Normal Airway Epithelial Cells Research Articles

Abstract 1761: PDE10, a novel therapeutic target for lung cancer

Abstract Phosphodiesterase 10A (PDE10) is a relatively new PDE isozyme that has been mostly studied in the brain as a therapeutic target for certain psychiatric and neurological conditions, but has not been associated with tumorigenesis. While other investigators have reported low levels of PDE10 in most adult peripheral tissues, we found a large percentage of human lung tumors express high levels of PDE10 compared with normal lung tissue as determined by real-time PCR analysis of cDNA arrays. Statistical analysis revealed that 68% of lung tumors (n=19) displayed more than two-fold increase compared with normal lung tissue, while 26% showed greater than a 10-fold difference. High levels of PDE10 mRNA and protein were also found in human non-small cell lung cancer (NSCLC) cell lines compared with normal airway epithelial cells. PDE10 knockdown by small interference RNA significantly suppressed lung tumor cell growth, as did PDE10 selective inhibitors, papaverine, PQ-10 and Pf-2545920. A novel PDE10 inhibitor, MCI-020 also inhibited the growth of NSCLC cells with a high degree of selectivity for tumor cells. Consistent with a mechanism involving PDE10 inhibition, PQ-10 and MCI-020 increased cGMP levels in a concentration-dependent manner as determined using a cGMP biosensor assay. MCI-020 also activated cGMP-dependent protein kinase (PKG) in NSCLCs as evident by increased phosphorylation of VASP-Serine239. With attractive pharmacokinetic properties, MCI-020 was studied further and found to display an unusual characteristic of achieving high concentrations in lung tissue compared with other tissues and plasma following oral administration. MCI-020 was well tolerated in mice at a dose of 250 mg/kg bid and strongly inhibited tumor formation in an orthotopic model of lung cancer at a dose of 150 mg/kg bid. These observations suggest that PDE10 is essential for lung tumor cell growth and represents a novel therapeutic target for lung cancer. Citation Format: Bing Zhu, Nan Li, Veronica Ramirez-Alcantara, Joshua Canzoneri, Evrim Gurpinar, Alexandra Fajardo, Kevin Lee, Sara Sigler, Bernard Gary, Meagan Thomas, Adam Keeton, Xi Chen, William Grizzle, Gary Piazza. PDE10, a novel therapeutic target for lung cancer. [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 1761. doi:10.1158/1538-7445.AM2014-1761

Functional and pharmacological characterization of volume-regulated anion channels in human normal and cystic fibrosis bronchial and nasal epithelial cells

Volume-regulated anion channels (VRACs) are widely present in various cell types and have important functions ranging from regulatory volume decrease to control of cell proliferation and apoptosis. Here we aimed to compare the biophysical features and pharmacological profiles of VRAC currents in healthy and cystic fibrosis (CF) respiratory epithelial cells in order to characterize these currents both functionally and pharmacologically. Whole-cell electrophysiology was used to characterize the VRAC current in normal (16HBE14o−; HBE) and CF cell lines (CFBE14o−; CFBE), as well as in native human nasal epithelial cells. Application of hypotonic solution produced current responses of similar sizes in both HBE and CFBE cells. Biophysical properties of VRACs, such as instantaneous activation and deactivation upon voltage step, some inactivation at potentials positive to 40mV and outwardly-rectifying I–V curves, were indistinguishable in both cell types. Extensive pharmacological analysis of the currents revealed a similar pharmacological profile in response to three blockers – NPPB, DCPIB and DIDS. Native primary human nasal epithelial cells from both healthy and CF volunteers also showed typical VRAC responses of comparable sizes. VRACs in these cells were more sensitive to external solution hypotonicity compared to HBE and CFBE cells. In all cell types studied robust VRAC currents could be induced at constant cell volume by G-protein activation with GTPγS infusion. This study provides the first extensive comparative functional and pharmacological analysis of VRAC currents in normal and CF airway epithelial cells and shows that VRACs are unimpaired molecularly or functionally in CF.

