RESUMO
Cytoglobin (CYGB) is frequently downregulated in many types of human malignancies, and its exogenous overexpression reduces proliferation of cancer cells. Despite its implied tumour suppressor (TSG) functions, its exact role in carcinogenesis remains unclear as CYGB upregulation is also associated with tumour hypoxia and aggressiveness. In this study, we explore the TSG role of CYGB, its influence on the phenotype of cancerous cells under stress conditions and the clinical significance of CYGB expression and promoter methylation in non-small cell lung cancer (NSCLC). DNA methylation-dependent expression silencing of CYGB is demonstrated in both clinical samples and cell lines. CYGB promoter was more frequently methylated in lung adenocarcinomas (P = 1.4 × 10(-4)). Demethylation by 5'-azadeoxycytidine partially restored CYGB expression in cell lines. Interestingly, trichostatin A triggered upregulation of CYGB expression in cancer cell lines and downregulation in non-tumourigenic ones. CYGB mRNA expression in NSCLC surgical specimens correlated with that of HIF1α and VEGFa (P < 1 × 10(-4)). Overexpression of CYGB in cancer cell lines reduced cell migration, invasion and anchorage-independent growth. Moreover, CYGB impaired cell proliferation, but only in the lung adenocarcinoma cell line (H358). Upon hydrogen peroxide treatment, CYGB protected cell viability, migratory potential and anchorage independence by attenuating oxidative injury. In hypoxia, CYGB overexpression decreased cell viability, augmented migration and anchorage independence in a cell-type-specific manner. In conclusion, CYGB revealed TSG properties in normoxia but promoted tumourigenic potential of the cells exposed to stress, suggesting a bimodal function in lung tumourigenesis, depending on cell type and microenvironmental conditions.
Assuntos
Adenocarcinoma/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Globinas/genética , Globinas/metabolismo , Neoplasias Pulmonares/genética , Oncogenes , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Carcinoma Pulmonar de Células não Pequenas/patologia , Hipóxia Celular/genética , Linhagem Celular Tumoral , Citoglobina , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genes Supressores de Tumor , Humanos , Ácidos Hidroxâmicos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Although the prognosis of chronic myeloid leukemia (CML) patients treated with imatinib is good, many fail to develop an optimal response or lose one. This heterogeneity could be attributed to the presence of human organic cation transporter-1 (hOCT1) single nucleotide polymorphisms (SNPs). In the present study, we analyzed the effect of 23 hOCT1 SNPs on imatinib treatment outcome in newly diagnosed CML patients using MassARRAY sequencing and pyrosequencing. The only SNP associated with outcome was M420del (rs35191146), with patients with the M420del demonstrating an increased probability of imatinib treatment failure. In CML cell lines transfected with M420del and/or M408V, M420del significantly decreased imatinib uptake, but this effect was countered if the M408V (rs628031) SNP was also present. A similar effect was seen for the uptake of the hOCT1 substrates TEA(+) and ASP(+). Finally, apparent hOCT1 mRNA levels were studied using both our earlier primers covering the M420del and another set that did not. Different mRNA expression was observed, explaining the disparity in published data on the prognostic importance of hOCT1 mRNA and highlighting the importance of avoiding common SNP sites in primer design. These data demonstrate that the common M420del SNP can modulate the outcome of imatinib treatment.
