Flavored E-cigarette product aerosols induce transformation of human bronchial epithelial cells.

OBJECTIVES: E-cigarettes are the most commonly used nicotine containing products among youth. In vitro studies support the potential for e-cigarettes to cause cellular stress in vivo; however, there have been no studies to address whether exposure to e-liquid aerosols can induce cell transformation, a process strongly associated with pre-malignancy. We examined whether weekly exposure of human bronchial epithelial cell (HBEC) lines to e-cigarette aerosols would induce transformation and concomitant changes in gene expression and promoter hypermethylation. MATERIALS AND METHODS: An aerosol delivery system exposed three HBEC lines to unflavored e-liquid with 1.2% nicotine, 3 flavored products with nicotine, or the Kentucky reference cigarette once a week for 12 weeks. Colony formation in soft agar, RNA-sequencing, and the EPIC Beadchip were used to evaluate transformation, genome-wide expression and methylation changes. RESULTS: Jamestown e-liquid aerosol induced transformation of HBEC2 and HBEC26, while unflavored and Blue Pucker transformed HBEC26. Cigarette smoke aerosol transformed HBEC4 and HBEC26 at efficiencies up to 3-fold greater than e-liquids. Transformed clones exhibited extensive reprogramming of the transcriptome with common and distinct gene expression changes observed between the cigarette and e-liquids. Transformation by e-liquids induced alterations in canonical pathways implicated in lung cancer that included axonal guidance and NRF2. Gene methylation, while prominent in cigarette-induced transformed clones, also affected hundreds of genes in HBEC2 transformed by Jamestown. Many genes with altered expression or epigenetic-mediated silencing were also affected in lung tumors from smokers. CONCLUSIONS: These studies show that exposure to e-liquid aerosols can induce a pre-malignant phenotype in lung epithelial cells. While the Food and Drug Administration banned the sale of flavored cartridge-based electric cigarettes, consumers switched to using flavored products through other devices. Our findings clearly support expanding studies to evaluate transformation potency for the major categories of e-liquid flavors to better inform risk from these complex mixtures.

Comparative Genotoxicity and Mutagenicity of Cigarette, Cigarillo, and Shisha Tobacco Products in Epithelial and Cardiac Cells.

Epidemiology studies link cigarillos and shisha tobacco (delivered through a hookah waterpipe) to increased risk for cardiopulmonary diseases. Here we performed a comparative chemical constituent analysis between 3 cigarettes, 3 cigarillos, and 8 shisha tobacco products. The potency for genotoxicity and oxidative stress of each product's generated total particulate matter (TPM) was also assessed using immortalized oral, lung, and cardiac cell lines to represent target tissues. Levels of the carcinogenic carbonyl formaldehyde were 32- to 95-fold greater, while acrolein was similar across the shisha aerosols generated by charcoal heating compared to cigarettes and cigarillos. Electric-mediated aerosol generation dramatically increased acrolein to levels exceeding those in cigarettes and cigarillos by up to 43-fold. Equivalent cytotoxic-mediated cell death and dose response for genotoxicity through induction of mutagenicity and DNA strand breaks was seen between cigarettes and cigarillos, while minimal to no effect was observed with shisha tobacco products. In contrast, increased potency of TPM from cigarillos compared to cigarettes for inducing oxidative stress via reactive oxygen radicals and lipid peroxidation across cell lines was evident, while positivity was seen for shisha tobacco products albeit at much lower levels. Together, these studies provide new insight into the potential harmful effects of cigarillos for causing tobacco-associated diseases. The high level of carbonyls in shisha products, that in turn is impacted by the heating mechanism, reside largely in the gas phase which will distribute throughout the respiratory tract and systemic circulation to likely increase genotoxic stress.

Chromatin remodeling by the histone methyltransferase EZH2 drives lung pre-malignancy and is a target for cancer prevention.

