Molecular events that drive the development of precancerous lesions in the bronchial epithelium, which are precursors of lung squamous cell carcinoma (LUSC), are poorly understood. We demonstrate that disruption of epithelial cellular polarity, via the conditional deletion of the apical determinant Crumbs3 (Crb3), initiates and sustains precancerous airway pathology. The loss of Crb3 in adult luminal airway epithelium promotes the uncontrolled activation of the transcriptional regulators YAP and TAZ, which stimulate intrinsic signals that promote epithelial cell plasticity and paracrine signals that induce basal-like cell growth. We show that aberrant polarity and YAP/TAZ-regulated gene expression associates with human bronchial precancer pathology and disease progression. Analyses of YAP/TAZ-regulated genes further identified the ERBB receptor ligand Neuregulin-1 (NRG1) as a key transcriptional target and therapeutic targeting of ERBB receptors as a means of preventing and treating precancerous cell growth. Our observations offer important molecular insight into the etiology of LUSC and provides directions for potential interception strategies of lung cancer.

Bronchial premalignant lesions (PMLs) are precursors of lung squamous cell carcinoma, but have variable outcome, and we lack tools to identify and treat PMLs at risk for progression to cancer. Here we report the identification of four molecular subtypes of PMLs with distinct differences in epithelial and immune processes based on RNA-Seq profiling of endobronchial biopsies from high-risk smokers. The Proliferative subtype is enriched with bronchial dysplasia and exhibits up-regulation of metabolic and cell cycle pathways. A Proliferative subtype-associated gene signature identifies subjects with Proliferative PMLs from normal-appearing uninvolved large airway brushings with high specificity. In progressive/persistent Proliferative lesions expression of interferon signaling and antigen processing/presentation pathways decrease and immunofluorescence indicates a depletion of innate and adaptive immune cells compared with regressive lesions. Molecular biomarkers measured in PMLs or the uninvolved airway can enhance histopathological grading and suggest immunoprevention strategies for intercepting the progression of PMLs to lung cancer.

The need to reduce per sample cost of RNA-seq profiling for scalable data generation has led to the emergence of highly multiplexed RNA-seq. These technologies utilize barcoding of cDNA sequences in order to combine multiple samples into a single sequencing lane to be separated during data processing. In this study, we report the performance of one such technique denoted as sparse full length sequencing (SFL), a ribosomal RNA depletion-based RNA sequencing approach that allows for the simultaneous sequencing of 96 samples and higher. We offer comparisons to well established single-sample techniques, including: full coverage Poly-A capture RNA-seq, microarrays, as well as another low-cost highly multiplexed technique known as 3' digital gene expression (3'DGE). Data was generated for a set of exposure experiments on immortalized human lung epithelial (AALE) cells in a two-by-two study design, in which samples received both genetic and chemical perturbations of known oncogenes/tumor suppressors and lung carcinogens. SFL demonstrated improved performance over 3'DGE in terms of coverage, power to detect differential gene expression, and biological recapitulation of patterns of differential gene expression from in vivo lung cancer mutation signatures.