Assuntos
Antineoplásicos/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Transportador 1 de Cátions Orgânicos/genética , Piperazinas/uso terapêutico , Polimorfismo de Nucleotídeo Único , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Antineoplásicos/farmacocinética , Benzamidas , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Genótipo , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Transportador 1 de Cátions Orgânicos/química , Transportador 1 de Cátions Orgânicos/metabolismo , Piperazinas/farmacocinética , Conformação Proteica , Inibidores de Proteínas Quinases/farmacocinética , Pirimidinas/farmacocinética , Resultado do Tratamento , Adulto JovemRESUMO
Lung cancer is the most common cause of cancer death worldwide, with over one million cases annually. To identify genetic factors that modify disease risk, we conducted a genome-wide association study by analysing 317,139 single-nucleotide polymorphisms in 1,989 lung cancer cases and 2,625 controls from six central European countries. We identified a locus in chromosome region 15q25 that was strongly associated with lung cancer (P = 9 x 10(-10)). This locus was replicated in five separate lung cancer studies comprising an additional 2,513 lung cancer cases and 4,752 controls (P = 5 x 10(-20) overall), and it was found to account for 14% (attributable risk) of lung cancer cases. Statistically similar risks were observed irrespective of smoking status or propensity to smoke tobacco. The association region contains several genes, including three that encode nicotinic acetylcholine receptor subunits (CHRNA5, CHRNA3 and CHRNB4). Such subunits are expressed in neurons and other tissues, in particular alveolar epithelial cells, pulmonary neuroendocrine cells and lung cancer cell lines, and they bind to N'-nitrosonornicotine and potential lung carcinogens. A non-synonymous variant of CHRNA5 that induces an amino acid substitution (D398N) at a highly conserved site in the second intracellular loop of the protein is among the markers with the strongest disease associations. Our results provide compelling evidence of a locus at 15q25 predisposing to lung cancer, and reinforce interest in nicotinic acetylcholine receptors as potential disease candidates and chemopreventative targets.
Assuntos
Cromossomos Humanos Par 15/genética , Predisposição Genética para Doença/genética , Neoplasias Pulmonares/genética , Subunidades Proteicas/genética , Receptores Nicotínicos/genética , Europa (Continente) , Genótipo , Humanos , Razão de Chances , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Since the discovery of cytoglobin (Cygb) a decade ago, growing amounts of data have been gathered to characterise Cygb biochemistry, functioning and implication in human pathologies. Its molecular roles remain under investigation, but nitric oxide dioxygenase and lipid peroxidase activities have been demonstrated. Cygb expression increases in response to various stress conditions including hypoxia, oxidative stress and fibrotic stimulation. When exogenously overexpressed, Cygb revealed cytoprotection against these factors. Cygb was shown to be upregulated in fibrosis and neurodegenerative disorders and downregulated in multiple cancer types. CYGB was also found within the minimal region of a hereditary tylosis with oesophageal cancer syndrome, and its expression was reduced in tylotic samples. Recently, Cygb has been shown to inhibit cancer cell growth in vitro, thus confirming its suggested tumour suppressor role. This article aims to review the biochemical and functional aspects of Cygb, its involvement in various pathological conditions and potential clinical utility.
Assuntos
Globinas/metabolismo , Terapia de Alvo Molecular , Animais , Citoglobina , Globinas/química , Globinas/genética , HumanosRESUMO
Mass spectrometric profiling approaches such as MALDI-TOF and SELDI-TOF are increasingly being used in disease marker discovery, particularly in the lower molecular weight proteome. However, little consideration has been given to the issue of sample size in experimental design. The aim of this study was to develop a protocol for the use of sample size calculations in proteomic profiling studies using MS. These sample size calculations can be based on a simple linear mixed model which allows the inclusion of estimates of biological and technical variation inherent in the experiment. The use of a pilot experiment to estimate these components of variance is investigated and is shown to work well when compared with larger studies. Examination of data from a number of studies using different sample types and different chromatographic surfaces shows the need for sample- and preparation-specific sample size calculations.
