RESUMO
BACKGROUND/AIMS: p21-activated Ser/Thr kinase 1 (PAK1) is essential for the genesis and development of many cancers. The purpose of this study was to investigate the role of the PAK1-cyclic AMP response element-binding (CREB) axis in non-small cell lung cancer (NSCLC) tumorigenesis and its related mechanisms. METHODS: Western blot assay and immunohistochemical staining were employed to investigate the PAK1 and CREB expression in the tissue microarray of human squamous NSCLC. Co-immunoprecipitation and immunofluorescence confocal assays were performed to determine the link between PAK1 and CREB. NSCLC xenograft models were used to study oncogenic function of PAK1 in vivo. RESULTS: We observed that PAK1 and CREB expression levels were significantly elevated in human squamous NSCLC-tissue specimens, compared with those in adjacent normal bronchial or bronchiolar epithelial-tissue specimens, as well as their phosphorylated forms, based on western blotting. We showed in vitro that PAK1 knockdown by small-interfering RNA (siRNA) blocked CREB phosphorylation, whereas plasmid-based PAK1 overexpression resulted in CREB phosphorylation at Ser133, based on western blotting. In addition, PAK1 interacted with CREB in co-immunoprecipitation assays. Additionally, our in vitro findings detected by flow cytometry revealed that PAK1 silencing attenuated cell cycle progression, inducing apoptosis. Inhibition of PAK1 expression reduced tumor sizes and masses by modulating CREB expression and activation in xenograft models. CONCLUSION: These results suggest a novel mechanism whereby the PAK1-CREB axis drives carcinogenesis of squamous-cell carcinomas, and have important implications in the development of targeted therapeutics for squamous-cell lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Neoplasias Pulmonares/patologia , Quinases Ativadas por p21/metabolismo , Idoso , Animais , Apoptose , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/uso terapêutico , Ativação Transcricional , Regulação para Cima , Quinases Ativadas por p21/genéticaRESUMO
Mucus secretion is an important protective mechanism for the luminal lining of open tubular organs, but mucin overproduction in the respiratory tract can exacerbate the inflammatory process and cause airway obstruction. Production of MUC5AC, a predominant gel-forming mucin secreted by airway epithelia, can be induced by various inflammatory mediators such as prostaglandins. The two major prostaglandins involved in inflammation are PGE(2) and PGF(2alpha). PGE(2)-induced mucin production has been well studied, but the effect of PGF(2alpha) on mucin production remains poorly understood. To elucidate the effect and underlying mechanism of PGF(2alpha) on MUC5AC production, we investigated the signal transduction of PGF(2alpha) associated with this effect using normal human tracheobronchial epithelial cells. Our results demonstrated that PGF(2alpha) induces MUC5AC overproduction via a signaling cascade involving protein kinase C, ERK, p90 ribosomal S6 protein kinase, and CREB. The regulation of PGF(2alpha)-induced MUC5AC expression by CREB was further confirmed by cAMP response element-dependent MUC5AC promoter activity and by interaction between CREB and MUC5AC promoter. The abrogation of all downstream signaling activities via suppression of each signaling molecule along the pathway indicates that a single pathway from PGF(2alpha) receptor to CREB is responsible for inducing MUC5AC overproduction. As CREB also mediates mucin overproduction induced by PGE(2) and other inflammatory mediators, our findings have important clinical implications for the management of airway mucus hypersecretion.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dinoprosta/metabolismo , Células Epiteliais/metabolismo , Mucina-5AC/biossíntese , Mucosa Respiratória/metabolismo , Transdução de Sinais/fisiologia , Western Blotting , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Dinoprosta/imunologia , Células Epiteliais/imunologia , Citometria de Fluxo , Imunofluorescência , Humanos , Mucina-5AC/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA , Mucosa Respiratória/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Evasion of the immune response is a hallmark of cancer, and programmed cell death 1 (PD-1) and PD-1 ligand 1 (PD-L1) are major mediators of this immunosuppression. Chitinase 3-like 1 (CHI3L1) is induced in many cancers, where it portends a poor prognosis and contributes to tumor metastasis and spread. However, the mechanism(s) that CHI3L1 uses in metastasis have not been defined. Here we demonstrate that CHI3L1 regulates the expression of PD-L1, PD-L2, PD-1, LAG3, and TIM3 and plays a critical role in melanoma progression and lymphatic spread. CHI3L1 also contributed to IFN-γ-stimulated macrophage PD-L1 expression, and RIG-like helicase innate immunity suppressed CHI3L1, PD-L1, and melanoma progression. Individual antibodies against CHI3L1 or PD-1 had discrete antitumor effects and additive antitumor responses in metastasis models and T cell-tumor cell cocultures when administered simultaneously. Synergistic cytotoxic tumor cell death was seen in T cell-tumor cell cocultures, and significantly enhanced antitumor responses were seen in in vivo tumor models treated with bispecific antibodies that simultaneously target CHI3L1 and PD-1. CHI3L1 contributes to tumor progression by stimulating the PD-1/PD-L1 axis and other checkpoint molecules. The simultaneous targeting of CHI3L1 and the PD-1/PD-L1 axis with individual and, more powerfully, with bispecific antibodies represents a promising therapy for pulmonary metastasis and progression.
