Bronchial Airway Epithelium Research Articles

Abstract 3077: Dysregulation of microRNA-mRNA regulatory networks in the bronchial airway epithelium of smokers with lung cancer

Abstract We have previously shown that gene expression alterations in cytologically normal epithelial cells from the bronchial airway can be used as an early detection biomarker for lung cancer in smokers. We hypothesize that bronchial epithelial expression of microRNAs, as regulators of gene expression, may also be affected by the presence of cancer and may regulate some of these gene expression differences. We propose a novel method to identify microRNAs functionally associated with disease that leverages the relationship between microRNA and mRNA expression by determining the differential connectivity (DC) of microRNA-mRNA association networks between disease and normal states. Bronchial epithelial brushes were collected from 220 former and current smokers who underwent bronchoscopy for suspicion of lung cancer (120 lung cancer patients and 100 healthy controls). For these subjects, we profiled microRNA expression via small RNA sequencing and gene expression via microarray. Each microRNA node is assigned a DC score, which captures the overall difference in the pairwise microRNA-gene correlation strengths between lung cancer and control subjects. We quantify the change in both the directionality and strength of the correlations between a microRNA and the gene nodes. Then, the observed DC scores are compared to the DC scores obtained with permuted class labels to identify microRNAs with signinficant disease-specific differences in microRNA-mRNA connectivity. The proposed DC method identifies 54 microRNAs which are significantly differentially connected in lung cancer cases compared to controls (FDR<0.1). Among the 54 microRNAs we find those previously associated with lung cancer, such as known dysregulated oncomirs, regulators of drug sensitivity to anticancer agents or targets for DNA methylation in non-small cell lung cancers. In addition, the genes that are significantly correlated with the DC microRNAs are enriched in cancer and immune system related pathways, such as membrane trafficking, apoptosis, mitotic cell cycle, hemostasis, FC gamma R mediated phagocytosis and signaling by Rho GTPases (FDR<0.1). We propose a novel approach for integrating microRNA and gene expression data to identify disease-associated changes in gene regulation by microRNAs and show that the microRNA-mRNA networks are significantly different between disease and normal states. These data suggest that changes in microRNA expression may drive some of the gene expression alterations observed in the cytologically normal epithelium from the proximal airway of patients with lung cancer and that airway microRNA-mRNA expression changes may ultimately serve as a biomarker for lung cancer detection. Citation Format: Ana Brandusa Pavel, Joshua D. Campbell, Gang Liu, Sherry Zhang, Hanqiao Liu, Lingqi Luo, Ji Xiao, Kate Porta, Duncan Whitney, Steven Dubinett, David Elashoff, Marc E. Lenburg, Avrum Spira. Dysregulation of microRNA-mRNA regulatory networks in the bronchial airway epithelium of smokers with lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3077. doi:10.1158/1538-7445.AM2015-3077

Identification and Functional Analysis of Novel Nonstructural Proteins of Human Bocavirus 1.

Human bocavirus 1 (HBoV1) is a single-stranded DNA parvovirus that causes lower respiratory tract infections in young children worldwide. In this study, we identified novel splice acceptor and donor sites, namely, A1' and D1', in the large nonstructural protein (NS1)-encoding region of the HBoV1 precursor mRNA. The novel small NS proteins (NS2, NS3, and NS4) were confirmed to be expressed following transfection of an HBoV1 infectious proviral plasmid and viral infection of polarized human airway epithelium cultured at an air-liquid interface (HAE-ALI). We constructed mutant pIHBoV1 infectious plasmids which harbor silent mutations (sm) smA1' and smD1' at the A1' and D1' splice sites, respectively. The mutant infectious plasmids maintained production of HBoV1 progeny virions at levels less than five times lower than that of the wild-type plasmid. Importantly, the smA1' mutant virus that does not express NS3 and NS4 replicated in HAE-ALI as effectively as the wild-type virus; however, the smD1' mutant virus that does not express NS2 and NS4 underwent an abortive infection in HAE-ALI. Thus, our study identified three novel NS proteins, NS2, NS3, and NS4, and suggests an important function of the NS2 protein in HBoV1 replication in HAE-ALI. Human bocavirus 1 infection causes respiratory diseases, including acute wheezing in infants, of which life-threatening cases have been reported. In vitro, human bocavirus 1 infects polarized human bronchial airway epithelium cultured at an air-liquid interface that mimics the environment of human lower respiratory airways. Viral nonstructural proteins are often important for virus replication and pathogenesis in infected tissues or cells. In this report, we identified three new nonstructural proteins of human bocavirus 1 that are expressed during infection of polarized human bronchial airway epithelium. Among them, we proved that one nonstructural protein is critical to the replication of the virus in polarized human bronchial airway epithelium. The creation of nonreplicating infectious HBoV1 mutants may have particular utility in vaccine development for this virus.