Abstract Background: Lung squamous cell carcinoma (SqCC) arises in the epithelial layer of the bronchial airways and is often preceded by the development of premalignant lesions. However, not all premalignant lesions will progress to lung SqCC and many of these lesions will regress without therapeutic intervention. Understanding the molecular events that contribute to progression of premalignant lesions in the airway will allow us to identify biomarkers for early detection and develop therapeutic strategies for early intervention. Methods: Bronchial brushings and biopsies were obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and CT at the Roswell Park Cancer Institute. For each subject (n = 30), both premalignant lesions (PMLs) and the cytologically normal mainstem bronchus were sampled repeatedly over time (n = 288 samples). DNA and RNA were isolated from a total of 197 bronchial biopsies of PML (average of 5 per subject) and 91 bronchial brushings. DNA was also isolated from the blood to serve as a matched normal. Exome capture was performed using the Agilent SureSelect Human All Exon+UTR 70MB kit and sequenced to a mean depth of coverage of 75x (n = 85 samples from 22 subjects). RNA libraries were prepared with Illumina TruSeq (mRNA-Seq: n = 288 samples from 30 subjects and miRNA-Seq: n = 183 samples from 26 subjects). Results: We identified gene and miRNA expression changes associated with histological grade as well as progressive/stable disease. The Hippo pathway, Wnt signaling, p53 signaling, and immune-related pathways are modulated with histological grade and disease progression. Genes associated with histological grade in the cytologically normal airway and in the biopsies were significantly concordantly enriched (FDR<0.05) demonstrating a strong relationship between the PMLs and the field of injury. The somatic mutation rate of PMLs displayed no significant association with histological grade (p = 0.65). Mutations in previously characterized lung cancer genes included TP53 (3%), CREBBP (3%), FAT1 (3%), and NOTCH1 (9%). Examining copy number alterations revealed a single metaplastic lesion with an arm-level amplification on chr5p containing TERT. The two lesions with the highest mutation rates (>3/Mb) were taken from adjacent sites over two time points in the same individual with a history of lung squamous cell carcinoma. These lesions had a significantly overlapping set of mutations (p = 2.2 × 10−17) indicating a common evolutionary ancestor, and contained mutations in CREBBP and FAT1, suggesting they are at increased risk for progressing to frank malignancy. Conclusions: We performed genomic profiling of PMLs in the airways of high-risk smokers. The gene expression and somatic alterations that were observed in known cancer genes may be among the earliest events in cancer development. Citation Format: Joshua D. Campbell, Catalina Perdomo, Sarah Mazzilli, Yaron Geshalter, Samjot S. Dhillon, Gang Liu, Sherry Zhang, Hangqio Lin, Jessica Vick, Christopher Moy, Evan Johnson, Matthew Meyerson, Suso Platero, Marc Lenburg, Mary Reid, Avrum Spira, Jennifer Beane. Genomic characterization of premalignant lung squamous cell carcinoma lesions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 895.

Abstract Background: Lung squamous cell carcinoma (SqCC) arises in the epithelial layer of the bronchial airway and is often preceded by the development of premalignant lesions. However, not all premalignant lesions progress to lung SqCC and many regress without therapeutic intervention. Understanding the somatic alterations that contribute to progression of premalignant lesions in the airway will allow us to identify biomarkers for early detection and develop therapeutic strategies for early intervention. Methods: Airway biopsies were obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and chest CT at the Roswell Park Cancer Institute. For each subject (n=30), multiple premalignant lesions were sampled repeatedly over time (n=144 samples). One biopsy from each region was sent for pathological review while another biopsy was taken for molecular studies. DNA was also isolated from the blood or cytologically normal bronchial brushings to serve as a matched normal control. Exome capture was performed using the Illumina TruSeq Rapid Exome kit and sequenced to a mean depth of coverage of 120x at Uniform Services University and Walter Reed National Military Medical Center. Results: The median number of somatic mutations across all premalignant lesions was 0.73 per megabase (range: 0.10 - 9.8 per Mb) and displayed a modest association with histological grade (p=0.07). The most frequently mutated lung cancer genes included KMT2C (12%), NOTCH1 (11%), FAT1 (6%), TP53 (5%), and CDKN2A (<1%). Known oncogenic hotspot mutations were observed in PIK3CA (1%) and KRAS (<1%). The majority of lesions did not have overlapping sets of mutations with other samples from the same patient, indicating that most of these lesions arose from clonally distinct populations. The two lesions with the relatively high mutation rates (>7/Mb) were taken from adjacent sites over two time points in the same individual with a prior history of lung squamous cell carcinoma. These lesions had a significantly overlapping set of mutations including FAT1 indicating a common evolutionary ancestor. Conclusions: The somatic alterations observed in known cancer genes such as TP53, KMT2C, NOTCH1, and FAT1 may be among the earliest driver events in lung SqCC development and may be useful as biomarkers for early detection as well as targets for lung cancer interception. Citation Format: Joshua Campbell, Xijun Zhang, Samjot S. Dhillon, Catalina Perdomo, Sarah Mazzilli, Yaron Geshalter, Gang Liu, Sherry Zhang, Hanqiao Lin, Jessica Vick, Christopher Moy, Stefano Monti, Evan Johnson, Matthew Meyerson, Steven Dubinett, Suso Platero, Matthew Wilkerson, Clifton Dalgard, Marc Lenburg, Mary Reid, Jennifer Beane, Avrum Spira. The genomic landscape of premalignant lung squamous cell carcinoma lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3259. doi:10.1158/1538-7445.AM2017-3259