Assuntos
Anticorpos Biespecíficos , Anticorpos Antineoplásicos , Antígeno B7-H1 , Proteína 1 Semelhante à Quitinase-3 , Neoplasias Pulmonares , Proteínas de Neoplasias , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Anticorpos Antineoplásicos/imunologia , Anticorpos Antineoplásicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Proteína 1 Semelhante à Quitinase-3/antagonistas & inibidores , Proteína 1 Semelhante à Quitinase-3/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Knockout , Metástase Neoplásica , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/imunologiaRESUMO
Mucus production is a primary defense mechanism for maintaining lung health. However, the overproduction of mucin (the chief glycoprotein component of mucus) is a common pathological feature in asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and lung cancer. Although it is associated with disease progression, effective therapies that directly target mucin overproduction and hypersecretion are lacking. Recent advances in our understanding of the control of mucin gene expression in the lungs, the cells that produce airway mucins, and the mechanisms used for releasing them into the airways have provided new potentials for the development of efficacious interventions that will be discussed in this review.
Assuntos
Mucinas/biossíntese , Muco/metabolismo , Sistema Respiratório/metabolismo , Fibrose Cística/tratamento farmacológico , Fibrose Cística/fisiopatologia , Exocitose , Regulação da Expressão Gênica , Humanos , Pulmão/metabolismo , Mucinas/genética , Mucinas/metabolismo , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Medicamentos para o Sistema Respiratório/uso terapêutico , Transdução de SinaisRESUMO
Vitamin A and its metabolite retinoic acid (RA) are essential elements for normal lung development and the differentiation of lung epithelial cells. We previously showed that RA rapidly activated cyclic AMP response element-binding protein (CREB) in a nonclassical manner in normal human tracheobronchial epithelial (NHTBE) cells. In the present study, we further demonstrated that this nonclassical signaling of RA on the activation of CREB plays a critical role in regulating the expression of airway epithelial cell differentiation markers, the MUC2, MUC5AC, and MUC5B genes. We found that RA rapidly activates the protein kinase Calpha isozyme and transmits the activation signal to CREB via the Raf/MEK/extracellular signal-regulated kinase/p90 ribosomal S6 kinase (RSK) pathway. Activated RSK translocated from the cytoplasm to the nucleus, where it phosphorylates CREB. Activated CREB then binds to a cis-acting replication element motif on the promoter (at nucleotides [nt] -878 to -871) of the MUC5AC gene. The depletion of CREB using small interfering RNA abolished not only the RA-induced MUC5AC but also RA-induced MUC2 and MUC5B. Taken together, our findings demonstrate that CREB activation via this nonclassical RA signaling pathway may play an important role in regulating the expression of mucin genes and mediating the early biological effects of RA during normal mucous differentiation in NHTBE cells.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Mucinas , Transdução de Sinais/fisiologia , Tretinoína/metabolismo , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Ativação Enzimática , Células Epiteliais/citologia , Células Epiteliais/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Isoenzimas/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Mucina-5AC , Mucina-2 , Mucina-5B , Mucinas/genética , Mucinas/metabolismo , Proteína Quinase C-alfa/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores do Ácido Retinoico/metabolismo , Mucosa Respiratória/citologia , Receptores X de Retinoides/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Quinases raf/metabolismoRESUMO
CREB [CRE (cAMP-response element)-binding protein] is an important transcription factor that is differentially regulated in cells of various types. We recently reported that RA (retinoic acid) rapidly activates CREB without using RARs (RA receptors) or RXRs (retinoid X receptors) in NHTBE cells (normal human tracheobronchial epithelial cells). However, little is known about the role of RA in the physiological regulation of CREB expression in the early mucous differentiation of NHTBE cells. In the present study, we report that RA up-regulates CREB gene expression and that, using 5'-serial deletion promoter analysis and mutagenesis analyses, two Sp1 (specificity protein 1)-binding sites located at nt -217 and -150, which flank the transcription initiation site, are essential for RA induction of CREB gene transcription. Furthermore, we found that CREs located at nt -119 and -98 contributed to basal promoter activity. Interestingly, RA also up-regulated Sp1 in a time- and dose-dependent manner. Knockdown of endogenous Sp1 using siRNA (small interfering RNA) decreased RA-induced CREB gene expression. However, the converse was not true: knockdown of CREB using CREB siRNA did not affect RA-induced Sp1 gene expression. We conclude that RA up-regulates CREB gene expression during the early stage of NHTBE cell differentiation and that RA-inducible Sp1 plays a major role in up-regulating human CREB gene expression. This result implies that co-operation of these two transcription factors plays a crucial role in mediating early events of normal mucous cell differentiation of bronchial epithelial cells.
