RESUMEN
RATIONALE: Despite shared environmental exposures, idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease are usually studied in isolation, and the presence of shared molecular mechanisms is unknown. OBJECTIVES: We applied an integrative genomic approach to identify convergent transcriptomic pathways in emphysema and IPF. METHODS: We defined the transcriptional repertoire of chronic obstructive pulmonary disease, IPF, or normal histology lungs using RNA-seq (n = 87). MEASUREMENTS AND MAIN RESULTS: Genes increased in both emphysema and IPF relative to control were enriched for the p53/hypoxia pathway, a finding confirmed in an independent cohort using both gene expression arrays and the nCounter Analysis System (n = 193). Immunohistochemistry confirmed overexpression of HIF1A, MDM2, and NFKBIB members of this pathway in tissues from patients with emphysema or IPF. Using reads aligned across splice junctions, we determined that alternative splicing of p53/hypoxia pathway-associated molecules NUMB and PDGFA occurred more frequently in IPF or emphysema compared with control and validated these findings by quantitative polymerase chain reaction and the nCounter Analysis System on an independent sample set (n = 193). Finally, by integrating parallel microRNA and mRNA-Seq data on the same samples, we identified MIR96 as a key novel regulatory hub in the p53/hypoxia gene-expression network and confirmed that modulation of MIR96 in vitro recapitulates the disease-associated gene-expression network. CONCLUSIONS: Our results suggest convergent transcriptional regulatory hubs in diseases as varied phenotypically as chronic obstructive pulmonary disease and IPF and suggest that these hubs may represent shared key responses of the lung to environmental stresses.
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Redes Reguladoras de Genes/genética , Fibrosis Pulmonar Idiopática/genética , Enfermedad Pulmonar Obstructiva Crónica/genética , Adulto , Enfisema/genética , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas I-kappa B/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Proteínas del Tejido Nervioso/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismoRESUMEN
RATIONALE: The relatively sparse but diverse microbiome in human lungs may become less diverse in chronic obstructive pulmonary disease (COPD). This article examines the relationship of this microbiome to emphysematous tissue destruction, number of terminal bronchioles, infiltrating inflammatory cells, and host gene expression. METHODS: Culture-independent pyrosequencing microbiome analysis was used to examine the V3-V5 regions of bacterial 16S ribosomal DNA in 40 samples of lung from 5 patients with COPD (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 4) and 28 samples from 4 donors (controls). A second protocol based on the V1-V3 regions was used to verify the bacterial microbiome results. Within lung tissue samples the microbiome was compared with results of micro-computed tomography, infiltrating inflammatory cells measured by quantitative histology, and host gene expression. MEASUREMENTS AND MAIN RESULTS: Ten operational taxonomic units (OTUs) was found sufficient to discriminate between control and GOLD stage 4 lung tissue, which included known pathogens such as Haemophilus influenzae. We also observed a decline in microbial diversity that was associated with emphysematous destruction, remodeling of the bronchiolar and alveolar tissue, and the infiltration of the tissue by CD4(+) T cells. Specific OTUs were also associated with neutrophils, eosinophils, and B-cell infiltration (P < 0.05). The expression profiles of 859 genes and 235 genes were associated with either enrichment or reductions of Firmicutes and Proteobacteria, respectively, at a false discovery rate cutoff of less than 0.1. CONCLUSIONS: These results support the hypothesis that there is a host immune response to microorganisms within the lung microbiome that appears to contribute to the pathogenesis of COPD.