Abstract Background: Squamous cell carcinoma (SCC) of the lung is a leading cause of cancer mortality in the US due to late stage diagnosis and lack of effective treatments. Lung SCC arises in the epithelial layer of the bronchial airways and is often preceded by the development of premalignant lesions (PMLs). The molecular events involved in the progression of PMLs to lung SCC are not clearly understood, and not all PMLs go on to form carcinoma. By molecularly characterizing PMLs and non-lesion areas in the airway of individuals with PMLs, we hypothesize that we will be able to identify subgroups of PMLs that are more likely to progress. Methods: We used mRNA sequencing to profile biopsies obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and CT at the Roswell Park Cancer Institute in Buffalo, NY. For each subject (n=30), we sampled bronchial biopsies repeatedly over time (394 +/- 170 days) with serial bronchoscopies (6 +/- 5 biopsies/subject) as the biopsied area progressed towards or regressed away from frank malignancy. mRNA-Seq (n=197 biopsies) data was aligned to hg19 using STAR, and gene/transcript levels were summarized using RNA-Seq using RSEM and Ensembl 74 annotation. Immune, stromal, and epithelial cells content were inferred using the ESTIMATE algorithm and pathway activity of in vitro derived oncogenic signatures was estimated using GSVA for each sample. Molecular subtypes were derived using non-negative matrix factorization (NMF) and consensus clustering. Linear modeling was used to associate PML outcome metrics and pathway activity scores with subtype membership. Results: We identified six distinct molecular subtypes of bronchial biopsies using NMF across the 10% most variable genes (n=2,322 gene). One subtype contained samples with stable high-grade histology (p<0.01). This subtype was also enriched for basal epithelial cell content and oncogenic pathway activity including AKT and EGFR (p<0.01). Another subtype was enriched for immune cell content and had elevated expression of immune-related pathways including chemokine, cytokine, and T-cell receptor signaling. Increased immune infiltrate was also observed in H&E stains from biopsies taken adjacent to the sequenced biopsy. Conclusions: Molecular classification of premalignant lesions may lead to biomarkers of disease progression that could be used to stratify patients into prevention trials and to monitor efficacy of the treatment. Additionally, the results suggest that personalized interventions targeting specific cancer-related pathways or the immune system may be have potential therapeutic benefits. Citation Format: Jennifer E. Beane, Sarah Mazzilli, Joshua Campbell, Christopher Moy, Michael Schaffer, Catalina Perdomo, David Jenkins, Gang Liu, Sherry Zhang, Hanqiao Liu, Jessica Vick, Evan Johnson, Suso Platero, Marc Lenburg, Mary Reid, Samjot S. Dhillon, Avrum Spira. Premalignant squamous cell lung carcinoma lesions have distinct molecular subtypes associated with histologic progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5002. doi:10.1158/1538-7445.AM2017-5002

Because the electronic cigarette (ECIG) is designed to deliver nicotine without combusting tobacco, they are widely advertised as a safer alternative to tobacco cigarettes (TCIGs). ECIGs are controversial due to the lack of quality control standards and the paucity of data on their safety and long–term health effects. The absence of product standards and regulation, leading to variability in product quality is a major concern. Studies analyzing the contents of the ECIG cartridge and/or vapor have revealed the presence of major tobacco-specific nitrosamines, volatile organic compounds, and metals. Multiple studies have detected inconsistent levels of nicotine in cartridges and refills between ECIG manufacturers compared to the content labeling. For this reason, each component of ECIGs is the subject of public health and safety concern. In this study, we assess the impact of ECIG exposure on the carcinogenic potential of immortalized human bronchial epithelial cells on a background of silenced p53 and activated KRAS, mutations often observed in the airway of current and former smokers at risk for lung cancer. Our preliminary results demonstrate that exposure to clinically relevant concentrations of ECIG vapor-conditioned media enhance the cancer-associated behavior of ‘at-risk’ airways with a demonstrated capacity for malignant transformation. We observed enhanced colony growth in anchorage independent assays and increased cell invasion-associated morphological changes in three-dimensional air-liquid interface models. In addition, we found that mutant epithelial cells exposed to ECIG vapor-conditioned media induces airway gene expression changes that are similar to those seen with TCIG exposure. Currently, we are defining an ECIG exposure signature. In addition, we will also evaluate the effects of chemical substances present in ECIGs such as tobacco-specific nitrosamines. These studies will identify the potential impact of ECIGs on airway epithelium carcinogenesis and add to our overall understanding of early disease pathogenesis in human lung cancer. These studies were supported by funding from the following: NIH/NCI #U01CA152751 (SMD, TCW), NCI #U01CA152751-S1 (SMD, TCW, SJP), NCI #U01CA152751-AS (SMD, KK), NCI #T32-CA009120-36 (SMD, SJP, PCP), NIH/NHLBI #T32HL072752 (SMD, EL), University of California Tobacco-Related Disease Research Program (TRDRP) #18FT-0060 (TCW), TRDRP #20KT-0055 (TCW), #23RT-0011 (SMD) Prevent Cancer Foundation (SJP), Texas Lung Cancer SPORE (P50CA70907, JDM, JEL).