Monosaccharide digitoxin derivative sensitize human non-small cell lung cancer cells to anoikis through Mcl-1 proteasomal degradation

Advanced stage cancers acquire anoikis resistance which provides metastatic potential to invade and form tumors at distant sites. Suppression of anoikis resistance by novel molecular therapies would greatly benefit treatment strategies for metastatic cancers. Recently, digitoxin and several of its novel synthetic derivatives, such as α-l-rhamnose monosaccharide derivative (D6-MA), have been synthesized and studied for their profound anticancer activity in various cancer cell lines. In this study, we investigated the anoikis sensitizing effect of D6-MA compared with digitoxin to identify their anti-metastatic mechanism of action. D6-MA sensitized NSCLC H460 cells to detachment-induced apoptosis with significantly greater cytotoxicity (IC50=11.9nM) than digitoxin (IC50=90.7nM) by activating caspase-9. Screening of the Bcl-2 protein family revealed that degradation of anti-apoptotic Mcl-1 protein is a favorable target. Mcl-1 over-expression and knockdown studies in D6-MA and digitoxin exposed cells resulted in rescue and enhancement, respectively, indicating a facilitative role for decreased Mcl-1 expression in NSCLC anoikis. Transfection with mutant Mcl-1S159 attenuated detachment-induced cell death and correlated with a remaining of Mcl-1 level. Furthermore, D6-MA suppressed Mcl-1 expression via ubiquitin proteasomal degradation that is dependent on activation of glycogen synthase kinase (GSK)-3β signaling. In addition, D6-MA also targeted Mcl-1 degradation causing an increased anoikis in A549 lung cancer cells. Anoikis sensitizing effect on normal small airway epithelial cells was not observed indicating the specificity of D6-MA and digitoxin for NSCLC. These results identify a novel cardiac glycoside (CG) sensitizing anoikis mechanism and provide a promising anti-metastatic target for lung cancer therapy.

CCDC65 Mutation Causes Primary Ciliary Dyskinesia with Normal Ultrastructure and Hyperkinetic Cilia

BackgroundPrimary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating.MethodsGenetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion.ResultsA single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells.ConclusionMutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable ultrastructural defects of the ciliary axoneme, emphasizing the role of the nexin-dynein regulatory complex and the limitations of certain methods for PCD diagnosis.

Human Airway Epithelial Secretions Inhibit the Formation of Pseudomonas aeruginosa Biofilms

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that rarely causes respiratory disease except in individuals with specific risk factors, such as cystic fibrosis (CF). The ability of P. aeruginosa to readily form biofilms in vivo is associated with airway colonization in susceptible individuals. Thus, we hypothesized that non‐CF airway epithelial cells secrete one or more compounds that inhibit P. aeruginosa biofilm formation. To test this, we modified and optimized a static biofilm assay and tested the ability of apical Calu‐3 cell secretions to inhibit biofilm formation by the P. aeruginosa strain, PA14. PA14 formed robust biofilms after 18–24 hours, and biofilm formation was significantly inhibited in the presence of Calu‐3 secretions. Preliminary experiments using secretions from a Calu‐3 cell line not expressing the CF transmembrane regulator (CFTR) suggest that CF‐like secretions do not inhibit PA14 biofilm formation. Thus, normal airway epithelial cells, but not CF‐like epithelial cells, appear to secrete one or more compounds that inhibit P. aeruginosa biofilm formation.This project was funded by the Augsburg College Undergraduate Research and Graduate Opportunities (URGO) Office, the Augsburg Sundquist Scholars Program, the Augsburg McNair Scholars Program, and the Augsburg Department of Biology.

Sevoflurane suppresses tumour necrosis factor-α-induced inflammatory responses in small airway epithelial cells after anoxia/reoxygenation

Lung ischaemia-reperfusion (I/R) injury is correlated with poor clinical outcome. The inflammatory cytokines interleukin (IL)-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) are produced by pulmonary epithelial cells during lung transplantation and are considered to be involved in I/R injury. The volatile anaesthetic sevoflurane has been shown to exert a protective effect on I/R injury in various organs. We investigated the effect of sevoflurane on the inflammatory functions of pulmonary epithelial cells in vitro. Human normal small airway epithelial cells (SAEC) were incubated under anoxic conditions for 24 h with or without sevoflurane and then stimulated with tumour necrosis factor (TNF)-α under hyperoxic conditions for 5 h with or without sevoflurane. After incubation, IL-6, IL-8, and MCP-1 mRNA expression was analysed by quantitative real-time RT-PCR. The production of IL-6, IL-8, and MCP-1 was assayed by enzyme-linked immunosorbent assay, the effects of sevoflurane on inflammatory gene expression were examined by DNA microarray analysis, and the effects of sevoflurane on NF-κB-mediated inflammatory cytokine production were examined by immunoblotting. Sevoflurane suppressed TNF-α-induced IL-6, IL-8, and MCP-1 gene expression and the production of IL-6 and IL-8 in SAEC under anoxia/reoxygenation conditions. DNA microarray analysis indicated that sevoflurane modulated NF-κB-related gene expression. Sevoflurane significantly inhibited TNF-α-induced translocation of p65 NF-κB into the nucleus. Sevoflurane enhanced TNF-α-induced gene expression of inhibitor κB (IκB) but not of NF-κB. Sevoflurane suppressed the NF-κB-mediated production of pulmonary epithelial cell-derived inflammatory cytokines, including IL-6 and IL-8, which are capable of causing I/R injury.