Heme oxygenase-1 protects corexit 9500A-induced respiratory epithelial injury across species.

The effects of Corexit 9500A (CE) on respiratory epithelial surfaces of terrestrial mammals and marine animals are largely unknown. This study investigated the role of CE-induced heme oxygenase-1 (HO-1), a cytoprotective enzyme with anti-apoptotic and antioxidant activity, in human bronchial airway epithelium and the gills of exposed aquatic animals. We evaluated CE-mediated alterations in human airway epithelial cells, mice lungs and gills from zebrafish and blue crabs. Our results demonstrated that CE induced an increase in gill epithelial edema and human epithelial monolayer permeability, suggesting an acute injury caused by CE exposure. CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production. Importantly, CE induced caspase-3 activation and subsequent apoptosis of epithelial cells. The expression of the intercellular junctional proteins, such as tight junction proteins occludin, zonula occludens (ZO-1), ZO-2 and adherens junctional proteins E-cadherin and Focal Adhesion Kinase (FAK), were remarkably inhibited by CE, suggesting that these proteins are involved in CE-induced increased permeability and subsequent apoptosis. The cytoskeletal protein F-actin was also disrupted by CE. Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis. Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK. These observations suggest that CE activates CRP and NOX4-mediated ROS production, alters permeability by inhibition of junctional proteins, and leads to caspase-3 dependent apoptosis of epithelial cells, while HO-1 and its reaction products protect against oxidative stress and apoptosis.

Abstract 1414: Race-associated variation in the airway transcriptome response to cigarette smoke

Abstract INTRODUCTION: Tobacco-induced lung cancer is the leading cause of cancer death. Lung cancer incidence is 15% higher in African Americans (AFA) than Caucasians (CAU) even after taking into account variations in smoking behavior and other environmental variables, and the reasons for this difference are unclear. As 90% of patients with lung cancer have a smoking history, we hypothesize that race-related differences in the response to tobacco smoke might contribute to the elevated lung cancer risk observed in AFA. To begin to test this hypothesis, we have used gene expression profiling to identify differences in the biologic response of the bronchial airway epithelium of AFA and CAU to tobacco smoke. METHODS: Cytologically normal bronchial epithelial cells obtained during bronchoscopy from current (C) and former (F) smokers with and without lung cancer were hybridized to Affymetrix Human Gene 1.0 ST microarrays (n=885). After quality control, n=810 samples (from n=710 subjects) were selected for subsequent analysis. RMA-normalized gene expression levels were batch-corrected using ComBat. Genes whose expression is altered by smoking differently in the airway epithelium of AFA (n=155; nC=76, nF=79) as compared to CAU (n=655; nC=283, nF=372), were identified via ANOVA using a linear model including terms for self-reported race, smoking status, and the interaction between race and smoking status, as well as other covariates including gender, cancer status, COPD status, age, cumulative cigarette smoke exposure, and RIN. Smoking-associated race-dependent pathways were identified using Enrichr. An independent dataset of cytologically normal bronchial epithelial cells hybridized to the Affymetrix HGU133A microarrays (nCAU=154, nAFA=58) was analyzed as detailed above and relationships between the two datasets were established using GSEA for the genes associated with race, and the interaction between race and smoking status. RESULTS: We identified 361 genes that displayed smoking-associated race-dependent patterns of expression (FDR<0.05) and were linked to multiple biological processes, pathways, and phenotypes, including DNA damage response, membrane organization, endoplasmic reticulum stress, and abnormal lung morphology and respiration. In addition, 2771 genes were found to be associated with race (FDR<0.01) and enriched in pathways related to metabolism of xenobiotics, integrin cell surface interactions, and response to chemical stimulus. (Enrichr; Fisher exact test p-value<0.05). Both sets of genes were significantly concordantly enriched in the independent dataset by GSEA (FDR<0.05). CONCLUSIONS: These findings suggest that race influences the airway epithelial response to cigarette smoke at the level of individual genes and pathways. Our findings also suggest that gene expression profiling of the airway epithelium might be useful to understand the molecular basis of the elevated risk of lung cancer among African Americans. Citation Format: Anna Tassinari, Duncan Whitney, Kate Porta, Marc Lenburg, Avrum Spira, Jennifer Beane. Race-associated variation in the airway transcriptome response to cigarette smoke. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1414. doi:10.1158/1538-7445.AM2014-1414

Abstract 3027: Stromal cells-derived paracrine factors promote dedifferentiation of human lung carcinoma cells into cancer stem cells