Little evidence is available regarding the physiological effects of exposure to electronic cigarette (ECIG) aerosol. We sought to determine the molecular impact of ECIG aerosol exposure in human bronchial epithelial cells (HBECs). Gene-expression profiling was conducted in primary grown at air liquid interface and exposed to 1 of 4 different ECIG aerosols, traditional tobacco cigarette (TCIG) smoke, or clean air. Findings were validated experimentally with quantitative polymerase chain reaction and a reactive oxygen species immunoassay. Using gene set enrichment analysis, signatures of in vitro ECIG exposure were compared with those generated from bronchial epithelial brushings of current TCIG smokers and former TCIG smokers currently using ECIGs. We found 546 genes differentially expressed across the ECIG, TCIG, and air-exposed groups of HBECs (ANOVA; FDR q < .05; fold change > 1.5). A subset of these changes were shared between TCIG- and ECIG-exposed HBECs. ECIG exposure induced genes involved in oxidative and xenobiotic stress pathways and increased a marker of reactive oxygen species production in a dose-dependent manner. ECIG exposure decreased expression of genes involved in cilia assembly and movement. Furthermore, gene-expression differences observed in vitro were concordant with differences observed in airway epithelium collected from ECIG users (q < .01). In summary, our data suggest that ECIG aerosol can induce gene-expression changes in bronchial airway epithelium in vitro, some of which are shared with TCIG smoke. These changes were generally less pronounced than the effects of TCIG exposure and were more pronounced in ECIG products containing nicotine than those without nicotine. Our data further suggest that the gene-expression alterations seen with the in vitro exposure system reflects the physiological effects experienced in vivo by ECIG users.

RATIONALEDespite 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.OBJECTIVESWe applied an integrative genomic approach to identify convergent transcriptomic pathways in emphysema and IPF.METHODSWe defined the transcriptional repertoire of chronic obstructive pulmonary disease, IPF, or normal histology lungs using RNA-seq (n = 87).MEASUREMENTS AND MAIN RESULTSGenes 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.CONCLUSIONSOur 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.

Electronic cigarettes (ECIGs) are an emerging alternative tobacco product thought by some to potentially be safer than traditional tobacco cigarettes (TCIGs). Despite the increasing prevalence of ECIG use, few studies have evaluated the potential physiological effects of ECIG exposure. In this study we aimed to determine the global gene expression effects of ECIG exposure on bronchial epithelium in vitro. Human bronchial epithelial cells (HBECs) grown at Air Liquid Interface (ALI) were exposed to TCIG smoke and ECIG vapor derived from tobacco or menthol flavored products with and without nicotine. We identified a number of gene expression alterations that were induced by both ECIG and TCIG exposure as well as a novel set of changes uniquely induced by ECIG exposure. ECIG exposure induced the expression of genes involved in oxidative and xenobiotic stress pathways and increased the production of reactive oxygen species, similar to, but generally lower in magnitude than, the effects of TCIGs. Furthermore, TCIG and ECIG exposure both decreased the expression of genes involved in cilia assembly and movement, suggesting that the integrity of the bronchial epithelium is concordantly impaired by both exposures. We additionally identified a number of ECIG-specific cell cycle and cell division pathway changes. Finally, we observed that ECIG-induced changes were dependent on both flavor and nicotine content. Together, these results indicate that ECIG vapor can induce cellular stress and molecular alterations within airway epithelium that share similarities with the effects of TCIG smoke. Based on these findings, further studies are warranted to determine whether ECIG use will lead to similar deleterious health outcomes as those caused by TCIGs. Citation Format: Elizabeth Moses, Teresa Wang, George R. Jackson, Sean Corbett, Eduard Drizik, Daniel Brooks, George O’Connor, Catalina Perdomo, Steven Dubinett, Patrick Hayden, Marc E. Lenburg, Avrum Spira. Molecular impact of in vitro exposure to electronic cigarette vapor in human bronchial epithelium. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4502.