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Microbiota , Enfermedad Pulmonar Obstructiva Crónica/microbiología , Enfermedad Pulmonar Obstructiva Crónica/patología , Bronquiolos/patología , Linfocitos T CD4-Positivos , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Infiltración Neutrófila , Enfermedad Pulmonar Obstructiva Crónica/inmunologíaRESUMEN
Chronic cigarette smoking exposes airway epithelial cells to thousands of carcinogens, oxidants and DNA-damaging agents, creating a field of molecular injury in the airway and altering gene expression. Studies of cytologically normal bronchial epithelial cells from smokers have identified transcription-based biomarkers that may prove useful in early diagnosis of lung cancer, including a number of p53-regulated genes. The ability of p53 to regulate transcription is critical for tumor suppression, and this suggests that single-nucleotide polymorphisms (SNPs) in functional p53 binding sites (p53 response elements, or p53REs) that affect gene expression could influence susceptibility to cancer. To connect p53RE SNP genotype with gene expression and cancer risk, we identified a set of 204 SNPs in putative p53REs, and performed cis expression quantitative trait loci (eQTL) analysis, assessing associations between SNP genotypes and mRNA levels of adjacent genes in bronchial epithelial cells obtained from 44 cigarette smokers. To further test and validate these genotype-expression associations, we searched published eQTL studies from independent populations and determined that 53% (39/74) of the bronchial epithelial eQTLs were observed in at least one of other studies. SNPs in p53REs were also evaluated for effects on p53-DNA binding using a quantitative in vitro protein-DNA binding assay. Last, based on linkage disequilibrium, we found 6 p53RE SNPs associated with gene expression were identified as cancer risk SNPs by either genome-wide association studies or candidate gene studies. We provide an approach for identifying and evaluating potentially functional SNPs that may modulate the airway gene expression response to smoking and may influence susceptibility to cancers.
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Células Epiteliales/metabolismo , Neoplasias Pulmonares/metabolismo , Elementos de Respuesta , Fumar/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Secuencia de Bases , Sitios de Unión , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Desequilibrio de Ligamiento , Neoplasias Pulmonares/etiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple , Unión Proteica , Sitios de Carácter Cuantitativo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Riesgo , Fumar/efectos adversos , TranscriptomaRESUMEN
Lung cancer is the leading cause of cancer death worldwide in part due to our inability to identify which smokers are at highest risk and the lack of effective tools to detect the disease at its earliest and potentially curable stage. Recent results from the National Lung Screening Trial have shown that annual screening of high-risk smokers with low-dose helical computed tomography of the chest can reduce lung cancer mortality. However, molecular biomarkers are needed to identify which current and former smokers would benefit most from annual computed tomography scan screening in order to reduce the costs and morbidity associated with this procedure. Additionally, there is an urgent clinical need to develop biomarkers that can distinguish benign from malignant lesions found on computed tomography of the chest given its very high false positive rate. This review highlights recent genetic, transcriptomic and epigenomic biomarkers that are emerging as tools for the early detection of lung cancer both in the diagnostic and screening setting.
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Epigénesis Genética/genética , Perfilación de la Expresión Génica/métodos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Tamizaje Masivo/métodos , Diagnóstico Precoz , Perfilación de la Expresión Génica/tendencias , Marcadores Genéticos/genética , Estudio de Asociación del Genoma Completo/métodos , Estudio de Asociación del Genoma Completo/tendencias , Humanos , Neoplasias Pulmonares/epidemiología , Tamizaje Masivo/tendencias , Factores de RiesgoRESUMEN
The tremendous expansion of data analytics and public and private big datasets presents an important opportunity for pre-clinical drug discovery and development. In the field of life sciences, the growth of genetic, genomic, transcriptomic and proteomic data is partly driven by a rapid decline in experimental costs as biotechnology improves throughput, scalability, and speed. Yet far too many researchers tend to underestimate the challenges and consequences involving data integrity and quality standards. Given the effect of data integrity on scientific interpretation, these issues have significant implications during preclinical drug development. We describe standardized approaches for maximizing the utility of publicly available or privately generated biological data and address some of the common pitfalls. We also discuss the increasing interest to integrate and interpret cross-platform data. Principles outlined here should serve as a useful broad guide for existing analytical practices and pipelines and as a tool for developing additional insights into therapeutics using big data.