Abstract Tumors are dynamic entities that act like organs in a perfect trading with the entire body. Through the employment of deceiving cellular and molecular strategies, they use the corporal machinery for the promotion of their survival and dissemination. The extensive crosstalk mediated by cytokines and chemokines overcome the inefficiency of the invasion-metastasis cascade, thus allowing the development of the often fatal metastatic disease. As heterogeneous entities, tumors are comprised of distinct cell populations. Cancer stem cells (CSCs) are a subset of notably chemo- and radiotherapy resistant malignant tumor cells with the capacity to drive tumorigenesis and metastases formation and eventually tumor relapse. Lung cancer is a leading cause of death worldwide and its prevalence is increasing mostly due to sustained smoking habits and the accumulation of atmosphere pollutants. In this work hexavalent chromium [Cr(VI)] was the carcinogenic agent used considering its increasing occupational relevance. The non-malignant human bronchial epithelial airway system 2B (BEAS-2B) was malignantly transformed into the RenG2 cell line using low density culture in the presence of Cr(VI). A parallel control cellular system (Cont1) was produced under the same conditions, though, in the absence of Cr(VI). Two additional derivative cell lines, DRenG2 and DDRenG2, were attained following serial rounds of injections in immunocompromised mice. A panoply of techniques was used to characterize the attained cellular systems leading to the hypothesis of CSCs involvement in Cr(VI)-driven BEAS-2B malignant transformation. The sphere-formation assay readily confirmed our hypothesis as CSCs spheres were isolated only from the derivative cell lines (SC-DRenG2 and SC-DDRenG2 cells, respectively). Tumorigenic and RT-qPCR studies further supported the stem potential of the attained systems, suggesting that a dedifferentiation process featured its emergence during RenG2 derivation in nude mice. Using an elegant cell culture model that encompassed the prolonged co-culture of surgically isolated mice lumbar stromal cells with the RenG2 cells, we proved that RenG2 cells’ dedifferentiation was driven by paracrine factors released by the mouse fibroblasts, as they lost the RenG2's molecular signature and acquired a new one more close to that of both DRenG2 and SC-DRenG2. Finally, the key cytokines released by the stromal fibroblasts were identified in the conditioned media of such cultures. CSCs formation boosted the malignant potential of our carcinoma cell population, providing them with increased resistance to therapeutic drugs. Also, the dedifferentiation process revealed to be essentially chemical and specie-unspecific, as human cells were able to dedifferentiate in response to mice cells-released cytokines. Work sponsored by FEDER, POFC-COMPETE and the FCT grants PTDC/BBB-BQB/2450/2012 and SFRH/BD/33884/2009. Citation Format: Carlos F D Rodrigues, Inês P. Rodrigues, Mariana Val, Lina Carvalho, Artur Paiva, Anatoly Zhitkovich, Isabel M. Carreira, Mª Carmen Alpoim. Stromal cells-derived paracrine factors promote dedifferentiation of human lung carcinoma cells into cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3027. doi:10.1158/1538-7445.AM2014-3027

Late-onset Radiologic Findings of Respiratory System Following Sulfur Mustard Exposure

Background: Sulfur mustard (SM) as a chemical warfare agent, increases permeability of bronchial vessels and damages airway epithelium. SM exposure causes debilitating respiratory complications. This study was designed to evaluate clinical respiratory manifestations, and to compare chest X ray (CXR) and high resolution computed tomography (HRCT) scan of chest in SM exposed patients with respiratory complaints. Methods:All patients with history of SM exposure who visited Imam Reza Specialized Clinic of Respiratory Diseases from September 2001 to March 2011 were included. Patients with other comorbidities which affect respiratory system were excluded. CXR and chest HRCT scan were performed on the same day and were repeated after 5 years. Clinical and radiologic findings were collected and were compared with each other. Results: In total, 62 male patients with mean age of 53 (6.9, 41-65) were studied. Dyspnea (61 cases; 100%), dry cough (40 cases; 66%), hemoptysis (21 cases; 35%) and productive cough (20 cases; 33%) were the most common respiratory manifestations. Pulmonary infiltration (51; 83%), pleural thickening (25; 40%) and emphysema (16; 26%) were the most common findings on CXR. According to HRCT scan, pulmonary infiltration (53; 85%), bronchiolitis obliterans (38; 61%) and pleural thickening (36; 58%) were the most common findings (Table 2). Repeated radiologic assessments after 5 years showed a few additional findings in HRCT scan, while in about one fifth of CXRs, new pathologic findings were found. Conclusion: Patients with SM exposure experience debilitating respiratory disorders in long term. Repeating CXR in patients who present with subjective symptoms may show new findings; however, repeating HRCT scan is probably not necessary.