Assuntos
Brônquios/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células Epiteliais/efeitos dos fármacos , Fator de Transcrição Sp1/fisiologia , Tretinoína/farmacologia , Regulação para Cima , Sequência de Bases , Brônquios/citologia , Brônquios/metabolismo , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Células Epiteliais/metabolismo , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido NucleicoRESUMO
Vitamin A (retinol) is essential for normal regulation of cell growth and differentiation. We have shown that the retinol metabolite retinoic acid (RA) induces mucous cell differentiation of normal human tracheobronchial epithelial (NHTBE) cells. However, early biological effects of RA in the differentiation of bronchial epithelia are largely unknown. Here, we showed that RA rapidly activated cAMP response element-binding protein (CREB). However, RA did not use the conventional retinoic acid receptor (RAR)/retinoid X receptor (RXR) to activate CREB. RA activated CREB in NHTBE and H1734 cells in which RARs/RXR were silenced with small interfering RNA (siRNA) targeting RAR/RXR expression or deactivated by antagonist. Inhibition of protein kinase C (PKC) or extracellular regulated kinase (ERK1/2) blocked the RA-mediated activation of CREB. In addition, depletion of p90 ribosomal S6 kinase (RSK) via siRSK1/2 completely abolished the activation, suggesting that PKC, ERK, and RSK are required for the activation. Altogether, this study provides the first evidence that RA rapidly activates CREB transcription factor via PKC, ERK, and RSK in a retinoid receptor-independent manner in normal bronchial epithelial cells. This noncanonical RA signaling pathway may play an important role in mediating early biological effects in the mucociliary differentiation of bronchial epithelia.
Assuntos
Brônquios/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Mucosa Respiratória/metabolismo , Tretinoína/fisiologia , Brônquios/citologia , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/efeitos dos fármacos , Receptores X de Retinoides/fisiologia , Proteínas Quinases S6 Ribossômicas , Transdução de Sinais , Ativação Transcricional , Tretinoína/farmacologiaRESUMO
Squamous cell carcinoma in the lung originates from bronchial epithelial cells that acquire increasingly abnormal phenotypes. Currently, no known biomarkers are clinically efficient for the early detection of premalignant lesions and lung cancer. We sought to identify secreted molecules produced from squamous bronchial epithelial cells cultured with organotypic culture methods. We analyzed protein expression patterns in the apical surface fluid (ASF) from aberrantly differentiated squamous metaplastic normal human tracheobronchial epithelial (NHTBE) and mucous NHTBE cells. Comparative two-dimensional PAGE analysis revealed 174 unique proteins in the ASF of squamous NHTBE cells compared with normal mucociliary differentiated NHTBE cells. Among them, 64 well-separated protein spots were identified by liquid chromatography-tandem mass spectrometry, revealing 22 different proteins in the ASF from squamous NHTBE cells. Expression of six of these proteins [SCC antigen 1 (SCCA1), SCC antigen 2 (SCCA2), S100A8, S100A9, Annexin I, and Annexin II] in the squamous NHTBE cells was further confirmed with immunoblot analysis. Notably, SCCA1 and SCCA2 were verified as being expressed in squamous metaplastic NHTBE cells but not in normal mucous NHTBE or normal bronchial epithelium. Moreover, SCCA1 and SCCA2 expression increased in in vitro lung carcinogenesis model cell lines with increasing malignancy. In summary, we identified proteins that are uniquely secreted from squamous metaplastic primary human bronchial epithelial cells cultured by the organotypic air-liquid interface method. These ASF proteins may be used to detect abnormal lesions in the lung without collecting invasive biopsy specimens.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Biologia Molecular/métodos , Proteínas/química , Proteômica/métodos , Neoplasias da Traqueia/metabolismo , Ar , Antígenos de Neoplasias/biossíntese , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Metástase Neoplásica , Análise Serial de Proteínas , Serpinas/biossíntese , Propriedades de SuperfícieRESUMO
