Carcinogens In Cigarette Smoke Research Articles

Benzene Uptake and Glutathione S-transferase T1 Status as Determinants of S-Phenylmercapturic Acid in Cigarette Smokers in the Multiethnic Cohort.

Research from the Multiethnic Cohort (MEC) demonstrated that, for the same quantity of cigarette smoking, African Americans and Native Hawaiians have a higher lung cancer risk than Whites, while Latinos and Japanese Americans are less susceptible. We collected urine samples from 2,239 cigarette smokers from five different ethnic groups in the MEC and analyzed each sample for S-phenylmercapturic acid (SPMA), a specific biomarker of benzene uptake. African Americans had significantly higher (geometric mean [SE] 3.69 [0.2], p<0.005) SPMA/ml urine than Whites (2.67 [0.13]) while Japanese Americans had significantly lower levels than Whites (1.65 [0.07], p<0.005). SPMA levels in Native Hawaiians and Latinos were not significantly different from those of Whites. We also conducted a genome-wide association study in search of genetic risk factors related to benzene exposure. The glutathione S-transferase T1 (GSTT1) deletion explained between 14.2–31.6% (p = 5.4x10-157) and the GSTM1 deletion explained between 0.2%-2.4% of the variance (p = 1.1x10-9) of SPMA levels in these populations. Ethnic differences in levels of SPMA remained strong even after controlling for the effects of these two deletions. These results demonstrate the powerful effect of GSTT1 status on SPMA levels in urine and show that uptake of benzene in African American, White, and Japanese American cigarette smokers is consistent with their lung cancer risk in the MEC. While benzene is not generally considered a cause of lung cancer, its metabolite SPMA could be a biomarker for other volatile lung carcinogens in cigarette smoke.

Fanconi anemia genes in lung adenocarcinoma- a pathway-wide study on cancer susceptibility.

BackgroundCarcinogens in cigarette smoke can induce the formation of DNA-DNA cross-links, which are repaired by the Fanconi anemia (FA) pathway, and it is tempting to speculate that this pathway is involved in lung tumorigenesis. This study is to determine whether genetic polymorphism of the FA genes is associated with an elevated risk of lung adenocarcinoma, and whether the association between genotypes and risk is modified by exposure to cigarette smoke.MethodsThis case–control study genotyped 53 single-nucleotide polymorphisms (SNPs) in FA genes in 709 patients (354 males and 355 females) with lung adenocarcinoma and in 726 cancer-free individuals (339 males and 387 females). Genotypic frequencies of SNPs were compared between cases and controls to identify important FA genes associated with cancer susceptibility. Joint effects in determining cancer risk contributed by genes and smoking-related risk factors and by multiple genes involved in different FA subpathways were evaluated by multivariate regression analysis and stratified analysis. All analyses were performed on males and females separately, and the comparison of results was considered a way of examining the validity of study findings.ResultsLung adenocarcinomas in both male and female patients were associated with (a) genotypic polymorphisms of FANCC and FANCD1; (b) a combined effect of harboring a higher number of high-risk genotypes and smoking/passive smoking; (c) specific interactions of multiple genes, proteins encoded by which have been known to work jointly within the FA pathway.ConclusionsGenetic polymorphism of the FA genes is associated with inter-individual susceptibility to lung adenocarcinoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-016-0240-9) contains supplementary material, which is available to authorized users.

Associations between Polymorphisms and Haplotypes in the UDPGlucuronosyl Transferase 1A Gene Family with Lung Cancer Risk