A Dynamic Bronchial Airway Gene Expression Signature of Chronic Obstructive Pulmonary Disease and Lung Function Impairment

Molecular phenotyping of chronic obstructive pulmonary disease (COPD) has been impeded in part by the difficulty in obtaining lung tissue samples from individuals with impaired lung function. We sought to determine whether COPD-associated processes are reflected in gene expression profiles of bronchial airway epithelial cells obtained by bronchoscopy. Gene expression profiling of bronchial brushings obtained from 238 current and former smokers with and without COPD was performed using Affymetrix Human Gene 1.0 ST Arrays. We identified 98 genes whose expression levels were associated with COPD status, FEV1% predicted, and FEV1/FVC. In silico analysis identified activating transcription factor 4 (ATF4) as a potential transcriptional regulator of genes with COPD-associated airway expression, and ATF4 overexpression in airway epithelial cells in vitro recapitulates COPD-associated gene expression changes. Genes with COPD-associated expression in the bronchial airway epithelium had similarly altered expression profiles in prior studies performed on small-airway epithelium and lung parenchyma, suggesting that transcriptomic alterations in the bronchial airway epithelium reflect molecular events found at more distal sites of disease activity. Many of the airway COPD-associated gene expression changes revert toward baseline after therapy with the inhaled corticosteroid fluticasone in independent cohorts. Our findings demonstrate a molecular field of injury throughout the bronchial airway of active and former smokers with COPD that may be driven in part by ATF4 and is modifiable with therapy. Bronchial airway epithelium may ultimately serve as a relatively accessible tissue in which to measure biomarkers of disease activity for guiding clinical management of COPD.

Abstract 112: Transcriptomic changes in the oral mucosal epithelium reflect the physiologic response to indoor burning of solid fuels.

Abstract RATIONALE: Human exposure to indoor smoke emissions from the domestic burning of solid fuels (e.g. coal, wood, plant materials) is a major cause of morbidity and mortality that affects nearly half of the world's population. While epidemiologic studies on the health impact of indoor air pollution (IAP) have yielded valuable insights from the use of various exposure assessments (e.g. personal and area air sampling, urinary metabolites), there is a need to connect these exposure measures to biomarkers of the host's physiologic response to exposure. We have previously shown that gene-expression (GE) profiles in the upper airway epithelium are affected by tobacco smoke. Thus, we sought to determine whether transcriptomic changes in the oral mucosal epithelium might also capture physiologic responses to IAP from the burning of solid fuels. METHODS: Buccal mucosa epithelial cell scrapings were collected from healthy, non-smoking female subjects in rural Xuanwei, China, where female lung cancer rates are among the highest in the world. Benzo(a)pyrene (BaP) exposure levels from indoor burning of solid fuels (primarily coal) for cooking and heating were measured by personal in-home air monitoring. RNA from 18 samples was extracted and processed onto Affymetrix Gene 1.0 ST arrays. Student's t-test and DAVID were used to identify and characterize genes that vary between relatively high levels of BaP exposure (n=8; BaP 99.0 +/- 45.9) and low (n=10; BaP 16.2 +/- 6.1) exposure. Using GSEA, results were compared to independent airway epithelial GE profiles that distinguish current smokers from never smokers and bronchial GE profiles in subjects with and without lung cancer. RESULTS: We identified 227 genes that were differentially expressed between subjects with high and low BaP exposure (p<0.01). Genes elevated with high BaP exposure were enriched for genes involved in the activation of apoptosis and the Toll-Like Receptor Signaling pathway. Genes that we have previously shown to have altered expression in the bronchial, buccal, and nasal epithelium of current smokers relative to never smokers were enriched among the genes associated with BaP exposure (GSEA; q<0.01). There was also a significant enrichment for bronchial epithelial airway genes that were altered in subjects with and without lung cancer in a previous report (GSEA; q<0.01). CONCLUSION: Buccal epithelial GE is affected by BaP exposure in a population that uses coal and other solid fuels for indoor cooking and heating among healthy women in Xuanwei, China. Solid fuel emissions may elicit some of the same transcriptomic responses as cigarette smoke and lung cancer. This demonstrates the feasibility of using non-invasively collected buccal GE profiling to assess physiologic responses to other inhaled toxins such as coal smoke in population-based studies. Citation Format: Teresa W. Wang, Qing Lan, Bozena Krystyna, Nathaniel Rothman, Roberta Florido, Wei Hu, Katrina Steiling, Gang Liu, Ji Xiao, Yuriy Alekseyev, Jun Xu, Fusheng Wei, H. Dean Hosgood, Boris Reiss, George Downward, Marc Lenburg, Roel Vermeulen, Avrum Spira. Transcriptomic changes in the oral mucosal epithelium reflect the physiologic response to indoor burning of solid fuels. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 112. doi:10.1158/1538-7445.AM2013-112