Bone morphogenetic proteins enhance an epithelial-mesenchymal transition in normal airway epithelial cells during restitution of a disrupted epithelium

BackgroundMechanisms of airway repair are poorly understood. It has been proposed that, following injury, progenitor populations such as club cells (Clara) become undifferentiated, proliferate and re-differentiate to re-epithelialise the airway. The exact phenotype of such cells during repair is unknown however. We hypothesised that airway epithelial cells (AECs) undergo some degree of epithelial-mesenchymal transition (EMT) in order to migrate over a denuded airway and effect re-epithelialisation. Furthermore, based on our previous findings that BMP signalling is an early event in AECs following injury in vivo and that BMP4 down-regulates E-cadherin expression and enhances migration in AECs in vitro, we hypothesised that BMPs could play a role in inducing such a phenotypic switch.MethodsNormal AECs were isolated from mouse lungs and analysed in a model of a disrupted epithelium. EMT marker expression and BMP signalling were examined by immunofluorescence, Western blotting and RT-PCR.ResultsFollowing generation of a wound area, AECs at the wound edge migrated and acquired a mesenchymal-like morphology. E-cadherin expression was reduced in migrating cells while vimentin and α-smooth muscle actin (α-SMA) expression was increased. Re-expression of membrane E-cadherin was subsequently observed in some cells in the wound area following re-establishment of the monolayer. A transient increase in the incidence of nuclear phosphorylated Smad1/5/8 was observed in migrating cells compared with confluent cells, indicating active BMP signalling during migration. BMP antagonists noggin and gremlin inhibited cell migration, confirming the involvement of BMP signalling in migration and indicating autocrine signalling, possibly involving BMP7 or BMP4 which were expressed in AECs. Exogenous BMP2, BMP4 and BMP7 induced a mesenchymal-like morphology in AECs, enhanced the rate of cell migration and increased α-SMA protein expression in AECs.ConclusionsFollowing disruption of an intact epithelium, migrating AECs at the wound edge acquire an EMT-like phenotype involving altered expression of E-cadherin, vimentin and α-SMA. BMP signalling is involved in AEC migration and is likely to mediate the switch towards an EMT-like phenotype by altering protein expression to facilitate cell migration and wound closure. We propose therefore that acquisition of an EMT-like phenotype by AECs is a normal aspect of wound repair. Furthermore, we suggest that diseases involving fibrosis may arise because the EMT phase of repair is prolonged by chronic injury/inflammation, rather than being caused by it, as is the current paradigm.

IL-17 induces type V collagen overexpression and EMT via TGF-β-dependent pathways in obliterative bronchiolitis.

Obliterative bronchiolitis (OB), a fibrotic airway lesion, is the leading cause of death after lung transplantation. Type V collagen [col(V)] overexpression and IL-17-mediated anti-col(V) immunity are key contributors to OB pathogenesis. Here, we report a previously undefined role of IL-17 in inducing col(V) overexpression, leading to epithelial mesenchymal transition (EMT) and subsequent OB. We observed IL-17-mediated induction of col(V) α1 chains [α1 (V)] in normal airway epithelial cells in vitro and detected α1 (V)-specific antibodies in bronchoalveolar lavage fluid of lung transplant patients. Overexpression of IL-17 and col(V) was detected in OB lesions in patient lung biopsies and in a murine OB model. IL-17 is shown to induce EMT, TGF-β mRNA expression, and SMAD3 activation, whereas downregulating SMAD7 expression in vitro. Pharmacological inhibition of TGF-βRI tyrosine kinase, p38 MAPK, or focal adhesion kinase prevented col(V) overexpression and EMT. In murine orthotopic lung transplants, neutralizing IL-17 significantly decreased TGF-β mRNA and protein expression and prevented epithelial repair/OB. Our findings highlight a feed-forward loop between IL-17 and TGF-β, leading to induction of col(V) and associated epithelial repair, thus providing one possible link between autoimmunity and OB after lung transplantation.

Time for TMEM?

Time for TMEM?

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.