Assuntos
Análise Serial de Proteínas/métodos , Proteoma/análise , Projetos de Pesquisa , Análise de Variância , Proteínas Sanguíneas/análise , Humanos , Espectrometria de Massas , Plasma/química , Tamanho da AmostraRESUMO
LINE-1 and Alu elements are non-LTR retrotransposons, constituting together over 30% of the human genome and they are frequently hypomethylated in human tumors. A relationship between global hypomethylation and genomic instability has been shown, however, there is little evidence to suggest active role for hypomethylation-mediated reactivation of retroelements in human cancer. In our study, we examined by Pyrosequencing the methylation levels of LINE-1 and Alu sequences in 48 primary nonsmall cell carcinomas and their paired adjacent tissues. We demonstrate a significant reduction of the methylation levels of both elements (p = 7.7 x 10(-14) and 9.6 x 10(-7), respectively). The methylation indices of the 2 elements correlated (p = 0.006), suggesting a possible common mechanism for their methylation maintenance. Genomic instability was measured utilizing 11 fluorescent microsatellite markers located on lung cancer hot-spot regions such as 3p, 5q 9p, 13q and 17p. Hypomethylation of both transposable elements was associated with increased genomic instability (LINE, p = 7.1 x 10(-5); Alu, p = 0.008). The reduction of the methylation index of LINE-1 and Alu following treatment of 3 lung cell lines with 5-aza-2'-deoxycitidine, consistently resulted in increased expression of both elements. Our study demonstrates the strong link between hypomethylation of transposable elements with genomic instability in non-small cell lung cancer and provides early evidence for a potential active role of these elements in lung neoplasia. As demethylating agents are now entering lung cancer trials, it is imperative to gain a greater insight into the potential reactivation of silent retrotransposons in order to advance for the clinical utilization of epigenetics in cancer therapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA , Instabilidade Genômica , Neoplasias Pulmonares/genética , Retroelementos/genética , Idoso , Idoso de 80 Anos ou mais , Elementos Alu , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Feminino , Genoma Humano , Humanos , Elementos Nucleotídeos Longos e Dispersos/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
Endothelin-1 is overexpressed in several tumor types. Activation of the endothelin-A (ETA) receptor may promote cell growth, angiogenesis and invasion, and inhibits the apoptotic process, while activation of the endothelin-B (ETB) receptor may induce cell death by apoptosis and inhibit tumor progression. Hypermethylation and subsequent silencing of the ETB receptor gene promoter has been reported in some cancer types. As the endothelin pathway is subject to research for pharmacological cancer treatment, we investigated the extent of epigenetic deregulation of the ETB receptor gene in non-small cell lung cancer (NSCLC). We scanned 64 NSCLC paired tumor/normal surgical specimens for the ETB receptor promoter for methylation by developing four pyrosequencing assays that covered 24 CpGs. The ETB receptor promoter was significantly hypermethylated in 31 (48%) of tumor samples, presenting considerably higher methylation in 22/24 CpG sites compared with the normal counterpart tissues. ETB receptor mRNA levels were reduced in all lung tumors compared with normal adjacent lung tissue, indicating the potentially important involvement of this gene in lung cancer development. Furthermore, tumor samples with ETB receptor gene methylation tended to have lower receptor mRNA levels compared with unmethylated tumor specimens, suggesting a primary epigenetic role in ETB receptor silencing. Our results point to a significant involvement of ETB receptor epigenetic deregulation in the pathogenesis of lung cancer making the gene a promising candidate biomarker for response to regimens modulating the endothelin axis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Inativação Gênica , Neoplasias Pulmonares/genética , Receptor de Endotelina B/genética , Sequência de Bases , Metilação de DNA , DNA de Neoplasias/genética , Repetições de Dinucleotídeos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Neoplásico/genéticaRESUMO
Aberrant promoter methylation is frequently observed in different types of lung cancer. Epigenetic modifications are believed to occur before the clinical onset of the disease and hence hold a great promise as early detection markers. Extensive analysis of DNA methylation has been impeded by methods that are either too labor intensive to allow large-scale studies or not sufficiently quantitative to measure subtle changes in the degree of methylation. We used a novel quantitative DNA methylation analysis technology to complete a large-scale cytosine methylation profiling study involving 47 gene promoter regions in 96 lung cancer patients. Each individual contributed a lung cancer specimen and corresponding adjacent normal tissue. The study identified six genes with statistically significant differences in methylation between normal and tumor tissue (P < 10(-6)). We explored the quantitative methylation data using an unsupervised hierarchical clustering algorithm. The data analysis revealed that methylation patterns differentiate normal from tumor tissue. For validation of our approach, we divided the samples to train a classifier and test its performance. We were able to distinguish normal from lung cancer tissue with >95% sensitivity and specificity. These results show that quantitative cytosine methylation profiling can be used to identify molecular classification markers in lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA , Neoplasias Pulmonares/genética , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Análise por Conglomerados , Ilhas de CpG , Citosina/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Feminino , Marcadores Genéticos/genética , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Estadiamento de NeoplasiasRESUMO
The molecular basis for alternative lengthening of telomeres (ALT), a prognostic marker for glioma patients, remains unknown. We examined TP53 status in relation to telomere maintenance mechanism (TMM) in 108 patients with glioblastoma multiforme and two patients with anaplastic astrocytoma from New Zealand and United Kingdom. Tumor samples were analyzed with respect to telomerase activity, telomere length, and ALT-associated promyelocytic leukemia nuclear bodies to determine their TMM. TP53 mutation was analyzed by direct sequencing of coding exons 2 to 11. We found an association between TP53 mutation and ALT mechanism and between wild-type TP53 and telomerase and absence of a known TMM (P < 0.0001). We suggest that TP53 deficiency plays a permissive role in the activation of ALT.