BICARBONATE PERMEABILITY OF PORCINE BRONCHIAL EPITHELIUM

CFTR mediates secretion of bicarbonate by airway epithelia. Since CFTR function is lost in cystic fibrosis (CF), there is speculation that bicarbonate concentration in mucous liquid is reduced, leading to increased mucus viscosity and reduced mucociliary clearance. Elevating bicarbonate in airway liquid is being considered for CF treatments. Thus, we sought to determine the permeability of airway epithelium to this anion. Pig bronchi were cannulated, filled with either isosmotic NaHCO3 solution (156 meq HCO3−/l, n=6) or isosmotic NaCl solution (0 meq HCO3−/l, n=5), and immersed in normal bicarbonate‐buffered Krebs solution (24 meq HCO3−/l) at 37ݸC. The bath solution contained acetazolamide to inhibit bicarbonate synthesis. Permeabilities were calculated from the mean change in luminal bicarbonate concentration over the first 7.5 min exposure. Bicarbonate permeabilities were 1.7 × 10−5 cm/sec and 8.6 × 10−5 cm/sec for NaHCO3‐filled and NaCl‐filled bronchi, respectively. We conclude that the permeability of bronchial airway epithelium to bicarbonate is relatively high, comparable to the permeabilities for other small solutes of similar molecular weights.

SIRT1 Pathway Dysregulation in the Smoke-Exposed Airway Epithelium and Lung Tumor Tissue

Cigarette smoke produces a molecular field of injury in epithelial cells lining the respiratory tract. However, the specific signaling pathways that are altered in the airway of smokers and the signaling processes responsible for the transition from smoking-induced airway damage to lung cancer remain unknown. In this study, we use a genomic approach to study the signaling processes associated with tobacco smoke exposure and lung cancer. First, we developed and validated pathway-specific gene expression signatures in bronchial airway epithelium that reflect activation of signaling pathways relevant to tobacco exposure, including ATM, BCL2, GPX1, NOS2, IKBKB, and SIRT1. Using these profiles and four independent gene expression datasets, we found that SIRT1 activity is significantly upregulated in cytologically normal bronchial airway epithelial cells from active smokers compared with nonsmokers. In contrast, this activity is strikingly downregulated in non-small cell lung cancer. This pattern of signaling modulation was unique to SIRT1, and downregulation of SIRT1 activity is confined to tumors from smokers. Decreased activity of SIRT1 was validated using genomic analyses of mouse models of lung cancer and biochemical testing of SIRT1 activity in patient lung tumors. Together, our findings indicate a role of SIRT1 in response to smoke and a potential role in repressing lung cancer. Furthermore, our findings suggest that the airway gene expression signatures derived in this study can provide novel insights into signaling pathways altered in the "field of injury" induced by tobacco smoke and thus may impact strategies for prevention of tobacco-related lung cancer.

Gene therapy for the treatment of cystic fibrosis

Gene therapy is being developed as a novel treatment for cystic fibrosis (CF), a condition that has hitherto been widely-researched yet for which no treatment exists that halts the progression of lung disease. Gene therapy involves the transfer of correct copies of cystic fibrosis transmembrane conductance regulator (CFTR) DNA to the epithelial cells in the airways. The cloning of the CFTR gene in 1989 led to proof-of-principle studies of CFTR gene transfer in vitro and in animal models. The earliest clinical trials in CF patients were conducted in 1993 and used viral and non-viral gene transfer agents in both the nasal and bronchial airway epithelium. To date, studies have focused largely on molecular or bioelectric (chloride secretion) outcome measures, many demonstrating evidence of CFTR expression, but few have attempted to achieve clinical efficacy. As CF is a lifelong disease, turnover of the airway epithelium necessitates repeat administration. To date, this has been difficult to achieve with viral gene transfer agents due to host recognition leading to loss of expression. The UK Cystic Fibrosis Gene Therapy Consortium (Imperial College London, University of Edinburgh and University of Oxford) is currently working on a large and ambitious program to establish the clinical benefits of CF gene therapy. Wave 1, which has reached the clinic, uses a non-viral vector. A single-dose safety trial is nearing completion and a multi-dose clinical trial is shortly due to start; this will be powered for clinically-relevant changes. Wave 2, more futuristically, will look at the potential of lentiviruses, which have long-lasting expression. This review will summarize the current status of translational research in CF gene therapy.

