduced basal ErbB3 phosphorylation most effectively in cancers possessing ligand-dependent activation of ErbB3. In those cancers, MM-121 treatment led to decreased ErbB3 phosphorylation and, in some instances, decreased ErbB3 expression. The efficacy of single-agent MM-121 was also examined in xenograft models. A machine learning algorithm found that MM-121 was most effective against xenografts with evidence of ligand-dependent activation of ErbB3. We subsequently investigated whether MM-121 treatment could abrogate resistance to anti-EGFR therapies by preventing reactivation of ErbB3. We observed that an EGFR mutant lung cancer cell line (HCC827), made resistant to gefitinib by exogenous heregulin, was resensitized by MM-121. In addition, we found that a de novo lung cancer mouse model induced by EGFR T790M-L858R rapidly became resistant to cetuximab. Resistance was associated with an increase in heregulin expression and ErbB3 activation. However, concomitant cetuximab treatment with MM-121 blocked reactivation of ErbB3 and resulted in a sustained and durable response. Thus, these results suggest that targeting ErbB3 with MM-121 can be an effective therapeutic strategy for cancers with ligand-dependent activation of ErbB3.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias/terapia , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Células CHO , Linhagem Celular Tumoral , Cetuximab , Cricetinae , Cricetulus , Receptores ErbB/antagonistas & inibidores , Gefitinibe , Humanos , Ligantes , Camundongos , Neoplasias/metabolismo , Neuregulina-1/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Receptor ErbB-3/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We have previously shown clinical activity of a mammalian target of rapamycin (mTOR) complex 1 inhibitor in Waldenstrom macroglobulinemia (WM). However, 50% of patients did not respond to therapy. We therefore examined mechanisms of activation of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR in WM, and mechanisms of overcoming resistance to therapy. We first demonstrated that primary WM cells show constitutive activation of the PI3K/Akt pathway, supported by decreased expression of phosphate and tensin homolog tumor suppressor gene (PTEN) at the gene and protein levels, together with constitutive activation of Akt and mTOR. We illustrated that dual targeting of the PI3K/mTOR pathway by the novel inhibitor NVP-BEZ235 showed higher cytotoxicity on WM cells compared with inhibition of the PI3K or mTOR pathways alone. In addition, NVP-BEZ235 inhibited both rictor and raptor, thus abrogating the rictor-induced Akt phosphorylation. NVP-BEZ235 also induced significant cytotoxicity in WM cells in a caspase-dependent and -independent manner, through targeting the Forkhead box transcription factors. In addition, NVP-BEZ235 targeted WM cells in the context of bone marrow microenvironment, leading to significant inhibition of migration, adhesion in vitro, and homing in vivo. These studies therefore show that dual targeting of the PI3K/mTOR pathway is a better modality of targeted therapy for tumors that harbor activation of the PI3K/mTOR signaling cascade, such as WM.