Assuntos
Astrocitoma/genética , Genes p53 , Glioblastoma/genética , Mutação , Telômero/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
PURPOSE: Collagen 1A1 (COL1A1), RNA-binding and pre-mRNA Processing Factor (PRPF40A), and Uncoupling Protein 2 (UCP2) were identified as downstream effectors of cytoglobin (CYGB), which was shown implicated in tumour biology. Although these three genes have been previously associated with cancer, little is known about their status in lung malignancies. METHODS: Hereby, we investigated the expression and promoter methylation of COL1A1, PRPF40A, and UCP2 in 156 non-small cell lung cancer (NSCLC) and adjacent normal tissues. RESULTS: We demonstrate that COL1A1 and PRPF40A mRNAs are significantly overexpressed in NSCLC (p < 1 × 10-4), while UCP2 exhibits a trend of upregulation (p = 0.066). Only COL1A1 promoter revealed hypermethylation in NSCLCs (36%), which was particularly evident in squamous cell carcinomas (p = 0.024) and in the tumours with moderate-to-good differentiation (p = 0.01). Transcript level of COL1A1, as well as PRPF40A and UCP2, exhibited striking association (p ≤ 0.001) with the expression of hypoxia markers. In addition, we demonstrate in lung cancer cell lines exposed to hypoxia or oxidative stress that COL1A1 transcription significantly responds to oxygen depletion, while other genes showed the modest upregulation in stress conditions. CONCLUSION: In conclusion, our data revealed that COL1A1, UCP2, and PRPF40A are novel players implicated in the complex network of hypoxia response in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Transporte/biossíntese , Colágeno Tipo I/biossíntese , Neoplasias Pulmonares/patologia , Proteína Desacopladora 2/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas de Transporte/genética , Hipóxia Celular/fisiologia , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Metilação de DNA , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Transcriptoma , Proteína Desacopladora 2/genética , Regulação para CimaRESUMO
AIM: Misoprostol, a prostaglandin analogue used for the treatment of postpartum hemorrhage and termination of pregnancy, can cause high fevers. Genetic susceptibility may play a role in misoprostol-induced fever. SUBJECTS & METHODS: Body temperature of women treated with misoprostol for termination of pregnancy in the UK (n = 107) and for postpartum hemorrhage in Ecuador (n = 50) was measured. Genotyping for 33 single nucleotide polymorphisms in 15 candidate genes was performed. Additionally, we investigated the transport of radiolabeled misoprostol acid across biological membranes in vitro. RESULTS: The ABCC4 single nucleotide polymorphism rs11568658 was associated with misoprostol-induced fever. Misoprostol acid was transported across a blood-brain barrier model by MRP4 and SLCO1B1. CONCLUSION: Genetic variability in ABCC4 may contribute to misoprostol-induced fever in pregnant women. Original submitted 21 January 2015; Revision submitted 24 April 2015.