BACKGROUND: Trimethylation of lysine 27 and dimethylation of lysine 9 of histone-H3 catalyzed by the histone methyltransferases EZH2 and G9a impede gene transcription in cancer. Our human bronchial epithelial (HBEC) pre-malignancy model studied the role of these histone modifications in transformation. Tobacco carcinogen transformed HBEC lines were characterized for cytosine DNA methylation, transcriptome reprogramming, and the effect of inhibiting EZH2 and G9a on the transformed phenotype. The effects of targeting EZH2 and G9a on lung cancer prevention was assessed in the A/J mouse lung tumor model. RESULTS: Carcinogen exposure induced transformation and DNA methylation of 12-96 genes in the four HBEC transformed (T) lines that was perpetuated in malignant tumors. In contrast, 506 unmethylated genes showed reduced expression in one or more HBECTs with many becoming methylated in tumors. ChIP-on-chip for HBEC2T identified 327 and 143 genes enriched for H3K27me3 and H3K9me2. Treatment of HBEC2T and HBEC13T with DZNep, a lysine methyltransferase inhibitor depleted EZH2, reversed transformation, and induced transcriptional reprogramming. The EZH2 small molecule inhibitor EPZ6438 also affected transformation and expression in HBEC2T, while a G9a inhibitor, UNC0642 was ineffective. Genetic knock down of EZH2 dramatically reduced carcinogen-induced transformation of HBEC2. Only DZNep treatment prevented progression of hyperplasia to adenomas in the NNK mouse lung tumor model through reducing EZH2 and affecting the expression of genes regulating cell growth and invasion. CONCLUSION: These studies demonstrate a critical role for EZH2 catalyzed histone modifications for premalignancy and its potential as a target for chemoprevention of lung carcinogenesis.

Cytotoxicity and Genotoxicity of E-Cigarette Generated Aerosols Containing Diverse Flavoring Products and Nicotine in Oral Epithelial Cell Lines.

Electronic cigarettes are the most commonly used nicotine containing product among teenagers. The oral epithelium is the first site of exposure and our recent work revealed considerable diversity among e-liquids for composition and level of chemical constituents that impact nicotine deposition in a human oral-trachea cast and affect the formation of reactive carbonyls. Here, we evaluate the dose response for cytotoxicity and genotoxicity of e-cigarette-generated aerosols from 10 diverse flavored e-liquid products with and without nicotine compared with unflavored in 3 immortalized oral epithelial cell lines. Three e-liquids, Blue Pucker, Love Potion, and Jamestown caused ≥20% cell toxicity assessed by the neutral red uptake assay. Nine products induced significant levels of oxidative stress up to 2.4-fold quantified by the ROS-Glo assay in at least 1 cell line, with dose response seen for Love Potion with and without nicotine across all cell lines. Lipid peroxidation detected by the thiobarbituric acid reactive substances assay was less common among products; however, dose response increases up to 12-fold were seen for individual cell lines. Micronuclei formation indicative of genotoxicity was increased up to 5-fold for some products. Blue Pucker was the most genotoxic e-liquid, inducing micronuclei across all cell lines irrespective of nicotine status. A potency score derived from all assays identified Blue Pucker and Love Potion as the most hazardous e-liquids. These in vitro acute exposure studies provide new insight about the potential for some flavored vaping products to induce significant levels of oxidative stress and genotoxicity.

Dietary Nutrient Intake, Ethnicity, and Epigenetic Silencing of Lung Cancer Genes Detected in Sputum in New Mexican Smokers.

Lung cancer gene methylation detected in sputum assesses field cancerization and predicts lung cancer incidence. Hispanic smokers have higher lung cancer susceptibility compared with non-Hispanic whites (NHW). We aimed to identify novel dietary nutrients affecting lung cancer gene methylation and determine the degree of ethnic disparity in methylation explained by diet. Dietary intakes of 139 nutrients were assessed using a validated Harvard food frequency questionnaire in 327 Hispanics and 1,502 NHWs from the Lovelace Smokers Cohort. Promoter methylation of 12 lung cancer genes was assessed in sputum DNA. A global association was identified between dietary intake and gene methylation (Ppermutation = 0.003). Seventeen nutrient measurements were identified with magnitude of association with methylation greater than that seen for folate. A stepwise approach identified B12, manganese, sodium, and saturated fat as the minimally correlated set of nutrients whose optimal intakes could reduce the methylation by 36% (Ppermutation < 0.001). Six protective nutrients included vitamin D, B12, manganese, magnesium, niacin, and folate. Approximately 42% of ethnic disparity in methylation was explained by insufficient intake of protective nutrients in Hispanics compared with NHWs. Functional validation of protective nutrients showed an enhanced DNA repair capacity toward double-strand DNA breaks, a mechanistic biomarker strongly linked to acquisition of lung cancer gene methylation in smokers. Dietary intake is a major modifiable factor for preventing promoter methylation of lung cancer genes in smokers' lungs. Complex dietary supplements could be developed on the basis of these protective nutrients for lung cancer chemoprevention in smokers. Hispanic smokers may benefit the most from this complex for reducing their lung cancer susceptibility. Cancer Prev Res; 11(2); 93-102. ©2017 AACR.