Abstract A27: Airway molecular alterations associated with premalignant lesion progression and lung cancer development

Abstract Recently, the National Lung Screening Trial reported a 20% reduction in lung cancer mortality and lung cancer chemoprevention trials targeting the arachidonic acid pathway have demonstrated decreases in lung cancer associated markers. These studies highlight possibilities for future reductions in lung cancer mortality through early detection and chemoprevention. Our group has previously identified smoking- and lung cancer-specific gene expression alterations in cytologically normal airway epithelial cells that can serve as a clinically-relevant biomarker for the early detection of lung cancer. Here, in an effort that could lead to markers of lung cancer risk, we identify changes in these cells associated with regression of premalignant airway lesions. Airway epithelial cells were collected via bronchoscopy from patients with bronchial dysplasia at baseline, on-treatment, and post-treatment with green tea extract (GTE) or placebo ranging from 2 to 6 months (n=27 patients, n=63 samples). RNA from the samples was processed and hybridized to Affymetrix Human Gene 1.0 ST microarrays. A linear mixed effect model was used to identify a gene expression signature predictive of subsequent dysplasia regression. Further a paired t-test was used to identify genes associated with dysplasia regression over time. Using gene set enrichment analysis (GSEA) we identified that the baseline dysplasia regression signature was enriched (FDR<0.05) among genes whose altered expression was associated with: dysplasia regression over time; the presence or future development of lung cancer; and human bronchial biopsies at successive morphological stages of lung squamous carcinogenesis. The genes associated with dysplasia regression were validated small cohorts of independent samples from chemoprevention trials testing Sulindac, Myo-inositol, and GTE. Analysis of the Connectivity Map identified compounds that reverse the dysplasia regression signature in vitro and are therefore candidate chemoprevention agents. Our studies suggest that the airway “field of injury” is modulated by bronchial premalignant lesions. The molecular signatures identified may be important tools for stratifying high-risk smokers for chemoprevention trials, as surrogate endpoints of efficacy in these trials, and for identification of novel molecular targets for chemoprevention. In addition, the molecular signature of regression of airway dysplasia may have additional utility as a biomarker predictive of the presence of or future lung cancer development. Citation Format: Jennifer Ebel Beane, Kahkeshan Hijazi, Katrina Steiling, Gang Liu, Sherry Zhang, Stephen Lam, Marc Lenburg. Airway molecular alterations associated with premalignant lesion progression and lung cancer development. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A27.

Elevated SP-1 Transcription Factor Expression and Activity Drives Basal and Hypoxia-induced Vascular Endothelial Growth Factor (VEGF) Expression in Non-Small Cell Lung Cancer

VEGF plays a central role in angiogenesis in cancer. Non-small cell lung cancer (NSCLC) tumors have increased microvascular density, localized hypoxia, and high VEGF expression levels; however, there is a lack of understanding of how oncogenic and tumor microenvironment changes such as hypoxia lead to greater VEGF expression in lung and other cancers. We show that NSCLC cells secreted higher levels of VEGF than normal airway epithelial cells. Actinomycin D inhibited all NSCLC VEGF secretion, and VEGF minimal promoter-luciferase reporter constructs were constitutively active until the last 85 base pairs before the transcription start site containing three SP-1 transcription factor-binding sites; mutation of these VEGF promoter SP-1-binding sites eliminated VEGF promoter activity. Furthermore, dominant negative SP-1, mithramycin A, and SP-1 shRNA decreased VEGF promoter activity, whereas overexpression of SP-1 increased VEGF promoter activity. Chromatin immunoprecipitation assays demonstrated SP-1, p300, and PCA/F histone acetyltransferase binding and histone H4 hyperacetylation at the VEGF promoter in NSCLC cells. Cultured NSCLC cells expressed higher levels of SP-1 protein than normal airway epithelial cells, and double-fluorescence immunohistochemistry showed a strong correlation between SP-1 and VEGF in human NSCLC tumors. In addition, hypoxia-driven VEGF expression in NSCLC cells was SP-1-dependent, with hypoxia increasing SP-1 activity and binding to the VEGF promoter. These studies are the first to demonstrate that overexpression of SP-1 plays a central role in hypoxia-induced VEGF secretion.