INTRODUCTION: This study aimed to characterize the tumor-infiltrating immune cells population in Kras/tumor protein 53 (Trp53)-driven lung tumors and to evaluate the combinatorial antitumor effect with MEK inhibitor (MEKi), trametinib, and immunomodulatory monoclonal antibodies (mAbs) targeting either programmed death -1 (PD-1) or programmed cell death ligand 1 (PD-L1) in vivo. METHODS: Trp53FloxFlox;KrasG12D/+;Rosa26LSL-Luciferase/LSL-Luciferase (PKL) genetically engineered mice were used to develop autochthonous lung tumors with intratracheal delivery of adenoviral Cre recombinase. Using these tumor-bearing lungs, tumor-infiltrating immune cells were characterized by both mass cytometry and flow cytometry. PKL-mediated immunocompetent syngeneic and transgenic lung cancer mouse models were treated with MEKi alone as well as in combination with either anti-PD-1 or anti-PD-L1 mAbs. Tumor growth and survival outcome were assessed. Finally, immune cell populations within spleens and tumors were evaluated by flow cytometry and immunohistochemistry. RESULTS: Myeloid-derived suppressor cells (MDSCs) were significantly augmented in PKL-driven lung tumors compared to normal lungs of tumor-free mice. PD-L1 expression appeared to be highly positive in both lung tumor cells and, particularly MDSCs. The combinatory administration of MEKi with either anti-PD-1 or anti-PD-L1 mAbs synergistically increased antitumor response and survival outcome compared with single-agent therapy in both the PKL-mediated syngeneic and transgenic lung cancer models. Theses combinational treatments resulted in significant increases of tumor-infiltrating CD8+ and CD4+ T cells, whereas attenuation of CD11b+/Gr-1high MDSCs, in particular, Ly6Ghigh polymorphonuclear-MDSCs in the syngeneic model. CONCLUSIONS: These findings suggest a potential therapeutic approach for untargetable Kras/p53-driven lung cancers with synergy between targeted therapy using MEKi and immunotherapies.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Antineoplásicos Imunológicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Supressora de Tumor p53/genética , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/patologia , Animais , Antineoplásicos Imunológicos/administração & dosagem , Antígeno B7-H1/biossíntese , Antígeno B7-H1/imunologia , Sinergismo Farmacológico , Feminino , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/patologia , Receptor de Morte Celular Programada 1/biossíntese , Receptor de Morte Celular Programada 1/imunologia , Inibidores de Proteínas Quinases/administração & dosagem , Piridonas/farmacologia , Pirimidinonas/farmacologia , Análise de SobrevidaRESUMO
PURPOSE: Human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCC) commonly bear disruptive mutations in TP53, resulting in treatment resistance. In these patients, direct targeting of p53 has not been successful, but synthetic lethal approaches have promise. Although Aurora A kinase (AURKA) is overexpressed and an oncogenic driver, its inhibition has only modest clinical effects in HPV-negative HNSCC. We explored a novel combination of AURKA and WEE1 inhibition to overcome intrinsic resistance to AURKA inhibition.Experimental Design: AURKA protein expression was determined by fluorescence-based automated quantitative analysis of patient specimens and correlated with survival. We evaluated treatment with the AURKA inhibitor alisertib (MLN8237) and the WEE1 inhibitor adavosertib (AZD1775), alone or in combination, using in vitro and in vivo HNSCC models. RESULTS: Elevated nuclear AURKA correlated with worse survival among patients with p16(-) HNSCC. Alisertib caused spindle defects, G2-M arrest and inhibitory CDK1 phosphorylation, and cytostasis in TP53 mutant HNSCC FaDu and UNC7 cells. Addition of adavosertib to alisertib instead triggered mitotic entry and mitotic catastrophe. Moreover, in FaDu and Detroit 562 xenografts, this combination demonstrated synergistic effects on tumor growth and extended overall survival compared with either vehicle or single-agent treatment. CONCLUSIONS: Combinatorial treatment with adavosertib and alisertib leads to synergistic antitumor effects in in vitro and in vivo HNSCC models. These findings suggest a novel rational combination, providing a promising therapeutic avenue for TP53-mutated cancers.