miR-196b Is Epigenetically Silenced during the Premalignant Stage of Lung Carcinogenesis.

miRNA silencing by promoter hypermethylation may represent a mechanism by which lung cancer develops and progresses, but the miRNAs involved during malignant transformation are unknown. We previously established a model of premalignant lung cancer wherein we treated human bronchial epithelial cells (HBEC) with low doses of tobacco carcinogens. Here, we demonstrate that next-generation sequencing of carcinogen-transformed HBECs treated with the demethylating agent 5-aza-2'deoxycytidine revealed miR-196b and miR-34c-5p to be epigenetic targets. Bisulfite sequencing confirmed dense promoter hypermethylation indicative of silencing in multiple malignant cell lines and primary tumors. Chromatin immunoprecipitation studies further demonstrated an enrichment in repressive histone marks on the miR-196b promoter during HBEC transformation. Restoration of miR-196b expression by transfecting transformed HBECs with specific mimics led to cell-cycle arrest mediated in part through transcriptional regulation of the FOS oncogene, and miR-196b reexpression also significantly reduced the growth of tumor xenografts. Luciferase assays demonstrated that forced expression of miR-196b inhibited the FOS promoter and AP-1 reporter activity. Finally, a case-control study revealed that methylation of miR-196b in sputum was strongly associated with lung cancer (OR = 4.7, P < 0.001). Collectively, these studies highlight miR-196b as a tumor suppressor whose silencing early in lung carcinogenesis may provide a selective growth advantage to premalignant cells. Targeted delivery of miR-196b could therefore serve as a preventive or therapeutic strategy for the management of lung cancer. Cancer Res; 76(16); 4741-51. ©2016 AACR.

Implication of a Chromosome 15q15.2 Locus in Regulating UBR1 and Predisposing Smokers to MGMT Methylation in Lung.

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that protects cells from carcinogenic effects of alkylating agents; however, MGMT is silenced by promoter hypermethylation during carcinogenesis. A single-nucleotide polymorphism (SNP) in an enhancer in the MGMT promoter was previously identified to be highly significantly associated with risk for MGMT methylation in lung cancer and sputum from smokers. To further genetic investigations, a genome-wide association and replication study was conducted in two smoker cohorts to identify novel loci for MGMT methylation in sputum that were independent of the MGMT enhancer polymorphism. Two novel trans-acting loci (15q15.2 and 17q24.3) that were identified acted together with the enhancer SNP to empower risk prediction for MGMT methylation. We found that the predisposition to MGMT methylation arising from the 15q15.2 locus involved regulation of the ubiquitin protein ligase E3 component UBR1. UBR1 attenuation reduced turnover of MGMT protein and increased repair of O6-methylguanine in nitrosomethylurea-treated human bronchial epithelial cells, while also reducing MGMT promoter activity and abolishing MGMT induction. Overall, our results substantiate reduced gene transcription as a major mechanism for predisposition to MGMT methylation in the lungs of smokers, and support the importance of UBR1 in regulating MGMT homeostasis and DNA repair of alkylated DNA adducts in cells.

GATA2 is epigenetically repressed in human and mouse lung tumors and is not requisite for survival of KRAS mutant lung cancer.

INTRODUCTION: GATA2 was recently described as a critical survival factor and therapeutic target for KRAS mutant non-small-cell lung cancer (NSCLC). However, whether this role is affected by epigenetic repression of GATA2 in lung cancer is unclear. METHODS: GATA2 expression and promoter CpG island methylation were evaluated using human and mouse NSCLC cell lines and tumor-normal pairs. In vitro assays were used to study GATA2 repression on cell survival and during tobacco carcinogen-induced transformation. RESULTS: GATA2 expression in KRAS wild-type (n = 15) and mutant (n = 10) NSCLC cell lines and primary lung tumors (n = 24) was significantly lower, 1.3- to 33.6-fold (p = 2.2 × 10(9)), compared with corresponding normal lung. GATA2 promoter was unmethylated in normal lung (0 of 10) but frequently methylated in lung tumors (96%, 159 of 165) and NSCLC cell lines (97%, 30 of 31). This highly prevalent aberrant methylation was independently validated using The Cancer Genome Atlas data for 369 NSCLC tumor-normal pairs. In vitro studies using an established carcinogen-induced premalignancy model revealed that GATA2 expression was initially repressed by chromatin remodeling followed by cytosine methylation during transformation. Similarly, expression of GATA2 in NNK-induced mouse lung tumors (n = 6) and cell lines (n = 5) was fivefold and 100-fold lower, respectively, than normal mouse lung. Finally, siRNA-mediated knockdown of GATA2 in KRAS mutant (human [n = 4] and murine [n = 5]) and wild-type (human [n = 4]) NSCLC cell lines showed that further reduction of expression (up to 95%) does not induce cell death. CONCLUSION: GATA2 is epigenetically repressed in human and mouse lung tumors and its further inhibition is not a valid therapeutic strategy for KRAS mutant lung cancer.