Title: Associations between polymorphisms and haplotypes in the UDP-Glucuronosyl transferase 1A gene family with lung cancer risk. Background: Over 5,000 compounds have been identified in cigarette smoke, of which 73 are considered carcinogenic to either laboratory animals or humans by the International Agency for Research on Cancer. The UDPglucuronsyl transferases (UGTs) are Phase II drug-metabolizing enzymes that catalyze the metabolism of several cigarette smoke carcinogens for excretion. There are hundreds of genetic variants that span the nine genes in the UGT1A gene cluster, but the effect of UGT1A polymorphisms on lung cancer risk has not been studied in American Caucasians. This study hypothesized that UGT1A variants, either individually or as a haplotype, would be associated with lung cancer risk in American Caucasians. Methods and Findings: To examine the effect of genetic variation in the nine UGT1A genes on lung cancer risk, a comprehensive association and haplotype study was conducted using American Caucasian lung cancer cases and matched healthy controls on ninety-six tagSNPs. Known lung cancer risk factors including age, sex, smoking status, and pack-years of smoking were controlled for in all analyses. Multiple SNPs in the UGT1A gene cluster were associated with lung cancer risk in various stratified analyses before a multiple testing correction was applied. A significant association was found between two haplotypes in the UGT1A haplotype block 2 (rs7569014, rs7421795, rs1817154) and small cell carcinoma risk in American Caucasians. Multiple SNPs were found to affect lung cancer risk that did not remain significant following multiple testing correction. The UGT1A haplotype block 2 may be associated with small cell lung carcinoma. However, single SNP association studies did not yield significant results after multiple testing correction. Conclusions: UGT1A variants may play only a minor role in other lung cancer risk in American Caucasians; however, this needs to be confirmed in larger studies.

Heterocyclic aromatic amines and their contribution to the bacterial mutagenicity of the particulate phase of cigarette smoke.

Heterocyclic aromatic amines (HAAs) rank among the strongest known mutagens. Approximately 30 HAAs have been found in cooked foods (broiled, fried, and grilled) and several HAAs have been characterized as animal carcinogens. Nine HAAs have also been reported to be constituents of cigarette smoke (CS) raising concerns that HAAs might contribute significantly to the known carcinogenicity of CS. As HAAs are found predominantly in the total particulate matter (TPM) of CS, an improved method for the quantification of HAAs in TPM is reported allowing detection and quantification of 8 HAAs in a single run. The mutagenic potency of these HAAs and that of TPM from the reference cigarette 2R4F was determined in the Salmonella Reverse Mutation Assay (Ames assay) with tester strain TA98 and a metabolic activation system. The 8 HAAs, when applied together in the Ames assay, showed a clear sub-additive response. Likewise, the combination of HAAs and TPM, if at all, gave rise to a slight sub-additive response. In both cases, however, the sub-additive response in the Ames assay was observed at HAA doses that are far above the amounts found in CS. The contribution of the individual HAAs to the total mutagenic activity of TPM was calculated and experimentally confirmed to be approximately 1% of the total mutagenic activity. Thus, HAAs do not contribute significantly to the bacterial in vitro mutagenicity of CS TPM.

Quercetin inhibits multiple pathways involved in interleukin 6 secretion from human lung fibroblasts and activity in bronchial epithelial cell transformation induced by benzo[a]pyrene diol epoxide.

The interaction between epithelial and stromal cells through soluble factors such as cytokines plays an important role in carcinogenesis. Breaking this cancer-promoting interaction poses an opportunity for cancer prevention. The tumor-promoting function of interleukin 6 (IL-6) has been documented; however, the underlying mechanisms of this function in lung carcinogenesis are not well elucidated. Here, we show that benzo[a]pyrene diol epoxide (BPDE, the active metabolite of cigarette smoke carcinogen benzo[a]pyrene)-induced human bronchial epithelial cell (HBEC) transformation was enhanced by IL-6 in vitro. The carcinogen/IL-6-transformed cells exhibited higher expression of STAT3 (signal transducer and activator of transcription 3) when compared with cells transformed by BPDE alone. Constitutive STAT3 activation drove cell proliferation and survival through anti-apoptosis gene expression. We further show that quercetin, a dietary compound having preventive properties for lung cancer, decreased BPDE-stimulated IL-6 secretion from human lung fibroblasts through inhibition of the NF-κB and ERK pathways. The inhibition was accomplished at clinically achievable concentrations of the compound. Finally, quercetin blocked IL-6-induced STAT3 activation in HBECs, and IL-6 enhancement of HBEC transformation by BPDE was abolished by quercetin treatment. Altogether, our data reveal novel mechanisms for IL-6 in lung carcinogenesis and for the preventive role of quercetin in the process. © 2015 Wiley Periodicals, Inc.

Base damage, local sequence context and TP53 mutation hotspots: a molecular dynamics study of benzo[a]pyrene induced DNA distortion and mutability.