Assuntos
Antineoplásicos/farmacologia , Aurora Quinase A/antagonistas & inibidores , Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Imunofluorescência , Expressão Gênica , Humanos , Masculino , Camundongos , Gradação de Tumores , Estadiamento de Neoplasias , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pancreatic adenocarcinoma (PDAC) is the fourth most common cause of cancer-related death in the United States. PDAC is difficult to manage effectively, with a five-year survival rate of only 5%. PDAC is largely driven by activating KRAS mutations, and as such, cannot be directly targeted with therapeutic agents that affect the activated protein. Instead, inhibition of downstream signaling and other targets will be necessary to effectively manage PDAC. Here, we describe a tiered single-agent and combination compound screen to identify targeted agents that impair growth of a panel of PDAC cell lines. Several of the combinations identified from the screen were further validated for efficacy and mechanism. Combination of the bromodomain inhibitor JQ1 and the neddylation inhibitor MLN4294 altered the production of reactive oxygen species in PDAC cells, ultimately leading to defects in the DNA damage response. Dual bromodomain/neddylation blockade inhibited in vivo growth of PDAC cell line xenografts. Overall, this work revealed novel combinatorial regimens, including JQ1 plus MLN4294, which show promise for the treatment of RAS-driven PDAC. Mol Cancer Ther; 16(6); 1041-53. ©2017 AACR.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Sinergismo Farmacológico , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Terapia de Alvo Molecular , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Superóxidos , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias PancreáticasRESUMO
Overexpression or activation of cyclic AMP-response element-binding protein (CREB) has been known to be involved in several human malignancies, including lung cancer. Genes regulated by CREB have been reported to suppress apoptosis, induce cell proliferation, inflammation, and tumor metastasis. However, the critical target genes of CREB in lung cancer have not been well understood. Here, we identified GSK-3α as one of the CREB target genes which is critical for the viability of lung cancer cells. The CREB knockdown significantly reduced the expression of GSK-3α and the direct binding of CREB on the promoter of GSK3A was identified. Kaplan-Meier analysis with a public database showed a prognostic significance of aberrant GSK-3α expression in lung cancer. Inhibition of GSK-3α suppressed cell viability, colony formation, and tumor growth. For the first time, we demonstrated that GSK-3α is regulated by CREB in lung cancer and is required for the cell viability. These findings implicate CREB-GSK-3α axis as a novel therapeutic target for lung cancer treatment.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Neoplasias Pulmonares/enzimologia , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , PrognósticoRESUMO
PURPOSE: By applying the principles of real-time biopsy, biomarker-based, adaptively randomized studies in non-small-cell lung cancer (NSCLC) established by the Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) trial, we conducted BATTLE-2 (BATTLE-2 Program: A Biomarker-Integrated Targeted Therapy Study in Previously Treated Patients With Advanced Non-Small Cell Lung Cancer), an umbrella study to evaluate the effects of targeted therapies focusing on KRAS-mutated cancers. PATIENTS AND METHODS: Patients with advanced NSCLC (excluding sensitizing EGFR mutations and ALK gene fusions) refractory to more than one prior therapy were randomly assigned, stratified by KRAS status, to four arms: (1) erlotinib, (2) erlotinib plus MK-2206, (3) MK-2206 plus AZD6244, or (4) sorafenib. Tumor gene expression profiling-targeted next-generation sequencing was performed to evaluate predictive and prognostic biomarkers. RESULTS: Two hundred patients, 27% with KRAS-mutated (KRAS mut+) tumors, were adaptively randomly assigned to erlotinib (n = 22), erlotinib plus MK-2206 (n = 42), MK-2206 plus AZD6244 (n = 75), or sorafenib (n = 61). In all, 186 patients were evaluable, and the primary end point of an 8-week disease control rate (DCR) was 48% (arm 1, 32%; arm 2, 50%; arm 3, 53%; and arm 4, 46%). For KRAS mut+ patients, DCR was 20%, 25%, 62%, and 44% whereas for KRAS wild-type patients, DCR was 36%, 57%, 49%, and 47% for arms 1, 2, 3, and 4, respectively. Median progression-free survival was 2.0 months, not different by KRAS status, 1.8 months for arm 1, and 2.5 months for arms 2 versus arms 3 and 4 in KRAS mut+ patients (P = .04). Median overall survival was 6.5 months, 9.0 and 5.1 months for arms 1 and 2 versus arms 3 and 4 in KRAS wild-type patients (P = .03). Median overall survival was 7.5 months in mesenchymal versus 5 months in epithelial tumors (P = .02). CONCLUSION: Despite improved progression-free survival on therapy that did not contain erlotinib for KRAS mut+ patients and improved prognosis for mesenchymal tumors, better biomarker-driven treatment strategies are still needed.