EMT and stem cell-like properties associated with miR-205 and miR-200 epigenetic silencing are early manifestations during carcinogen-induced transformation of human lung epithelial cells.

Epithelial-to-mesenchymal transition (EMT) is strongly associated with cancer progression, but its potential role during premalignant development has not been studied. Here, we show that a 4-week exposure of immortalized human bronchial epithelial cells (HBEC) to tobacco carcinogens can induce a persistent, irreversible, and multifaceted dedifferentiation program marked by EMT and the emergence of stem cell-like properties. EMT induction was epigenetically driven, initially by chromatin remodeling through H3K27me3 enrichment and later by ensuing DNA methylation to sustain silencing of tumor-suppressive microRNAs (miRNA), miR-200b, miR-200c, and miR-205, which were implicated in the dedifferentiation program in HBECs and also in primary lung tumors. Carcinogen-treated HBECs acquired stem cell-like features characterized by their ability to form spheroids with branching tubules and enrichment of the CD44(high)/CD24(low), CD133, and ALDH1 stem cell-like markers. miRNA overexpression studies indicated that regulation of the EMT, stem-like, and transformed phenotypes in HBECs were distinct events. Our findings extend present concepts of how EMT participates in cancer pathophysiology by showing that EMT induction can participate in cancer initiation to promote the clonal expansion of premalignant lung epithelial cells.

Dual promoter regulation of death-associated protein kinase gene leads to differentially silenced transcripts by methylation in cancer.

Death-associated protein kinase (DAPK), a mediator of apoptotic systems, is silenced by promoter hypermethylation in lung and breast tumors. This gene has a CpG island extending 2500 bp from the translational start site; however, studies characterizing its transcriptional regulation have not been conducted. Two transcripts for DAPK were identified that code for a single protein, while being regulated by two promoters. The previously identified DAPK transcript designated as exon 1 transcript was expressed at levels 3-fold greater than the alternate exon 1b transcript. Deletion constructs of promoter 1 identified a 332 bp region containing a functional CP2-binding site important for expression of the exon 1 transcript. While moderate reporter activity was seen in promoter 2, the region comprising intron 1 and containing a HNF3B-binding site sustained expression of the alternate transcript. Sequencing the DAPK CpG island in tumor cell lines revealed dense, but heterogenous methylation of CpGs that blocked access of the CP2 and HNF3B proteins that in turn, was associated with loss of transcription that was restored by treatment with 5-aza-2'-deoxycytidine. Prevalences were similar for methylation of promoter 1 and 2 and intron 1 in lung tumors, but significantly greater in promoter 2 and intron 1 in breast tumors, indicative of tissue-specific differences in silencing these two transcripts. These studies show for the first time dual promoter regulation of DAPK, a tumor suppressor gene silenced in many cancers, and substantiate the importance of screening for silencing of both transcripts in tumors.

Identification of a novel Bcl-2 promoter region that counteracts in a p53-dependent manner the inhibitory P2 region.

Expression of the anti-apoptotic proto-oncogene bcl-2 is negatively affected by the pro-apoptotic p53. To understand the regulation of bcl-2 expression by p53, we studied the bcl-2 promoter regions individually and in the context of the full-length promoter. While the P1 promoter displayed the highest p53-independent activity, the P2 promoter activity was suppressed in p53-sufficient cancer cell lines. In addition, P2 activity was higher in primary airway epithelial cells from p53(-/-) mice compared to those from p53(+/+) mice. Chromatin immunoprecipitation assays confirmed that p53 interacts within a 140 bp sequence of P2 that contained the CCAAT- and TATA-elements. However, when P1 and P2 are linked in one construct, P2 suppressed P1 activity independent of p53. A potential novel promoter with a p53-dependent activity was identified located between P1 and P2, and was designated M. In the context of the full-length bcl-2 promoter, M counteracted in a p53-dependent manner the suppressive activity of P2 on P1. Collectively, these data suggest that P1 promoter is the main driving force for transcribing the bcl-2 gene and P1 activity is modulated by M and P2 in a p53-dependent and -independent manner. These findings may have implications for therapies that are geared towards inhibiting bcl-2 gene expression and inducing cell death.