Assuntos
Abortivos não Esteroides/efeitos adversos , Febre/induzido quimicamente , Febre/genética , Misoprostol/efeitos adversos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Transportadores de Ânions Orgânicos/genética , Polimorfismo Genético/genética , Abortivos não Esteroides/metabolismo , Adulto , Barreira Hematoencefálica/metabolismo , Temperatura Corporal , Linhagem Celular , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , América Latina , Transportador 1 de Ânion Orgânico Específico do Fígado , Misoprostol/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Hemorragia Pós-Operatória/complicações , Hemorragia Pós-Operatória/tratamento farmacológico , Hemorragia Pós-Operatória/genética , Gravidez , População BrancaRESUMO
BACKGROUND: Darunavir is a potent protease inhibitor of HIV. To enhance its pharmacokinetic profile, darunavir must be co-administered with ritonavir. There is wide inter-patient variability in darunavir pharmacokinetics among HIV-infected individuals, however. Darunavir is a known substrate for influx transporters, such as the 1A2 and the 1B1 members of the solute carrier organic anion transporter family (SLCO1A2, SLCO1B1), as well as for efflux transporters such as the multi-drug resistance protein 1 (MRP1). OBJECTIVE: The aim of this study was to develop a semi-mechanistic population pharmacokinetic model for darunavir and ritonavir administered in HIV-infected adults. The desired model would incorporate patient characteristics and pharmacogenetic data contributing to variability in drug concentrations and also take into account the interaction between the two compounds. METHODS: A population pharmacokinetic analysis was performed with 705 plasma samples from 75 Caucasian individuals receiving darunavir/ritonavir (600/100 mg twice daily) for at least 4 weeks. At least one full pharmacokinetic profile was obtained for each participant, and darunavir and ritonavir concentrations in plasma were determined by high performance liquid chromatography. Genotyping for 148 polymorphisms in genes coding for transporters or metabolizing enzymes was conducted by two methods: MALDI-TOF mass spectrometry and real-time polymerase chain reaction-based allelic discrimination. A population pharmacokinetic model was developed for darunavir and for ritonavir. The effect of single nucleotide polymorphisms on the post hoc individual pharmacokinetic parameters was first explored using graphic methods and regression analysis. Those covariates related to changes in darunavir or ritonavir pharmacokinetic parameters were then further evaluated using non-linear mixed effects modeling (NONMEM version VII). RESULTS: Darunavir and ritonavir pharmacokinetics were best described by a two- and one-compartment model, respectively, both with first-order absorption and elimination. The darunavir peripheral volume of distribution decreased as α1-acid glycoprotein concentrations increased. Darunavir clearance was 12 % lower in patients with SLCO3A1 rs8027174 GT/TT genotypes, while homozygosity for the rs4294800 A allele was associated with 2.5-fold higher central volume of distribution. Body weight influenced ritonavir clearance. Ritonavir inhibited darunavir clearance following a maximum-effect model. CONCLUSION: A population pharmacokinetic model to simultaneously describe the pharmacokinetics of darunavir and ritonavir was developed in HIV-infected patients. The model provides better understanding of the interaction between darunavir and ritonavir and suggests an association between SLCO3A1 polymorphisms and darunavir pharmacokinetics. Bayesian estimates of individual darunavir parameters and ritonavir may be useful to predict darunavir exposure.
Assuntos
Infecções por HIV/genética , Inibidores da Protease de HIV/farmacocinética , Transportadores de Ânions Orgânicos/genética , Ritonavir/farmacocinética , Sulfonamidas/farmacocinética , Adulto , Darunavir , Quimioterapia Combinada , Feminino , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/administração & dosagem , Inibidores da Protease de HIV/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Polimorfismo de Nucleotídeo Único , Ritonavir/administração & dosagem , Ritonavir/sangue , Sulfonamidas/administração & dosagem , Sulfonamidas/sangueRESUMO