RESUMO
BACKGROUND: The E2F members have been divided into transcription activators (E2F1-E2F3) and repressors (E2F4-E2F8). E2F8 with E2F7 has been known to play an important physiologic role in embryonic development and cell cycle regulation by repressing E2F1. However, the function of E2F8 in cancer cells is unknown. METHODS: E2F8 expression was assessed by immunoblotting or immunofluorescence staining in human lung cancer (LC) cells and tissues from LC patients (n = 45). Cell proliferation, colony formation, and invasion analysis were performed to evaluate the role of E2F8 in LC. Microarray analysis was used to determine the target genes of E2F8. The regulation of E2F8 on the expression of ubiquitin-like PHD and RING domain-containing 1 (UHRF1), one of E2F8 target genes, was determined using chromatin immunoprecipitation and promoter activity assays. Human LC xenograft models were used to determine the effects of inhibiting E2F8 by siRNAs (n = 7 per group) or antisense morpholino (n = 8 per group) on tumor growth. Survival was analyzed using the Kaplan-Meier method and group differences by the Student's t test. All statistical tests were two-sided. RESULTS: LC tumors overexpressed E2F8 compared with normal lung tissues. Depletion of E2F8 inhibited cell proliferation and tumor growth. E2F8 knockdown statistically significantly reduced the expression of UHRF1 (~60%-70%, P < .001), and the direct binding of E2F8 on the promoter of UHRF1 was identified. Kaplan-Meier analysis with a public database showed prognostic significance of aberrant E2F8 expression in LC (HR = 1.91 95% CI = 1.21 to 3.01 in chemo-naïve patients, P = .0047). CONCLUSIONS: We demonstrated that E2F8 is overexpressed in LC and is required for the growth of LC cells. These findings implicate E2F8 as a novel therapeutic target for LC treatment.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/antagonistas & inibidores , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Imunoprecipitação da Cromatina , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Immunoblotting , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Terapia de Alvo Molecular , Células-Tronco Neoplásicas , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Análise Serial de Tecidos , Ubiquitina-Proteína Ligases , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Lung adenocarcinoma, the most common subtype of lung cancer, is the leading cause of cancer death worldwide. Despite attempts for the treatment of lung cancer which have been accumulating, promising new therapies are still needed. Here, we found that cyclic-AMP response element-binding protein (CREB)-CREB binding protein (CBP) transcription factors complex inhibitor, Naphthol AS-TR phosphate (NASTRp), is a potential therapeutic agent for lung cancer. We show that NASTRp inhibited oncogenic cell properties through cell cycle arrest with concomitant suppression of tumor-promoting autophagy with down-regulations of Atg5-12 and Atg7, and accumulation of p62 in human lung cancer cell lines. In addition, NASTRp induced expression of endoplasmic reticulum stress markers such as DDIT3/CHOP, and led to apoptosis along with Bim induction. These findings suggest that transcription factor/co-activator complex, CREB-CBP, can be a potential therapeutic target and its inhibition could be a novel therapeutic strategy for lung cancer.