Interferon γ Stimulates Accumulation of Gas Phase Nitric Oxide in Differentiated Cultures of Normal and Cystic Fibrosis Airway Epithelial Cells

Exhaled nitric oxide (NO) levels have been reported to be lower in patients with cystic fibrosis (CF) than in controls; however the mechanism(s) responsible and the effect on pathogenesis are unclear. The objective of these studies was to determine if the low levels of gas phase NO (gNO) could be reproduced in well-differentiated air-liquid interface (ALI) cultures of normal and CF cells. Human bronchial epithelial (HBE) cells from CF and control tissues were cultured under ALI conditions that promote differentiation into a mostly ciliated, pseudostratified epithelium similar to that of the in vivo airway. Cultures were incubated in gas tight chambers and the concentration of gNO was measured using a Sievers nitric oxide analyzer. In CF and control cultures the level of accumulated gNO under baseline conditions was low (<20ppb). Treatment with interferon gamma (IFNγ) induced iNOS expression and increased gNO significantly in differentiated cultures, while having no significant effect on undifferentiated cultures. Submersion of the apical surface with fluid drastically reduced the level of gNO. Importantly, the average level of gNO measured after IFNγ treatment of control cells (576ppb) was threefold greater than that from CF cells (192ppb). The results demonstrate that the lower level of exhaled NO observed in CF patients is reproduced in well-differentiated primary cultures of HBE cells treated with IFNγ, supporting the hypothesis that the regulation of NO production is altered in CF. The results also demonstrate that IFNγ treatment of differentiated cells results in higher levels of gNO than treatment of undifferentiated cells, and that a layer of fluid on the apical surface drastically reduces the amount of gNO, possibly by limiting the availability of oxygen.

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

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 (&amp;lt;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 &amp;gt; 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&amp;lt;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

Increased proteinase inhibitor-9 (PI-9) and reduced granzyme B in lung cancer: Mechanism for immune evasion?

Cytotoxic CD8+ T-cells mount immune responses to cancer via cytotoxic pathways including granzyme B. Cancer cells are also known to develop immune evasion mechanisms. We hypothesised that lung cancer cells would over-express the granzyme B-inhibitor, proteinase inhibitor-9 (PI-9) and down-regulate granzyme B expression by neighbouring CD8+ T-cells.We investigated PI-9 expression in lung cancer cell lines, and primary lung cancer cells obtained at curative lung resection from cancer patients with/without chronic obstructive pulmonary disease (COPD). Granzyme B and PI-9 expression was also determined in CD8+ T-cells from the cancer and non-cancer areas of resected lung tissue and from bronchoalveolar lavage (BAL). We then evaluated the effects of conditioned media from lung cancer cell lines on granzyme B expression and the cytotoxic activity of CD8+ T-cells.PI-9 was highly expressed in lung cancer cell lines. Increased PI-9 expression was also observed in primary cancer cells vs. epithelial cells from non-cancer tissue or bronchial brushing-derived normal primary large airway epithelial cells. Expression significantly correlated with cancer stage. Significantly reduced granzyme B was noted in CD8+ T-cells from cancer vs. non-cancer tissue. Granzyme B production by CD8+ T-cells was reduced in the presence of conditioned media from lung cancer cell lines.Our data suggest that lung cancer cells utilise their increased PI-9 expression to protect from granzyme B-mediated cytotoxicity as an immune evasion mechanism, a function that increases with lung cancer stage.

Genetic and Epigenetic Inactivation of Extracellular Superoxide Dismutase Promotes an Invasive Phenotype in Human Lung Cancer by Disrupting ECM Homeostasis

Extracellular superoxide dismutase (EcSOD) is an important superoxide scavenger in the lung in which its loss, sequence variation, or abnormal expression contributes to lung diseases; however, the role of EcSOD in lung cancer has yet to be studied. We hypothesized that EcSOD loss could affect malignant progression in lung, and could be either genetic or epigenetic in nature. To test this, we analyzed EcSOD expression, gene copy number, promoter methylation, and chromatin accessibility in normal lung and carcinoma cells. We found that normal airway epithelial cells expressed abundant EcSOD and had an unmethylated promoter, whereas EcSOD-negative lung cancer cells displayed aberrant promoter hypermethylation and decreased chromatin accessibility. 5-aza-dC induced EcSOD suggesting that cytosine methylation was causal, in part, to silencing. In 48/50 lung tumors, EcSOD mRNA was significantly lower as early as stage I, and the EcSOD promoter was hypermethylated in 8/10 (80%) adenocarcinomas compared with 0/5 normal lung samples. In addition, 20% of the tumors showed loss of heterozygosity (LOH) of EcSOD. Reexpression of EcSOD attenuated the malignant phenotype of lung carcinoma cells by significantly decreasing invasion and survival. Finally, EcSOD decreased heparanase and syndecan-1 mRNAs in part by reducing NF-κB. By contrast, MnSOD and CuZnSOD showed no significant changes in lung tumors and had no effect on heparanase expression. Taken together, the loss of EcSOD expression is unique among the superoxide dismutases in lung cancer and is the result of EcSOD promoter methylation and LOH, suggesting that its early loss may contribute to ECM remodeling and malignant progression.