p73 possesses an extrinsic P1 promoter and an intrinsic P2 promoter controlling the expression of the pro-apoptotic TAp73 isoforms and the anti-apoptotic ΔΝp73 isoforms respectively. In this study, we investigated the DNA methylation status of both promoters as a means of epigenetic transcriptional control of their corresponding isoforms in 102 primary non-small cell lung carcinomas (NSCLCs). We demonstrated that while P1 hypermethylation-associated reduction of TAp73 mRNA levels is relatively infrequent, the P2 hypomethylation-associated over-expression of ΔΝp73 mRNA is a frequent event, particularly among squamous cell carcinomas. P2 hypomethylation strongly correlated with LINE-1 element hypomethylation, indicating that ΔΝp73 over-expression may be a passive consequence of global DNA hypomethylation.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Neoplasias Pulmonares/genética , Proteínas Nucleares/genética , Ativação Transcricional , Proteínas Supressoras de Tumor/genética , Adenocarcinoma/genética , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Proteína Tumoral p73RESUMO
Globins are respiratory proteins involved in oxygen metabolism, which is a critical factor in tumor growth and progression. The status of neuroglobin and myoglobin is largely unknown in human malignancies, including lung cancer. The aim of this study was to explore mRNA expression profiles, potential regulatory mechanisms and clinicopathological associations of neuroglobin and myoglobin in non-small cell lung cancer (NSCLC). We screened 208 surgically resected NSCLC specimens and a panel of lung normal and cancer cell lines. The mRNA expression of neuroglobin, myoglobin and hypoxia markers (HIF1α and VEGFa) was measured with qRTPCR, while neuroglobin promoter methylation was assessed with Pyrosequencing. Neuroglobin and myoglobin were upregulated in the tumor samples compared to normal tissue (p=1.3×10(-22) and p=1.9×10(-9), respectively). Neuroglobin was more frequently overexpressed in squamous cell carcinomas (SqCCL) than adenocarcinomas. Overexpression of myoglobin was more profound in adenocarcinomas, which correlated with poor survival (p=0.013). Neuroglobin promoter was hypermethylated in 30.8% of NSCLC cases, which correlated with neuroglobin mRNA downregulation. The epigenetic regulation of neuroglobin was confirmed by treating lung cell lines with 5'azadeoxycytidine and/or trichostatin A. Expression of both genes correlated with the expression of HIF1α (neuroglobin: p=3.8×10(-5), myoglobin: p=1.1×10(-11)). Myoglobin expression was also associated to that of VEGFa (p=2.1×10(-7)). Hypoxia-dependent upregulation of both globins was validated in vitro. In summary, neuroglobin and myoglobin overexpression in NSCLC is associated with histological subtype, hypoxia and, in case of neuroglobin - epigenetic regulation. Myoglobin expression may have potential significance in the prognostication of lung adenocarcinomas.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Regulação da Expressão Gênica , Globinas/metabolismo , Neoplasias Pulmonares/diagnóstico , Mioglobina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Adulto , Idoso , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Metilação de DNA , Feminino , Globinas/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mioglobina/genética , Proteínas do Tecido Nervoso/genética , Neuroglobina , PrognósticoRESUMO
BACKGROUND: The UHRF1 gene possesses an essential role in DNA methylation maintenance, but its contribution to tumor suppressor gene hypermethylation in primary human cancers currently remains unclear. METHODS: mRNA expression levels of UHRF1, DNMT1, DNMT3A, DNMT3B, and E2F1 were evaluated in 105 primary nonsmall cell lung carcinomas by quantitative polymerase chain reaction. The methylation status of CDKN2A and RASSF1 promoters was examined by pyrosequencing. UHRF1 was knocked down by short hairpin RNA in A549 lung adenocarcinoma cells. RESULTS: All 4 genes were overexpressed in a coordinated manner in the lung tumor tissues, and their expression correlated with that of E2F1. Higher UHRF1 expression in tumor tissues correlated with the hypermethylation of CDKN2A (P = .005) and RASSF1 promoters (P = .034), and the relationship with a combined epigenotype was even stronger (P = 2.3 × 10(-4) ). When UHRF1 was knocked down in A549 lung adenocarcinoma cells, lower methylation levels of RASSF1, CYGB, and CDH13 promoters were observed. Also, UHRF1 knockdown clones demonstrated reduced proliferation and decreased cell migration properties. CONCLUSIONS: Our data demonstrate that UHRF1 is a key epigenetic switch, which controls cell cycle in nonsmall cell lung carcinoma through its ability to sustain the transcriptional silencing of tumor suppressor genes by maintaining their promoters in a hypermethylated status. Thus, UHRF1 should be considered, along with DNMTs, among the potential targets for cancer treatment and/or therapeutic stratification.