Assuntos
Anilidas/farmacologia , Antineoplásicos/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Organofosfatos/farmacologia , Fragmentos de Peptídeos/antagonistas & inibidores , Sialoglicoproteínas/antagonistas & inibidores , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Anilidas/química , Antineoplásicos/química , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Proteína 7 Relacionada à Autofagia , Proteína 11 Semelhante a Bcl-2 , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Ensaios de Seleção de Medicamentos Antitumorais , Estresse do Retículo Endoplasmático , Humanos , Concentração Inibidora 50 , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Proteínas de Membrana/metabolismo , Simulação de Acoplamento Molecular , Organofosfatos/química , Fragmentos de Peptídeos/química , Modelos de Riscos Proporcionais , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Sialoglicoproteínas/química , Enzimas Ativadoras de Ubiquitina/metabolismoRESUMO
The prototypic chitinase-like protein Chi3l1 is induced in cancers and portends a poor prognosis, but whether it contributes to cancer progression is unknown. To address this gap in knowledge, we investigated the production of Chi3l1 in melanoma lung metastases. We found that Chi3l1 was induced during pulmonary melanoma metastasis and that this induction was regulated by the semaphorin Sema7a, interacting in stimulatory or inhibitory ways with its ß1 integrin or Plexin C1 receptors, respectively. In mouse strains with genetic deletions of Chi3l1 or Sema7a, there was a significant reduction in pulmonary metastasis. Notably, antiserum raised against Chi3l1 or Sema7a phenocopied the reduction produced by genetic deletions. Melanoma lung metastasis was also decreased in the absence of IL13Rα2, a recently identified receptor for Chi3l1, consistent with a key role for Chi3l1 in melanoma spread. We confirmed roles for Sema7a and Chi3l1 in pulmonary metastasis of EMT6 breast cancer cells. Taken together, our studies establish a novel pathway through which Sem7a and its receptors regulate Chi3l1, revealing a host axis involving IL13Rα2 that plays a critical role in generating a pulmonary microenvironment that is critical to license metastasis.
Assuntos
Antígenos CD/metabolismo , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/metabolismo , Neoplasias Pulmonares/secundário , Melanoma/patologia , Semaforinas/metabolismo , Animais , Linhagem Celular Tumoral , Proteína 1 Semelhante à Quitinase-3 , Deleção de Genes , Inativação Gênica , Imuno-Histoquímica , Neoplasias Pulmonares/metabolismo , Melanoma/metabolismo , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
BACKGROUND: To generate specific transcript profiles, one must isolate homogenous cell populations using techniques that often yield small amounts of RNA, requiring researchers to employ RNA amplification methods. The data generated by using these methods must be extensively evaluated to determine any technique dependent distortion of the expression profiles. RESULTS: High-density oligonucleotide microarrays were used to perform experiments for comparing data generated by using two protocols, an in vitro transcription (IVT) protocol that requires 5 microg of total RNA and a double in vitro transcription (dIVT) protocol that requires 200 ng of total RNA for target preparation from RNA samples extracted from a normal and a cancer cell line. In both cell lines, about 10% more genes were detected with IVT than with dIVT. Genes were filtered to exclude those that were undetected on all arrays. Hierarchical clustering using the 9,482 genes that passed the filter showed that the variation attributable to biological differences between samples was greater than that introduced by differences in the protocols. We analyzed the behavior of these genes separately for each protocol by using a statistical model to estimate the posterior probability of various levels of fold change. At each level, more differentially expressed genes were detected with IVT than with dIVT. When we checked for genes that had a posterior probability greater than 99% of fold change greater than 2, in data generated by IVT but not dIVT, more than 60% of these genes had posterior probabilities greater than 90% in data generated by dIVT. Both protocols identified the same functional gene categories to be differentially expressed. Differential expression of selected genes was confirmed using quantitative real-time PCR. CONCLUSION: Using nanogram quantities on total RNA, the usage of dIVT protocol identified differentially expressed genes and functional categories consistent with those detected by the IVT protocol. There was a loss in sensitivity of about 10% when detecting differentially expressed genes using the dIVT protocol. However, the lower amount of RNA required for this protocol, as compared to the IVT protocol, renders this methodology a highly desirable one for biological systems where sample amounts are limiting.