Despite a strong correlation between cigarette smoking and the onset of lung cancer, the prevalence of smoking still remains high. The electronic cigarette (ECIG) is designed to deliver nicotine without combusting tobacco. Since nicotine is widely considered the addictive component in tobacco with limited ability to initiate cancer, ECIGs have been advertised to be a safer alternative to tobacco cigarettes. However, the potential health risks and carcinogenicity of ECIGs have not previously been evaluated. In this study, we assess the impact of ECIG exposure on the carcinogenic potential of immortalized human bronchial epithelial cells on a background of silenced p53 and activated KRAS (H3mut-P53/KRAS). This model is utilized because p53 and KRAS mutations are often observed in the airway of current and former smokers at risk for lung cancer. In anchorage independent growth assays, the in vitro correlate of malignant transformation, we found enhanced colony growth in the HBEC-P53/KRAS cells following a 12-day treatment with high concentrations of ECIG-conditioned media compared to the untreated and low concentration treatment groups. We next assessed the effect of ECIG and exposure on cell invasion using three-dimensional air-liquid interface (ALI) models. HBEC-P53/KRAS cells exhibited invasion-associated morphological changes following a 12-day treatment with the high conditioned media, including increased proliferation, diminished cell-cell cohesion and the appearance of cells percolating out of and breaching the modified basement membrane. Finally, to identify the biological impact of in vitro ECIG exposure in HBECs, we profiled the gene expression of P53/KRAS cells following a 96-hour exposure to ECIG- or tobacco cigarette (TCIG)-conditioned media. We found that epithelial cells exposed to clinically relevant concentrations of ECIG vapor-conditioned media have a gene expression pattern similar to those exposed to TCIG smoke-conditioned media and whole cigarette smoke. Rank-rank hyper-geometric overlap (RRHO) analysis indicated that differential expression - based ranked genes in TCIG and ECIG exposed groups were consistently overlapped at significant levels. There were 263 differentially expressed genes in the cells treated with high ECIG media versus untreated control. Annotations of the identified genes by the Molecular Signature database revealed several enriched biological pathways involved in malignant transformation and epithelial-mesenchymal transition (EMT). We have compared the resulting list of genes to publicly available microarray datasets and identified several transformation-related gene candidates. We are in the process of evaluating their contribution to ECIG-induced dissemination and carcinogenesis in vitro and in vivo. These studies will determine the impact of ECIG exposure on lung carcinogenicity and provide needed scientific guidance to the FDA regarding the physiologic effects of ECIGs. These studies were supported by funding from the following: NIH/NCI #U01CA152751 (SMD, TCW), NCI #U01CA152751-S1 (SMD, TCW, SJP), NCI #U01CA152751-AS (SMD, KK), NCI #T32-CA009120-36 (SMD, SJP, PCP), NIH/NHLBI #T32HL072752 (SMD, EL), University of California Tobacco-Related Disease Research Program (TRDRP) #18FT-0060 (TCW), TRDRP #20KT-0055 (TCW), Prevent Cancer Foundation (SJP), Lung Cancer SPORE (P50CA70907, JDM, JEL) Citation Format: Stacy J. Park, Tonya C. Walser, Linh M. Tran, Catalina Perdomo, Teresa Wang, Long-Sheng Hong, Paul C. Pagano, Elvira L. Liclican, Jill E. Larsen, Kostyantyn Krysan, Michael C. Fishbein, John D. Minna, Marc E. Lenburg, Spira Avrum, Steven Dubinett. The role of e-cigarette exposure on pulmonary epithelial cell transformation. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B22.