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Carcinoma Pulmonar de Células não Pequenas/genética , Inativação Gênica , Neoplasias Pulmonares/genética , Proteínas Estimuladoras de Ligação a CCAAT/antagonistas & inibidores , Proteínas Estimuladoras de Ligação a CCAAT/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica/fisiologia , Genes Supressores de Tumor/fisiologia , Humanos , Neoplasias Pulmonares/patologia , Masculino , Transfecção , Células Tumorais Cultivadas , Ubiquitina-Proteína Ligases , DNA Metiltransferase 3BRESUMO
Killer immunoglobulin-like receptor (KIR) and human leukocyte antigen (HLA) genotypes were analyzed from panels of lung (non-small-cell lung cancer [NSCLC] and small-cell lung cancer [SCLC]), colon, and kidney cancer patients and compared with normal control subjects. No significant differences were noted between KIR gene frequencies in patients compared with normal subjects. When combinations of KIR genes and their HLA ligands were considered, there were significant decreases in frequencies of both KIR2DL2 and KIR2DL3 in homozygotes for their ligand HLA-C1, and an increase in the frequency of KIR3DL1 and its ligand HLA-Bw4 in kidney cancer patients compared with controls. Both associations were partly attributable to changes in ligand frequencies alone. NSCLC patients showed a significant increase in the frequency of KIR2DL1 and its ligand HLA-C2 and a corresponding decrease in frequency of KIR2DL3 and its ligand HLA-C1 in homozygotes. In NSCLC, the Ile80 form of HLA-Bw4 was decreased in KIR3DL1+ HLA-Bw4+ patients, whereas in SCLC the Ile80 form was increased and the Thr80 form decreased in KIR3DS1+ HLA-Bw4+ patients. These findings are consistent with increased co-expression of high-affinity inhibitory KIRs and their ligands, potentially resulting in decreased natural killer cell function, and hence with natural killer cells having a protective role in lung and kidney cancer but not colon cancer.
Assuntos
Antígenos HLA-C/genética , Neoplasias/genética , Neoplasias/imunologia , Receptores KIR2DL2/genética , Receptores KIR2DL3/genética , Frequência do Gene , Estudos de Associação Genética , Antígenos HLA-C/imunologia , Haplótipos , Teste de Histocompatibilidade , Humanos , Neoplasias/patologia , Neoplasias/fisiopatologia , Receptores KIR2DL2/imunologia , Receptores KIR2DL3/imunologia , Reino UnidoRESUMO
BACKGROUND: Genetic variants in 15q25 have been identified as potential risk markers for lung cancer (LC), but controversy exists as to whether this is a direct association, or whether the 15q variant is simply a proxy for increased exposure to tobacco carcinogens. METHODS: We performed a detailed analysis of one 15q single nucleotide polymorphism (SNP) (rs16969968) with smoking behaviour and cancer risk in a total of 17 300 subjects from five LC studies and four upper aerodigestive tract (UADT) cancer studies. RESULTS: Subjects with one minor allele smoked on average 0.3 cigarettes per day (CPD) more, whereas subjects with the homozygous minor AA genotype smoked on average 1.2 CPD more than subjects with a GG genotype (P < 0.001). The variant was associated with heavy smoking (>20 CPD) [odds ratio (OR) = 1.13, 95% confidence interval (CI) 0.96-1.34, P = 0.13 for heterozygotes and 1.81, 95% CI 1.39-2.35 for homozygotes, P < 0.0001]. The strong association between the variant and LC risk (OR = 1.30, 95% CI 1.23-1.38, P = 1 x 10(-18)), was virtually unchanged after adjusting for this smoking association (smoking adjusted OR = 1.27, 95% CI 1.19-1.35, P = 5 x 10(-13)). Furthermore, we found an association between the variant allele and an earlier age of LC onset (P = 0.02). The association was also noted in UADT cancers (OR = 1.08, 95% CI 1.01-1.15, P = 0.02). Genome wide association (GWA) analysis of over 300 000 SNPs on 11 219 subjects did not identify any additional variants related to smoking behaviour. CONCLUSIONS: This study confirms the strong association between 15q gene variants and LC and shows an independent association with smoking quantity, as well as an association with UADT cancers.