Assuntos
Imidazóis/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteína Oncogênica v-akt/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Quinolinas/uso terapêutico , Macroglobulinemia de Waldenstrom/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Células Cultivadas , Sistemas de Liberação de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Humanos , Imidazóis/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteína Oncogênica v-akt/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TORRESUMO
Non-small cell lung cancers with activating mutations in the epidermal growth factor receptor (EGFR) are highly responsive to EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. Such cancers are "addicted" to EGFR, and treatment with a TKI invariably leads to down-regulation of the PI3K-AKT-mTOR and MEK-ERK signaling pathways, resulting in apoptosis. Using a dual PI3K-mTOR inhibitor, NVP-BEZ235, we evaluated whether PI3K-mTOR inhibition alone induced apoptosis in these cancers. In contrast to HER2-amplified breast cancers, we found that PI3K-mTOR inhibition did not promote substantial apoptosis in the EGFR mutant lung cancers. However, blocking both PI3K-mTOR and MEK simultaneously led to apoptosis to similar levels as the EGFR TKIs, suggesting that down-regulation of these pathways may account for much of the apoptosis promoted by EGFR inhibition. In EGFR mutant lung cancers, down-regulation of both intracellular pathways converged on the BH3 family of proteins regulating apoptosis. PI3K inhibition led to down-regulation of Mcl-1, and MEK inhibition led to up-regulation of BIM. In fact, down-regulation of Mcl-1 by siRNA was sufficient to sensitize these cancers to single-agent MEK inhibitors. Surprisingly, an AKT inhibitor did not decrease Mcl-1 levels, and when combined with MEK inhibitors, failed to induce apoptosis. Importantly, we observed that the combination of PI3K-mTOR and MEK inhibitors effectively shrunk tumors in a transgenic and xenograft model of EGFR T790M-L858R cancers. These data indicate simultaneous inhibition of PI3K-mTOR and MEK signaling is an effective strategy for treating EGFR mutant lung cancers, including those with acquired resistance to EGFR TKIs.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Receptores ErbB/genética , Cloridrato de Erlotinib , Feminino , Gefitinibe , Humanos , Imidazóis/farmacologia , Neoplasias Pulmonares/genética , MAP Quinase Quinase Quinases/antagonistas & inibidores , Camundongos , Proteínas Quinases/metabolismo , Quinazolinas/farmacologia , Quinolinas/farmacologia , Receptor ErbB-2/genética , Serina-Treonina Quinases TOR , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Genome-wide copy number analyses of human cancers identified a frequent 5p13 amplification in several solid tumour types, including lung (56%), ovarian (38%), breast (32%), prostate (37%) and melanoma (32%). Here, using integrative analysis of a genomic profile of the region, we identify a Golgi protein, GOLPH3, as a candidate targeted for amplification. Gain- and loss-of-function studies in vitro and in vivo validated GOLPH3 as a potent oncogene. Physically, GOLPH3 localizes to the trans-Golgi network and interacts with components of the retromer complex, which in yeast has been linked to target of rapamycin (TOR) signalling. Mechanistically, GOLPH3 regulates cell size, enhances growth-factor-induced mTOR (also known as FRAP1) signalling in human cancer cells, and alters the response to an mTOR inhibitor in vivo. Thus, genomic and genetic, biological, functional and biochemical data in yeast and humans establishes GOLPH3 as a new oncogene that is commonly targeted for amplification in human cancer, and is capable of modulating the response to rapamycin, a cancer drug in clinical use.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Proteínas de Membrana/metabolismo , Neoplasias/fisiopatologia , Proteínas Quinases/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Animais , Linhagem Celular Tumoral/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Nus , Proteínas Quinases/genética , Saccharomyces cerevisiae/genética , Serina-Treonina Quinases TOR , Fatores de Transcrição/genéticaRESUMO
Germline mutation in serine/threonine kinase 11 (STK11, also called LKB1) results in Peutz-Jeghers syndrome, characterized by intestinal hamartomas and increased incidence of epithelial cancers. Although uncommon in most sporadic cancers, inactivating somatic mutations of LKB1 have been reported in primary human lung adenocarcinomas and derivative cell lines. Here we used a somatically activatable mutant Kras-driven model of mouse lung cancer to compare the role of Lkb1 to other tumour suppressors in lung cancer. Although Kras mutation cooperated with loss of p53 or Ink4a/Arf (also known as Cdkn2a) in this system, the strongest cooperation was seen with homozygous inactivation of Lkb1. Lkb1-deficient tumours demonstrated shorter latency, an expanded histological spectrum (adeno-, squamous and large-cell carcinoma) and more frequent metastasis compared to tumours lacking p53 or Ink4a/Arf. Pulmonary tumorigenesis was also accelerated by hemizygous inactivation of Lkb1. Consistent with these findings, inactivation of LKB1 was found in 34% and 19% of 144 analysed human lung adenocarcinomas and squamous cell carcinomas, respectively. Expression profiling in human lung cancer cell lines and mouse lung tumours identified a variety of metastasis-promoting genes, such as NEDD9, VEGFC and CD24, as targets of LKB1 repression in lung cancer. These studies establish LKB1 as a critical barrier to pulmonary tumorigenesis, controlling initiation, differentiation and metastasis.