Squamous cell cancer (SCC) of the lung is a leading cause of cancer mortality in the US, due to late stage diagnosis and lack of effective treatments. Lung SCC arises in the epithelial layer of the bronchial airways and is often preceded by the development of premalignant lesions (PMLs). The molecular events involved in the progression of PMLs to lung SCC are not clearly understood and not all PMLs go on to form carcinoma. By molecularly characterizing PMLs and non-lesion areas in the airway of individuals with PMLs we hypothesize that we will be able to identify early events in the process of lung carcinogenesis that lead to SCC. We used next-generation sequencing to profile bronchial brushings and biopsies obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and CT at the Roswell Park Cancer Institute in Buffalo, NY. For each subject (n = 26), we sampled the PML(s) and the mainstem bronchus repeatedly over time (394 +/- 170 days) with serial bronchoscopies (5 +/- 3 biopsies/subject) as the PML progressed towards or regressed away from frank malignancy. mRNA-Seq (n = 192) and miRNA-Seq (n = 183) were performed on the endobronchial biopsies and brushings and exome-Seq was performed on blood DNA from these subjects. RNA-seq data was aligned to the hg19 and gene/transcript levels were summarized using RSEM/Ensembl 74 or Bedtools/ mirBase 18. Single nucleotide variants were quantified using a modified PRADA pipeline and GATK. We identified gene and miRNA expression changes as well as pathways that are associated with biopsy histological grade as well as progressive/stable disease. HE 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2878. doi:10.1158/1538-7445.AM2015-2878

Lung cancer is the leading cause of cancer deaths in the United States. Despite a strong correlation between cigarette smoking and the onset of lung cancer, the prevalence of smoking still remains high. Significant reduction or elimination of the use of combustible tobacco products would substantially lower tobacco-caused morbidity and mortality. The electronic cigarette (ECIG) is a battery-powered electronic nicotine delivery system (ENDS) designed to deliver nicotine without combusting tobacco. ECIGs are widely believed to be a safer alternative to tobacco cigarettes (TCIG), however, the toxicity and potential carcinogenicity of ECIGs have not previously been evaluated. In this study, we assess the impact of ECIG exposure on the carcinogenic potential of immortalized human bronchial epithelial cells on a background of silenced p53 and activated KRAS (H3mut-P53/KRAS). This model is utilized because p53 and KRAS mutations are often observed in the airway of current and former smokers at risk for lung cancer. The epithelial cells were exposed to a low and high nicotine concentration of ECIG vapor- or TCIG smoke-conditioned media. The lower nicotine concentration was selected to mimic plasma nicotine levels in ENDS users and did not demonstrate toxic or anti-proliferative effects on the cells. The higher concentration was chosen to represent the anticipated nicotine levels to which the epithelial cells of smokers are actually exposed. In anchorage independent growth assays, the in vitro correlate of malignant transformation, we found enhanced colony growth in the H3mut-P53/KRAS cells following a 10-day treatment with the high nicotine concentration of ECIG- and TCIG-conditioned media compared to the untreated and low nicotine treatment groups. We next assessed the effect of ECIG and TCIG exposure on cell invasion using three-dimensional air-liquid interface (ALI) models. While treatment with the low nicotine concentration of ECIG-conditioned media did not induce invasion-associated morphological changes, cells treated with the low nicotine concentration of TCIG-conditioned media exhibited slightly invasive behavior by breaching the modified basement membrane and invading through the field of fibroblasts/collagen. At baseline, H3mut-P53/KRAS cells exhibit invasive behavior in the ALI model, due to the downstream effects of P53 silencing and KRAS activation. Treatment of H3mut-P53/KRAS cells with the low nicotine concentration of ECIG- and TCIG-conditioned media did not further enhance the degree of invasion observed in the untreated group. We will next examine the effect of the high nicotine concentration on cell invasion. In addition, we are currently evaluating gene expression profiles by microarray of H3mut-P53/KRAS cells exposed to high and low nicotine concentrations of ECIG-conditioned media. This will directly evaluate the impact of ECIGs on carcinogenicity-related gene expression signatures established in previous and ongoing clinical investigations. These studies will determine the impact of ECIG exposure on lung carcinogenicity, which will provide needed scientific guidance to the FDA regarding the physiologic effects of ECIGs. These studies were supported by the NIH/NCI #U01CA152751-S1 (SMD, AS, MEL, TCW) and NIH/NHLBI #T32HL072752 (SJP). Citation Format: Stacy J. Park, Tonya C. Walser, Catalina Perdomo, Paul P. Pagano, Daniel Brass, Elvira L. Liclican, Kostyantyn Krysan, Marc E. Lenburg, Avrum Spira, Steven M. Dubinett. The gene expression and functional impact of E-cigarette exposure on pulmonary epithelial transformation. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr C58.