Abstract 3154: Sequencing of the small RNA transcriptome in cytologically normal bronchial airway epithelium of smokers with and without lung cancer

Abstract Introduction: Based on the concept that cigarette smoke creates a molecular field of injury in epithelial cells throughout the respiratory tract, we have previously shown that mRNA expression differences in cytologically normal airway epithelial cells in patients with and without lung cancer can serve as a clinically-relevant lung cancer biomarker. MicroRNAs are short, non-coding RNAs that can each regulate the expression of hundreds of target genes and can be reliably detected in clinical samples with variable quality. The goal of this study is to generate a microRNA biomarker in the airway epithelium of smokers for the diagnosis of lung cancer. Methods: Cytologically-normal bronchial airway epithelial cells were collected via brushings of the mainstem bronchus of smokers undergoing fiberoptic bronchoscopy for suspicion of lung cancer (n=128). Subjects were followed after their bronchoscopy until a final diagnosis of lung cancer (n=75) or an alternate benign diagnosis (n=53) was made. The small RNA transcriptome (<40 nucleotides) was sequenced using the Illumina HiSeq 2000 with multiplexing (8 samples/lane). Reads were aligned to hg19 using Bowtie allowing up to 3 mismatches and 10 locations to the genome. Linear models were used to associate expression profiles with disease-related phenotypes while controlling for differences in the total number of reads and other clinical covariates. Results: An average of 10.6 million reads was sequenced per sample with 77% of these reads having at least one alignment to the human genome. In addition, 75% of the aligned reads mapped to known microRNA precursors. 508 novel microRNA precursors were predicted using the miRDeep algorithm. 362 mature microRNAs were considered consistently expressed (Median Absolute Deviation > 20). 98 of these microRNAs were differentially expressed between current and former smokers and 56 microRNAs differentially expressed in the airway epithelium of smokers with and without lung cancer (p<0.05; 18 expected by chance at this threshold). Conclusions: We have sequenced the small RNA transcriptome in the cytologically-normal proximal airway epithelium of smokers and have identified microRNA expression profiles associated with smoking status and/or a final diagnosis of lung cancer. In the future, a microRNA biomarker will be developed on these samples using procedures outlined in the MicroArray Quality Control (MAQC)-II project and tested on an independent cohort in order to confirm their ability to serve as an early diagnostic tool for lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3154. doi:1538-7445.AM2012-3154

Abstract 3173: Identification of miR-4423 as a primate-specific microRNA highly expressed in airway epithelium and associated with lung cancer

Abstract Smoking is a significant risk factor for lung cancer, the leading cause of cancer-related deaths worldwide. Our group has previously shown that epithelial gene expression is altered throughout the airway of smokers and that some of these changes are regulated by microRNAs. Moreover, we have previously identified gene expression differences in cytologically normal bronchial airway epithelial cells between smokers with and without lung cancer that can serve as an early diagnostic biomarker for lung cancer. Here, we use next-generation sequencing of small RNAs to identify novel microRNAs expressed in airway epithelium and associated with lung cancer. We identify miR-4423 as a primate-specific microRNA highly expressed in the airway epithelium. In vitro, the expression of miR-4423 increases as Normal Human Bronchial Epithelial cells are differentiated into mucociliary epithelium at an Air Liquid Interface, while its mRNA targets decrease in expression. Furthermore, the expression of miR-4423 is reduced in lung tumors and in the cytologically normal bronchial airway epithelium of smokers with lung cancer. In gain-of-function experiments, ectopic expression of miR-4423 in lung cancer cell lines resulted in reduced colony formation in soft agar. Taken together, these data support the power of next-generation sequencing in identifying novel cell type- specific transcripts and provides evidence that this newly characterized microRNA may play a role in promoting the differentiation and/or maintenance of airway epithelium, and can reduce anchorage-independent lung cancer cell growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3173. doi:1538-7445.AM2012-3173

Functional Apical Large Conductance, Ca2+-activated, and Voltage-dependent K+ Channels Are Required for Maintenance of Airway Surface Liquid Volume

Large conductance, Ca(2+)-activated, and voltage-dependent K(+) (BK) channels control a variety of physiological processes in nervous, muscular, and renal epithelial tissues. In bronchial airway epithelia, extracellular ATP-mediated, apical increases in intracellular Ca(2+) are important signals for ion movement through the apical membrane and regulation of water secretion. Although other, mainly basolaterally expressed K(+) channels are recognized as modulators of ion transport in airway epithelial cells, the role of BK in this process, especially as a regulator of airway surface liquid volume, has not been examined. Using patch clamp and Ussing chamber approaches, this study reveals that BK channels are present and functional at the apical membrane of airway epithelial cells. BK channels open in response to ATP stimulation at the apical membrane and allow K(+) flux to the airway surface liquid, whereas no functional BK channels were found basolaterally. Ion transport modeling supports the notion that apically expressed BK channels are part of an apical loop current, favoring apical Cl(-) efflux. Importantly, apical BK channels were found to be critical for the maintenance of adequate airway surface liquid volume because continuous inhibition of BK channels or knockdown of KCNMA1, the gene coding for the BK α subunit (KCNMA1), lead to airway surface dehydration and thus periciliary fluid height collapse revealed by low ciliary beat frequency that could be fully rescued by addition of apical fluid. Thus, apical BK channels play an important, previously unrecognized role in maintaining adequate airway surface hydration.