Assuntos
Diferenciação Celular , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos/genética , Genes Supressores de Tumor/fisiologia , Genes p16 , Genes p53/genética , Genes ras/genética , Humanos , Camundongos , Metástase Neoplásica/genética , Proteínas Serina-Treonina Quinases/deficiênciaRESUMO
The EGFR T790M mutation has been identified in tumors from lung cancer patients that eventually develop resistance to erlotinib. In this study, we generated a mouse model with doxycycline-inducible expression of a mutant EGFR containing both L858R, an erlotinib-sensitizing mutation, and the T790M resistance mutation (EGFR TL). Expression of EGFR TL led to development of peripheral adenocarcinomas with bronchioloalveolar features in alveoli as well as papillary adenocarcinomas in bronchioles. Treatment with an irreversible EGFR tyrosine kinase inhibitor (TKI), HKI-272, shrunk only peripheral tumors but not bronchial tumors. However, the combination of HKI-272 and rapamycin resulted in significant regression of both types of lung tumors. This combination therapy may potentially benefit lung cancer patients with the EGFR T790M mutation.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Brônquicas/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Mutação , Animais , Neoplasias Brônquicas/tratamento farmacológico , Linhagem Celular Tumoral , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Quinolinas/administração & dosagem , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sirolimo/administração & dosagemRESUMO
Mutations in the BRAF and KRAS genes occur in approximately 1% to 2% and 20% to 30% of non-small-cell lung cancer patients, respectively, suggesting that the mitogen-activated protein kinase (MAPK) pathway is preferentially activated in lung cancers. Here, we show that lung-specific expression of the BRAF V600E mutant induces the activation of extracellular signal-regulated kinase (ERK)-1/2 (MAPK) pathway and the development of lung adenocarcinoma with bronchioloalveolar carcinoma features in vivo. Deinduction of transgene expression led to dramatic tumor regression, paralleled by dramatic dephosphorylation of ERK1/2, implying a dependency of BRAF-mutant lung tumors on the MAPK pathway. Accordingly, in vivo pharmacologic inhibition of MAPK/ERK kinase (MEK; MAPKK) using a specific MEK inhibitor, CI-1040, induced tumor regression associated with inhibition of cell proliferation and induction of apoptosis in these de novo lung tumors. CI-1040 treatment also led to dramatic tumor shrinkage in murine lung tumors driven by a mutant KRas allele. Thus, somatic mutations in different signaling intermediates of the same pathway induce exquisite dependency on a shared downstream effector. These results unveil a potential common vulnerability of BRAF and KRas mutant lung tumors that potentially affects rational deployment of MEK targeted therapies to non-small-cell lung cancer patients.