Abstract Lung cancer is the leading cause of cancer-related mortality in the United States. Despite a strong correlation between cigarette smoking and the onset of lung cancer, the prevalence of smoking still remains high. Strategies to eliminate cigarette smoking have led to the emergence of new tobacco-related products as alternatives for cigarette smoking or tools for smoking cessation. The electronic cigarette (ECIG) is a battery-powered electronic nicotine delivery system (ENDS) designed to deliver nicotine without combusting tobacco. Since nicotine is widely considered the addictive component in tobacco with limited ability to initiate cancer, ECIGs have been advertised to be a safer alternative to tobacco cigarettes (TCIGs). However, the toxicity and potential carcinogenicity of ECIGs have not previously been evaluated. In this study, we assess the impact of ECIG exposure on the carcinogenic potential of immortalized human bronchial epithelial cells on a background of silenced p53 and activated KRAS (H3mut-P53/KRAS). This model is utilized because p53 and KRAS mutations are often observed in the airway of current and former smokers at risk for lung cancer. The epithelial cells were exposed to both a low and high concentration of nicotine in the ECIG vapor- or TCIG smoke-conditioned media. The lower nicotine concentration was selected to mimic the average plasma nicotine levels in ENDS users and did not demonstrate toxic or anti-proliferative effects on the cells. The higher concentration was chosen to represent the anticipated nicotine levels to which the epithelial cells of smokers are actually exposed. In anchorage independent growth assays, the in vitro correlate of malignant transformation, we found enhanced colony growth in the H3mut-P53/KRAS cells following a 10-day treatment with the high nicotine ECIG- and TCIG-conditioned media compared to the untreated and low nicotine treatment groups. We next assessed the effect of ECIG and TCIG exposure on cell invasion using a three-dimensional air-liquid interface (ALI) model. At baseline, H3mut-P53/KRAS cells exhibit invasive behavior in the ALI model, due to the downstream effects of P53 silencing and KRAS activation. Treatment of H3mut-P53/KRAS cells with low nicotine ECIG- and TCIG-conditioned media did not further enhance the degree of invasion observed in the untreated group. We will next examine the effects of high nicotine conditioned media on cell invasion. Finally, gene expression studies show 263 differentially expressed genes following in vitro exposure to ECIG-conditioned media for 96hrs. The high nicotine ECIG-conditioned media induced a gene expression pattern similar to TCIG- conditioned media and whole cigarette smoke exposure in the H3mut-P53/KRAS cells. Preliminary analyses indicate the observed ECIG-specific gene expression changes were concordantly changed following TCIG-conditioned media exposure. We will next compare the ECIG-induced gene expression signature to carcinogenicity-related gene signatures established in previous and ongoing clinical investigations and test ECIG-altered candidate genes for their ability to drive the malignant transformation of airway epithelial cells. These studies will determine the impact of ECIG exposure on lung carcinogenicity and provide needed scientific guidance to the FDA regarding the physiologic effects of ECIGs. These studies were supported by funding from the following: NIH/NCI #U01CA152751 (SMD, TCW), NCI #U01CA152751-S1 (SMD, TCW, SJP), NCI #U01CA152751-AS (SMD, KK), NCI #T32-CA009120-36 (SMD, SJP, PCP), NIH/NHLBI #T32HL072752 (SMD, EL), University of California Tobacco-Related Disease Research Program (TRDRP) #18FT-0060 (TCW), TRDRP #20KT-0055 (TCW), Lung Cancer SPORE P50CA70907 (JDM, JEL) Citation Format: Stacy J. Park, Tonya C. Walser, Catalina Perdomo, Teresa Wang, Paul C. Pagano, Elvira L. Liclican, Kostyantyn Krysan, Jill E. Larsen, John D. Minna, Marc E. Lenburg, Avrum Spira, Steven M. Dubinett. The effect of e-cigarette exposure on airway epithelial cell gene expression and transformation. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B16.

RATIONALE: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.OBJECTIVES:We sought to determine whether COPD-associated processes are reflected in gene expression profiles of bronchial airway epithelial cells obtained by bronchoscopy.METHODS: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.MEASUREMENTS AND MAIN RESULTS: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.CONCLUSIONS: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.