The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia

Organotypic cultures of primary human airway epithelial cells have been used to investigate the morphology, ion and fluid transport, innate immunity, transcytosis, infection, inflammation, signaling, cilia, and repair functions of this complex tissue. However, we do not know how closely these cultures resemble the airway surface epithelium in vivo. In this study, we examined the genome-wide expression profile of tracheal and bronchial human airway epithelia in vivo and compared it with the expression profile of primary cultures of human airway epithelia grown at the air-liquid interface. For comparison, we also investigated the expression profile of Calu-3 cells grown at the air-liquid interface and primary cultures of human airway epithelia submerged in nutrient media. We found that the transcriptional profile of differentiated primary cultures grown at the air-liquid interface most closely resembles that of in vivo airway epithelia, suggesting that the use of primary cultures and the presence of an air-liquid interface are important to recapitulate airway epithelia biology. We describe a high level of similarity between cells of tracheal and bronchial origin within and between different human donors, which suggests a very robust expression profile that is specific to airway cells.

Theoretical approach to the hit probability of lung-cancer-sensitive epithelial cells by mineral fibers with various aspect ratios.

Inhalation of fibers may lead to the damage and, as a further consequence, to the malignant transformation of specific (most of all non-ciliated) cells of the bronchial and bronchiolar airway epithelium. In order to accurately estimate the cancer risk induced by inhaled fibers, hit probabilities of non-ciliated (secretory) cells by mineral fibers (asbestos and chrysotile) were computed. Besides the use of a stochastic lung geometry and a particle transport/deposition model being based upon the random-walk algorithm, histological data of cell distributions in the human lungs were applied for the theoretical calculations. Diameters of computer-generated fibers ranged from 0.1 µm to 10 µm, whilst two values (3 and 100) were selected for the aspect ratios (ratios of fiber length to fiber diameter), thereby simulating the behavior of short and very long fibrous particles. According to the modeling results, the highest regional hit probabilities (up to 10%) are available for fibers with a diameter of 1.0 µm, whereby cells of the bronchiolar compartment represent a preferential target of these particles. For fibers with a diameter of 0.1 µm, bronchiolar hit probabilities reach 2-3%, whereas fibers with a diameter of 10 µm penetrate to the peripheral lung parts with only low amounts (<0.1%). A change of the inhalation conditions from sitting to light-work breathing enhances the extrathoracic and bronchial filtering of large particles (diameter ≥1 µm), whilst penetration of small particles towards distal lung airways is subject to a reinforcement. Further refinement of hit probabilities by considering single airway generations results in the circumstance that fibers with diameters ≤1.0 µm preferably collide with cancer-sensitive cells of airway generations 12-15. In contrast, fibers with a diameter of 10 µm mainly represent a hazard for cancer-sensitive cells being located in the uppermost airways. A change of breathing conditions both supports the effect of short fibers and enhances the filtering of long ones. Based upon the results of this contribution it can be concluded that highest cancer risk is generated by the inhalation of mineral fibers with diameters ≤1.0 µm, whereby fiber length has to be classified as a parameter with lower importance.

Genetic Variation and Antioxidant Response Gene Expression in the Bronchial Airway Epithelium of Smokers at Risk for Lung Cancer

Prior microarray studies of smokers at high risk for lung cancer have demonstrated that heterogeneity in bronchial airway epithelial cell gene expression response to smoking can serve as an early diagnostic biomarker for lung cancer. As a first step in applying functional genomic analysis to population studies, we have examined the relationship between gene expression variation and genetic variation in a central molecular pathway (NRF2-mediated antioxidant response) associated with smoking exposure and lung cancer. We assessed global gene expression in histologically normal airway epithelial cells obtained at bronchoscopy from smokers who developed lung cancer (SC, n = 20), smokers without lung cancer (SNC, n = 24), and never smokers (NS, n = 8). Functional enrichment analysis showed that the NRF2-mediated, antioxidant response element (ARE)-regulated genes, were significantly lower in SC, when compared with expression levels in SNC. Importantly, we found that the expression of MAFG (a binding partner of NRF2) was correlated with the expression of ARE genes, suggesting MAFG levels may limit target gene induction. Bioinformatically we identified single nucleotide polymorphisms (SNPs) in putative ARE genes and to test the impact of genetic variation, we genotyped these putative regulatory SNPs and other tag SNPs in selected NRF2 pathway genes. Sequencing MAFG locus, we identified 30 novel SNPs and two were associated with either gene expression or lung cancer status among smokers. This work demonstrates an analysis approach that integrates bioinformatics pathway and transcription factor binding site analysis with genotype, gene expression and disease status to identify SNPs that may be associated with individual differences in gene expression and/or cancer status in smokers. These polymorphisms might ultimately contribute to lung cancer risk via their effect on the airway gene expression response to tobacco-smoke exposure.