Assuntos
Adenocarcinoma Bronquioloalveolar/genética , Adenocarcinoma/genética , Neoplasias Pulmonares/genética , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenocarcinoma/enzimologia , Adenocarcinoma/metabolismo , Adenocarcinoma Bronquioloalveolar/enzimologia , Adenocarcinoma Bronquioloalveolar/metabolismo , Animais , Benzamidas/farmacologia , Modelos Animais de Doenças , Doxiciclina/farmacologia , Genes ras , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/metabolismo , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/biossíntese , Proteínas Proto-Oncogênicas p21(ras)/biossínteseRESUMO
Activating EGFR mutations occur in human non-small cell lung cancer (NSCLC), with 5% of human lung squamous cell carcinomas having EGFRvIII mutations and approximately 10%-30% of lung adenocarcinomas having EGFR kinase domain mutations. An EGFR-targeting monoclonal antibody, mAb806, recognizes a conformational epitope of WT EGFR as well as the truncated EGFRvIII mutant. To explore the anticancer spectrum of this antibody for EGFR targeted cancer therapy, mAb806 was used to treat genetically engineered mice with lung tumors that were driven by either EGFRvIII or EGFR kinase domain mutations. Our results demonstrate that mAb806 is remarkably effective in blocking EGFRvIII signaling and inducing tumor cell apoptosis, resulting in dramatic tumor regression in the EGFRvIII-driven murine lung cancers. Another EGFR-targeting antibody, cetuximab, failed to show activity in these lung tumors. Furthermore, treatment of murine lung tumors driven by the EGFR kinase domain mutation with mAb806 also induced significant tumor regression, albeit to a less degree than that observed in EGFRvIII-driven tumors. Taken together, these data support the hypothesis that mAb806 may lead to significant advancements in the treatment of the population of NSCLC patients with these 2 classes of EGFR mutations.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Mutação , Animais , Anticorpos Monoclonais Humanizados , Apoptose , Cetuximab , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Inibidor p16 de Quinase Dependente de Ciclina/genética , Receptores ErbB/imunologia , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Neoplasias Hormônio-Dependentes/terapia , FosforilaçãoRESUMO
The NF-kappaB transcription factor signaling pathway is constitutively active in many human cancers, and inhibition of this pathway can often kill cancer cells by inducing apoptosis. In this study, we show that two synthetic epoxyquinoids, jesterone dimer (JD) and epoxyquinone A monomer (EqM), are equally effective at inhibiting the growth of two human lymphoma cell lines that have constitutively nuclear REL (human c-Rel) DNA-binding complexes, but either express (SUDHL-4 cells) or do not express (RC-K8 cells) the NF-kappaB inhibitor IkappaBalpha. Furthermore, in these cells, both JD and EqM dose-dependently induced apoptosis, inhibited REL DNA-binding activity, and converted REL to a high molecular weight form. In A293 cells, JD and EqM inhibited the DNA-binding activity of overexpressed REL, but not p50. Replacement of Cys-27 with Ser in REL reduced JD- and EqM-mediated inhibition of REL DNA-binding activity. These results suggest that JD and EqM can induce apoptosis in IkappaBalpha-deficient lymphoma cells through a mechanism involving direct inhibition of transcription factor REL.
Assuntos
Alcenos/farmacologia , Apoptose/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Compostos de Epóxi/farmacologia , Proteínas I-kappa B/fisiologia , Linfoma de Células B/patologia , Linfoma Difuso de Grandes Células B/patologia , Quinonas/farmacologia , Sequência de Bases , Divisão Celular , Linhagem Celular Tumoral , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Proteínas I-kappa B/genética , Inibidor de NF-kappaB alfaRESUMO
Transcription factor NF-kappaB is constitutively active in many human chronic inflammatory diseases and cancers. Epoxyquinone A monomer (EqM), a synthetic derivative of the natural product epoxyquinol A, has previously been shown to be a potent inhibitor of tumor necrosis factor-alpha (TNF-alpha)-induced activation of NF-kappaB, but the mechanism by which EqM inhibits NF-kappaB activation was not known. In this report, we show that EqM blocks activation of NF-kappaB by inhibiting two molecular targets: IkappaB kinase IKKbeta and NF-kappaB subunit p65. EqM inhibits TNF-alpha-induced IkappaBalpha phosphorylation and degradation by targeting IKKbeta, and an alanine substitution for Cys179 in the activation loop of IKKbeta makes it resistant to EqM-mediated inhibition. EqM also directly inhibits DNA binding by p65, but not p50; moreover, replacement of Cys38 in p65 with Ser abolishes EqM-mediated inhibition of DNA binding. Pretreatment of cells with reducing agent dithiothreitol dose-dependently reduces EqM-mediated inhibition of NF-kappaB, further suggesting that EqM directly modifies the thiol group of Cys residues in protein targets. Modifications of the exocyclic alkene of EqM substantially reduce EqM's ability to inhibit NF-kappaB activation. In the human SUDHL-4 lymphoma cell line, EqM inhibits both proliferation and NF-kappaB DNA binding, and activates caspase-3 activity. EqM also effectively inhibits the growth of human leukemia, kidney, and colon cancer cell lines in the NCI's tumor cell panel. Among six colon cancer cell lines, those with low amounts of constitutive NF-kappaB DNA-binding activity are generally more sensitive to growth inhibition by EqM. Taken together, these results suggest that EqM inhibits growth and induces cell death in tumor cells through a mechanism that involves inhibition of NF-kappaB activity at multiple steps in the signaling pathway.