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Macrodatos , Investigación Biomédica/normas , Descubrimiento de Drogas , Control de CalidadRESUMEN
The introduction of microCT has made it possible to show that the terminal bronchioles are narrowed and destroyed before the onset of emphysematous destruction in COPD. This report extends those observations to the cellular and molecular level in the centrilobular phenotype of emphysematous destruction in lungs donated by persons with very severe COPD (n = 4) treated by lung transplantation with unused donor lungs (n = 4) serving as controls. These lung specimens provided companion samples to those previously examined by microCT (n = 61) that we examined using quantitative histology (n = 61) and gene expression profiling (n = 48). The histological analysis showed that remodeling and destruction of the bronchiolar and alveolar tissue is associated with macrophage, CD4, CD8, and B cell infiltration with increased formation of tertiary lymphoid organs. Moreover, gene set enrichment analysis showed that genes known to be expressed by natural killer (NK), lymphoid tissue inducer (LTi), and innate lymphoid cell 1 (ILC1) cells, but not ILC2 or ILC3 cells, were enriched in the expression profiles associated with CD4, CD8, and B cell infiltration. Based on these findings, we postulate that the centrilobular phenotype of emphysematous destruction COPD is driven by a Th1 response activated by infiltrating ILC1, NK, and LTi cells.
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Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Enfisema Pulmonar/etiología , Enfisema Pulmonar/patología , Biomarcadores , Biopsia , Femenino , Perfilación de la Expresión Génica , Humanos , Sistema Inmunológico/inmunología , Sistema Inmunológico/metabolismo , Sistema Inmunológico/patología , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfisema Pulmonar/metabolismo , Pruebas de Función Respiratoria , Factores de Riesgo , Microtomografía por Rayos XRESUMEN
PURPOSE: The EGFR tyrosine kinase inhibitors (TKIs) demonstrate efficacy in NSCLC patients whose tumors harbor activating EGFR mutations. However, patients who initially respond to EGFR TKI treatment invariably develop resistance to the drugs. Known mechanisms account for approximately 70% of native and acquired EGFR TKI resistance. In the current study we investigated a novel mechanism of NSCLC resistance to erlotinib. EXPERIMENTAL DESIGN: The mechanisms of acquired erlotinib resistance were evaluated by microarray analysis in thirteen NSCLC cell lines and in vivo in mice. Correlations between plasma neutrophil gelatinase associated lipocalin (NGAL) levels, erlotinib response and the EGFR mutational status were assessed in advanced stage NSCLC patients treated with erlotinib. RESULTS: In 5 of 13 NSCLC cell lines NGAL was significantly upregulated. NGAL knockdown in erlotinib-resistant cells increased erlotinib sensitivity in vitro and in vivo. NGAL overexpression in erlotinib-sensitive cells augmented apoptosis resistance. This was mediated by NGAL-dependent modulation of the pro-apoptotic protein Bim levels. Evaluation of the plasma NGAL levels in NSCLC patients that received erlotinib revealed that patients with lower baseline NGAL demonstrated a better erlotinib response. Compared to patients with wild type EGFR, patients with activating EGFR mutations had lower plasma NGAL at baseline and weeks 4 and 8. CONCLUSIONS: Our studies uncover a novel mechanism of NGAL-mediated modulation of Bim levels in NSCLC that might contribute to TKI resistance in lung cancer patients. These findings provide the rationale for the further investigations of the utility of NGAL as a potential therapeutic target or diagnostic biomarker.
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The "field of injury" hypothesis proposes that exposure to an inhaled insult such as cigarette smoke elicits a common molecular response throughout the respiratory tract. This response can therefore be quantified in any airway tissue, including readily accessible epithelial cells in the bronchus, nose, and mouth. High-throughput technologies, such as whole-genome gene expression microarrays, can be employed to catalog the physiological consequences of such exposures in the airway epithelium. Pulmonary diseases such as chronic obstructive pulmonary disease, lung cancer, and asthma are also thought to be associated with a field of injury, and in patients with these diseases, airway epithelial cells can be a useful surrogate for diseased tissue that is often difficult to obtain. Global measurement of mRNA and microRNA expression in these cells can provide useful information about the molecular pathogenesis of such diseases and may be useful for diagnosis and for predicting prognosis and response to therapy. In this review, our aim is to summarize the history and state of the art of such "transcriptomic" studies in the human airway epithelium, especially in smoking and smoking-related lung diseases, and to highlight future directions for this field.