Similarities and differences between smoking-related gene expression in nasal and bronchial epithelium

Previous studies have shown that physiological responses to cigarette smoke can be detected via bronchial airway epithelium gene expression profiling and that heterogeneity in this gene expression response to smoking is associated with lung cancer. In this study, we sought to determine the similarity of the effects of tobacco smoke throughout the respiratory tract by determining patterns of smoking-related gene expression in paired nasal and bronchial epithelial brushings collected from 14 healthy nonsmokers and 13 healthy current smokers. Using whole genome expression arrays, we identified 119 genes whose expression was affected by smoking similarly in both bronchial and nasal epithelium, including genes related to detoxification, oxidative stress, and wound healing. While the vast majority of smoking-related gene expression changes occur in both bronchial and nasal epithelium, we also identified 27 genes whose expression was affected by smoking more dramatically in bronchial epithelium than nasal epithelium. Both common and site-specific smoking-related gene expression profiles were validated using independent microarray datasets. Differential expression of select genes was also confirmed by RT-PCR. That smoking induces largely similar gene expression changes in both nasal and bronchial epithelium suggests that the consequences of cigarette smoke exposure can be measured in tissues throughout the respiratory tract. Our findings suggest that nasal epithelial gene expression may serve as a relatively noninvasive surrogate to measure physiological responses to cigarette smoke and/or other inhaled exposures in large-scale epidemiological studies.

Reduced microRNA‐218 expression is associated with high nuclear factor kappa B activation in gastric cancer

Poor expression of microRNAs (miRs) reportedly plays an important role in gastric carcinogenesis. Large-scale microarray assays have indicated that there is significant down-regulation of miR-218 in gastric cancer. miR-218 also was decreased specifically in human papillomavirus-positive cell lines, cervical lesions, and cervical cancer tissues and in bronchial airway epithelium in smokers. However, its role in carcinogenesis remains unclear, especially in Helicobacter pylori (H. pylori)-associated gastric cancer. miR-218 levels were evaluated in 20 noncardia gastric cancer tissues, in 10 H. pylori-infected and 8 uninfected normal gastric biopsies, and in the human gastric epithelial cancer cell line AGS using TaqMan quantitative real-time polymerase chain reaction analysis. Pre-miR-218 and anti-miR-218 inhibitors were used to examine the effects of miR-218 expression on cell proliferation and apoptosis. A luciferase reporter assay was used to examine the potential target genes and related pathways. miR-218 expression was reduced significantly in gastric cancer tissues, in H. pylori-infected gastric mucosa, and in H. pylori-infected AGS cells. Overexpression of miR-218 inhibited cell proliferation and increased apoptosis in vitro. Epidermal growth factor receptor-coamplified and overexpressed protein (ECOP), which regulates nuclear factor kappa B (NF-kappaB) transcriptional activity and is associated with apoptotic response, was a direct target of miR-218. Overexpression of miR-218 also inhibited NF-kappaB transcriptional activation and transcription of cyclooxygenase -2, a proliferative gene regulated by NF-kappaB. H. pylori infection resulted in a decrease in miR-218 expression. The down-regulation of miR-218 has the potential to increase carcinogenesis by losing control of its targets, and it may be correlated with the high transcriptional activity of NF-kappaB that results from H. pylori infection.

CELLULAR EXPRESSION OF RENAL, CARDIAC AND PULMONARY INDUCIBLE NITRIC OXIDE SYNTHASE IN DOUBLE‐TRANSGENIC MICE EXPRESSING HUMAN RENIN AND ANGIOTENSINOGEN GENES

1. Hypertensive mice expressing the human renin (REN) and angiotensinogen (AGT) genes are used as a model for human hypertension. 2. The aim of the present study was to investigate the cellular expression and distribution of inducible nitric oxide synthase (iNOS) using immunohistochemistry in lung, heart and kidney tissues from a model of human hypertension using male and female double-transgenic (h-Ang 204/1h-Ren6) mice and wild-type C57/BI6J mice as controls. 3. In the kidney, the pattern of iNOS expression in various renal microanatomical regions during hypertension was similar to that of age-matched controls, except in the medullary ascending limb (MAL). In hypertension, iNOS expression was downregulated in the MAL. No significant differences in iNOS expression were seen between control or hypertensive mice in various cardiac microanatomical locations. In the lungs of hypertensive mice, iNOS expression was upregulated in bronchial airway epithelium and bronchial and vascular smooth muscle cells, but downregulated in alveolar macrophages, alveolar septa and pulmonary vascular endothelial cells. Expression of iNOS was similar between male and female mice in the kidney, heart and lungs. 4. In conclusion, iNOS regulation in hypertension is complex and depends on the cell type in which it is expressed and the localization of the cell type in the cardiorenal and pulmonary systems.