Assuntos
Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Cisteína/efeitos dos fármacos , Proteínas I-kappa B/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Quinonas/farmacologia , Fator de Transcrição RelA/antagonistas & inibidores , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Camundongos , Mutagênese Sítio-DirigidaRESUMO
Activation of the Rel/NF-kappaB signal transduction pathway has been associated with a variety of animal and human malignancies. However, among the Rel/NF-kappaB family members, only c-Rel has been consistently shown to be able to malignantly transform cells in culture. In addition, c-rel has been activated by a retroviral promoter insertion in an avian B-cell lymphoma, and amplifications of REL (human c-rel) are frequently seen in Hodgkin's lymphomas and diffuse large B-cell lymphomas, and in some follicular and mediastinal B-cell lymphomas. Phenotypic analysis of c-rel knockout mice demonstrates that c-Rel has a normal role in B-cell proliferation and survival; moreover, c-Rel nuclear activity is required for B-cell development. Few mammalian model systems are available to study the role of c-Rel in oncogenesis, and it is still not clear what features of c-Rel endow it with its unique oncogenic activity among the Rel/NF-kappaB family. In any event, REL may provide an appropriate therapeutic target for certain human lymphoid cell malignancies.
Assuntos
Linfócitos B/fisiologia , Divisão Celular/fisiologia , Proteínas Proto-Oncogênicas c-rel/fisiologia , Animais , Humanos , Leucemia de Células B/genética , NF-kappa B/fisiologia , Proteínas Proto-Oncogênicas c-rel/genética , Transdução de Sinais/fisiologiaRESUMO
Rel/nuclear factor-kappaB (NF-kappaB) transcription factors control a variety of cellular processes, such as cell growth and apoptosis, and are continually activated in many human diseases, including chronic inflammatory diseases and cancer. Jesterone dimer (JD) is a synthetic derivative of the natural fungal metabolite jesterone, and JD has previously been shown to be cytotoxic in select tumor cell lines. In this report, we demonstrate that JD is a potent inhibitor of the activation of transcription factor NF-kappaB. Namely, JD inhibits tumor necrosis factor-alpha-induced activation of NF-kappaB in mouse 3T3 and human HeLa cells. JD seems to block the induction of the NF-kappaB pathway by inhibiting the inhibitor of kappaB kinase (IKK); that is, treatment of cells with JD blocks phosphorylation of IkappaBalpha, inhibits the activity of a constitutively active form of the IKKbetacatalytic subunit, and converts IKKbetato stable high molecular mass forms. Like JD, a JD-related epoxyquinoid (isotorreyanic acid) inhibits activation of NF-kappaB at 20 microM, whereas several other epoxyquinoids that are related to JD, including its parent compound jesterone, do not block activation of NF-kappaB at this concentration. Finally, JD inhibits both proliferation and DNA binding by REL-containing complexes in the human lymphoma SUDHL-4 cell line, and JD activates caspase-3 activity in these cells. In summary, these results suggest that JD induces apoptosis in tumor cells through a mechanism that involves the inhibition of Rel/NF-kappaB activity and demonstrate the usefulness of assessing the bioactivity of synthetic derivatives of natural products.