Smoking is a significant risk factor for lung cancer, the leading cause of cancer-related deaths worldwide. Although microRNAs are regulators of many airway gene-expression changes induced by smoking, their role in modulating changes associated with lung cancer in these cells remains unknown. Here, we use next-generation sequencing of small RNAs in the airway to identify microRNA 4423 (miR-4423) as a primate-specific microRNA associated with lung cancer and expressed primarily in mucociliary epithelium. The endogenous expression of miR-4423 increases as bronchial epithelial cells undergo differentiation into mucociliary epithelium in vitro, and its overexpression during this process causes an increase in the number of ciliated cells. Furthermore, expression of miR-4423 is reduced in most lung tumors and in cytologically normal epithelium of the main-stem bronchus of smokers with lung cancer. In addition, ectopic expression of miR-4423 in a subset of lung cancer cell lines reduces their anchorage-independent growth and significantly decreases the size of the tumors formed in a mouse xenograft model. Consistent with these phenotypes, overexpression of miR-4423 induces a differentiated-like pattern of airway epithelium gene expression and reverses the expression of many genes that are altered in lung cancer. Together, our results indicate that miR-4423 is a regulator of airway epithelium differentiation and that the abrogation of its function contributes to lung carcinogenesis.

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 The goal of this study was to characterize the small RNA transcriptome and discover novel microRNAs (miRNAs) associated with lung cancer in human bronchial epithelial cells using massively parallel sequencing. To this end, small RNA (10-40 nts) was isolated from bronchial brushings obtained via bronchoscopy. Samples pooled from healthy non-smokers, healthy smokers, and smokers with and without lung cancer (n=3 per pool) were sequenced with ABI SOLiD, using extremely deep coverage for small RNA in human tissue, 60 million 36nt-long reads/pool. Reads were aligned to the human genome with Bowtie and novel miRNAs were predicted using the miRDeep algorithm. One of the predicted novel miRNAs was selected for experimental validation. In vitro expression of the putative novel microRNA precursor was assayed with and without the siRNA knock-down of Dicer, an enzyme that processes miRNA precursors into mature miRNAs. Tissue specificity of the putative novel miRNA was profiled across 23 human tissue types using qRT-PCR. Potential microRNA targets were identified using transient overexpression of the miRNA in a cell line combined with in silico algorithms. We identified 143 miRNA that are differentially expressed in the airways of smokers with lung cancer (FC > 2) in our cohort. The miRDeep algorithm identified 131 putative novel miRNAs across all samples, including 68 differentially expressed putative miRNA in the airways of lung cancer subjects (FC > 2). In vitro expression of the putative miRNA was reduced after siRNA knockdown of DICER, providing strong evidence that the novel transcript is truly a miRNA. Out of 23 human tissue types assayed, the microRNA is expressed almost exclusively in the respiratory tract, with highest expression in the bronchus and nose and moderate expression in lung. Importantly, the expression of the miRNA is decreased significantly in lung tumors compared to adjacent normal tissue. In addition, the expression of the novel miRNA is decreased in the bronchial epithelium of smokers with lung cancer as compared to smokers without lung cancer. Overexpression of the novel miRNA in lung cancer cell line has identified putative mRNA targets that are associated with apoptosis and cell proliferation. In summary, using next generation sequencing, we have identified airway miRNA expression profiles associated with lung cancer, and we discovered a novel miRNA whose expression is reduced in the airway and lung tissue of smokers with lung cancer. This miRNA may serve as a novel biomarker to identify smokers at high risk of developing lung cancer and may also provide new insights into the genomic regulatory networks that underlie lung cancer pathogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3989. doi:10.1158/1538-7445.AM2011-3989

miRNAs are a class of small, noncoding RNAs averaging 22 nucleotides in length that down-regulate gene expression by complimentary binding to the 3′ UTR of target genes. A growing body of research suggests that these small RNA species play significant roles in modulating the cellular response to a variety of types of stress. In this review, we summarize the available literature regarding the general response of miRNA to cellular stress, and then specifically focus on the miRNA response to inhaled toxins. These miRNA responses to inhaled toxins appear to be recapitulated in lung carcinogenesis, opening the possibility that modulation of the miRNA response could be a novel strategy for chemoprevention.