Assuntos
Cromossomos Humanos Par 15/genética , Neoplasias Pulmonares/genética , Polimorfismo de Nucleotídeo Único , Receptores Nicotínicos/genética , Fumar/efeitos adversos , Tabagismo/genética , Idoso , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Razão de ChancesRESUMO
Cytoglobin (CYGB) is a recently discovered vertebrate globin distantly related to myoglobin with unknown function. CYGB is assigned to chromosomal region 17q25, which is frequently lost in multiple malignancies. Previous studies failed to detect evidence for mutations in the CYGB gene. Recent studies provided preliminary evidence for increased methylation of the gene in lung cancer. Our study was aimed at investigating the role of CYGB as a tumor suppressor gene. By nested methylation-specific DNA sequencing analysis of lung and breast cancer cell lines and bronchial and mammary epithelial cell lines, we identified that methylation of a 110-bp CpG-rich segment of the CYGB promoter was correlated with gene silencing. We specifically targeted this sequence and developed a quantitative methylation-specific PCR assay, suitable for high-throughput analysis. We showed that the tumor specificity of CYGB methylation in discriminating patients with and without lung cancer, using biopsies and sputum samples. We further showed the tumor specificity of this assay with multiple other epithelial and hematologic malignancies. To show tumor suppressor activity of CYGB, we performed the following: (a) RNA interference-mediated knockdown of CYGB gene on colony formation in a CYGB expression-positive lung cancer cell line, resulting in increased colony formation; (b) enforced gene expression in CYGB expression-negative lung and breast cancer cell lines, reducing colony formation; and (c) identification of potential proximate targets down-stream of the CYGB genes. Our data constitute the first direct functional evidence for CYGB, the newest member of the globin family, as a tumor suppressor gene.
Assuntos
Genes Supressores de Tumor , Globinas/genética , Neoplasias/genética , Neoplasias da Mama/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Citoglobina , Metilação de DNA , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Neoplasias Pulmonares/genética , Reação em Cadeia da Polimerase , TransfecçãoRESUMO
We carried out a genome-wide association study of lung cancer (3,259 cases and 4,159 controls), followed by replication in 2,899 cases and 5,573 controls. Two uncorrelated disease markers at 5p15.33, rs402710 and rs2736100 were detected by the genome-wide data (P = 2 x 10(-7) and P = 4 x 10(-6)) and replicated by the independent study series (P = 7 x 10(-5) and P = 0.016). The susceptibility region contains two genes, TERT and CLPTM1L, suggesting that one or both may have a role in lung cancer etiology.
Assuntos
Cromossomos Humanos Par 5/genética , Predisposição Genética para Doença , Neoplasias Pulmonares/genética , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Telomerase/genética , Apoptose , Estudos de Casos e Controles , Estudo de Associação Genômica Ampla , Humanos , Análise de Componente Principal , FumarRESUMO
Lung cancer demonstrates the highest mortality in the UK. Previous studies have implicated allelic loss at chromosome 17q in the development of non-small cell lung carcinoma (NSCLC), and a number of known and putative tumour-suppressor genes reside within this region. One candidate tumour-suppressor gene is cytoglobin (CYGB), which is contained entirely within the 42.5 kb tylosis with oesophageal cancer (TOC) minimal region. CYGB abnormalities have been demonstrated only in sporadic head and neck cancers. In this study, we investigated the expression, promoter methylation and allelic imbalance status of this gene in 52 paired (normal/tumour) surgically excised lung tissue samples from patients with NSCLC. CYGB expression in tumour tissue was significantly reduced compared with corresponding adjacent normal in 54% of the examined cases (paired t-test, P<0.001). The CYGB promoter was shown by pyrosequencing to be significantly hypermethylated [2-fold increase of methylation index (MtI) in tumours] in 25/52 (48%) tumour samples compared with normal samples. MtI of the CYGB promoter was associated with CYGB mRNA expression (linear regression analysis, P=0.009), suggesting a primary role for the epigenetic events in CYGB silencing. In addition, frequent LOH was detected at the locus 17q25 in 32/48 (67%) tumours examined. It is of note that the loss of expression intensified when both LOH and hypermethylation coincided in samples (Mann-Whitney, P=0.049). These findings provide the first evidence to suggest the implication of CYGB in the pathogenesis of NSCLCs.