Interferon γ Stimulates Accumulation of Gas Phase Nitric Oxide in Differentiated Cultures of Normal and Cystic Fibrosis Airway Epithelial Cells

Interferon γ Stimulates Accumulation of Gas Phase Nitric Oxide in Differentiated Cultures of Normal and Cystic Fibrosis Airway Epithelial Cells

Abstract A54: Mithramycin inhibits cigarette smoke-mediated induction of ABCG2 in lung and esophageal cancer cells: Implications for targeting cancer stem cells in thoracic malignancies.

Abstract Limited information is available regarding epigenetic mechanisms by which cigarette smoke mediates initiation and progression of thoracic malignancies. In order to examine this issue, lung and esophageal cancer cells (A549, Calu-6, NCI-SB-ESC1 and NCI-SB-ESC2), were cultured in normal media (NM) with or without cigarette smoke condensate (CSC) under clinically relevant exposure conditions. Microarray analysis revealed that five day CSC exposure significantly up-regulated ABCG2, encoding a xenobiotic pump highly expressed in cancer stem cells. Quantitative reverse transcription-PCR (qRT-PCR), western blot, and immunohistochemistry experiments confirmed up-regulation of ABCG2 in cultured cancer lines, but not normal small airway epithelial cells (SAEC) or immortalized esophageal squamous cells (HET1A) exposed to CSC. Flow cytometry experiments demonstrated that CSC increased the side population (SP) of cultured cancer cells. Transient transfection experiments using ABCG2 promoter reporter constructs revealed that deletion of xenobiotic response elements as well as SP-1 sites markedly attenuated ABCG2 induction by CSC. Chromatin immunoprecipitation (ChIP) experiments revealed that CSC-mediated induction of ABCG2 coincided with increased occupancy of aryl hydrocarbon receptor (AHR), SP-1, and Nrf2, as well as increased levels of RNA pol II and H3K9Ac within the ABCG2 promoter. Under conditions potentially achievable in clinical settings, mithramycin diminished basal as well as CSC-mediated increases in AHR, SP-1, and Nrf2 levels within the ABCG2 promoter, down-regulated ABCG2 expression, decreased S P, inhibited in-vitro proliferation of lung and esophageal cancer cells, and markedly diminished growth of established tumor xenografts. Microarray analysis revealed significant down-regulation of numerous stem-cell related pathways in lung cancer cells following mithramycin exposure. Collectively, these findings provide a potential mechanistic link between smoking status and outcome of patients with lung and esophageal cancers, and support clinical evaluation of mithramycin for targeting cancer stem cells in thoracic malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A54.

Human Alveolar Type II Cells Secrete and Absorb Liquid in Response to Local Nucleotide Signaling

A balance sheet describing the integrated homeostasis of secretion, absorption, and surface movement of liquids on pulmonary surfaces has remained elusive. It remains unclear whether the alveolus exhibits an intra-alveolar ion/liquid transport physiology or whether it secretes ions/liquid that may communicate with airway surfaces. Studies employing isolated human alveolar type II (AT2) cells were utilized to investigate this question. Human AT2 cells exhibited both epithelial Na(+) channel-mediated Na(+) absorption and cystic fibrosis transmembrane conductance regulator-mediated Cl(-) secretion, both significantly regulated by extracellular nucleotides. In addition, we observed in normal AT2 cells an absence of cystic fibrosis transmembrane conductance regulator regulation of epithelial Na(+) channel activity and an absence of expression/activity of reported calcium-activated chloride channels (TMEM16A, Bestrophin-1, ClC2, and SLC26A9), both features strikingly different from normal airway epithelial cells. Measurements of alveolar surface liquid volume revealed that normal AT2 cells: 1) achieved an extracellular nucleotide concentration-dependent steady state alveolar surface liquid height of ∼4 μm in vitro; 2) absorbed liquid when the lumen was flooded; and 3) secreted liquid when treated with UTP or forskolin or subjected to cyclic compressive stresses mimicking tidal breathing. Collectively, our studies suggest that human AT2 cells in vitro have the capacity to absorb or secrete liquid in response to local alveolar conditions.