Assuntos
Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Algoritmos , Linhagem Celular , Linhagem Celular Tumoral , Análise por Conglomerados , Eletroforese em Gel de Ágar , Humanos , Hibridização de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/normas , RNA/genética , RNA/isolamento & purificação , RNA/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , RNA Neoplásico/metabolismoRESUMO
Polyps are believed to be the source of mucus hypersecretion in chronic inflammation of the sinus. However, it is not clear which mucins are responsible for the hypersecretion of mucus by nasal polyps. We describe the over-expression of MUC8 mRNA in nasal polyps and the upregulation of MUC8 mRNA expression and downregulation of MUC5AC mRNA expression by inflammatory mediators. We found that the level of MUC8 mRNA, but not the level of MUC5AC mRNA, increased in nasal polyps. We also found that there was an increase in intracellular mucin in nasal polyps, compared to the normal nasal inferior turbinate. A mixture of inflammatory mediators increased MUC8 mRNA expression and decreased MUC5AC mRNA expression in cultured normal human nasal epithelial cells. Among inflammatory mediators, IL-4 is responsible for the decrease in MUC5AC mRNA and MUC5AC mucin secretion. These results indicate that MUC8 may be one of the major mucins secreted from the polyp epithelium and that it may play an important role in the pathogenesis of mucus hypersecretion in chronic sinusitis with polyps.
Assuntos
Mucinas/genética , Pólipos Nasais/genética , Regulação para Cima/genética , Células Cultivadas , Doença Crônica , Humanos , Mucina-5AC , Mucinas/metabolismo , Muco/metabolismo , Mucosa Nasal/metabolismo , Mucosa Nasal/patologia , Pólipos Nasais/metabolismo , Pólipos Nasais/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sinusite/patologiaRESUMO
Recently, it was found that IL-13 is involved in allergic inflammation and mucus hypersecretion in in vivo animal models. However, the role of IL-13 in in vitro cellular models has yet to be determined. This study aimed to investigate the direct effect of IL-13 on mucin gene expression and mucin secretion in cultured normal human nasal epithelial cells. After treatment with IL-13, total mucin and MUC5AC mucin levels were measured using an immunoblotting assay. MUC2, MUC5AC and MUC8 mRNA expression were determined using reverse transcription-polymerase chain reaction. Also, immunostaining was performed using a MUC5AC antibody on histologic and cytospin slides. After treatment with > or = 5 ng/ml IL-13, the level of total mucin and MUC5AC mucin secretion decreased substantially. The expression of MUC2 and MUC8 mRNA increased with higher concentrations of IL-13, but the expression of MUC5AC mRNA decreased. On the 7th day after IL-13 treatment, a significant decrease in the number of MUC5AC-positive cells was confirmed by immunostaining. These findings indicate that IL-13 suppresses MUC5AC mucin gene expression and mucin secretion in cultured normal human nasal epithelial cells in vitro.
Assuntos
Interleucina-13/farmacologia , Mucinas/genética , Mucinas/metabolismo , Mucosa Nasal/fisiopatologia , Contagem de Células , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Imuno-Histoquímica , Interleucina-13/fisiologia , Mucina-5AC , Mucina-2 , Mucinas/biossíntese , Mucosa Nasal/citologia , Mucosa Nasal/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
CXCR2 in non-small cell lung cancer (NSCLC) has been studied mainly in stromal cells and is known to increase tumor inflammation and angiogenesis. Here, we examined the prognostic importance of CXCR2 in NSCLC and the role of CXCR2 and its ligands in lung cancer cells. The effect of CXCR2 expression on tumor cells was studied using stable knockdown clones derived from a murine KRAS/p53-mutant lung adenocarcinoma cell line with high metastatic potential and an orthotopic syngeneic mouse model and in vitro using a CXCR2 small-molecule antagonist (SB225002). CXCR2 protein expression was analyzed in tumor cells from 262 NSCLC. Gene expression profiles for CXCR2 and its ligands (CXCR2 axis) were analyzed in 52 human NSCLC cell lines and 442 human lung adenocarcinomas. Methylation of CXCR2 axis promoters was determined in 70 human NSCLC cell lines. Invasion and metastasis were decreased in CXCR2 knockdown clones in vitro and in vivo. SB225002 decreased invasion in vitro. In lung adenocarcinomas, CXCR2 expression in tumor cells was associated with smoking and poor prognosis. CXCR2 axis gene expression profiles in human NSCLC cell lines and lung adenocarcinomas defined a cluster driven by CXCL5 and associated with smoking, poor prognosis, and RAS pathway activation. Expression of CXCL5 was regulated by promoter methylation. The CXCR2 axis may be an important target in smoking-related lung adenocarcinoma.