The mutational pattern for the TP53 tumour suppressor gene in lung tumours differs to other cancer types by having a higher frequency of G:C>T:A transversions. The aetiology of this differing mutation pattern is still unknown. Benzo[a]pyrene,diol epoxide (BPDE) is a potent cigarette smoke carcinogen that forms guanine adducts at TP53 CpG mutation hotspot sites including codons 157, 158, 245, 248 and 273. We performed molecular modelling of BPDE-adducted TP53 duplex sequences to determine the degree of local distortion caused by adducts which could influence the ability of nucleotide excision repair. We show that BPDE adducted codon 157 has greater structural distortion than other TP53 G:C>T:A hotspot sites and that sequence context more distal to adjacent bases must influence local distortion. Using TP53 trinucleotide mutation signatures for lung cancer in smokers and non-smokers we further show that codons 157 and 273 have the highest mutation probability in smokers. Combining this information with adduct structural data we predict that G:C>T:A mutations at codon 157 in lung tumours of smokers are predominantly caused by BPDE. Our results provide insight into how different DNA sequence contexts show variability in DNA distortion at mutagen adduct sites that could compromise DNA repair at well characterized cancer related mutation hotspots.

Bcl2 inhibition of mitochondrial DNA repair.

BackgroundAccumulation of mitochondrial DNA (mtDNA) damage could enhance the frequency of mitochondrial mutations and promote a variety of mitochondria-related diseases, including cancer. However, the mechanism(s) involved are not fully understood.MethodsQuantitative extended length PCR was used to compare mtDNA and nDNA damage in human lung H1299 cells expressing WT Bcl2 or vector-only control. mtAPE1 endonuclease activity was analyzed by AP oligonucleotide assay. mtDNA mutation was measured by single molecule PCR. Subcellular localization of Bcl2 and APE1 was analyzed by subcellular fractionation.ResultsBcl2, an anti-apoptotic molecule and oncoprotein, effectively inhibits the endonuclease activity of mitochondrial APE1 (mtAPE1), leading to significant retardation of mtDNA repair and enhanced frequency of mtDNA mutations following exposure of cells to hydrogen peroxide (H2O2) or nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, a carcinogen in cigarette smoke). Inversely, depletion of endogenous Bcl2 by RNA interference increases mtAPE1 endonuclease activity leading to accelerated mtDNA repair and decreased mtDNA mutation. Higher levels of mtAPE1 were observed in human lung cancer cells than in normal human bronchial epithelial cells (i.e. BEAS-2B). Bcl2 partially co-localizes with APE1 in the mitochondria of human lung cancer cells. Bcl2 directly interacts with mtAPE1 via its BH domains. Removal of any of the BH domains from Bcl2 abolishes Bcl2’s capacity to interact with mtAPE1 as well as its inhibitory effects on mtAPE1 activity and mtDNA repair.ConclusionsBased our findings, we propose that Bcl2 suppression of mtDNA repair occurs through direct interaction with mtAPE1 and inhibition of its endonuclease activity in mitochondria, which may contribute to enhanced mtDNA mutations and carcinogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1594-1) contains supplementary material, which is available to authorized users.

SIRT1 in B[a]P-induced lung tumorigenesis

Benzo[a]pyrene (B[a]P) is a carcinogen in cigarette smoke. We found that B[a]P induced SIRT1 in human bronchial epithelial BEAS-2B cell. SIRT1 was overexpressed in the lung of B[a]P-exposed mice and in human lung cancer biopsies. SIRT1 up-regulated TNF-α and β-catenin and down-regulated the membrane fraction of E-cadherin. In addition, SIRT1 promoted invasion, migration and tumorigenesis of BEAS-2B cells in nude mice upon B[a]P exposure. Thus, SIRT1 is involved in B[a]P-induced transformation associated with activation of the TNF-α/β-catenin axis and is as a potential therapeutic target for lung cancer.

Binding Pattern Elucidation of NNK and NNAL Cigarette Smoke Carcinogens with NER Pathway Enzymes: an Onco- Informatics Study.

Cigarette smoke derivatives like NNK (4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone) and NNAL (4-(methylnitrosamino)-1-(3-pyridyl)-1-butan-1-ol) are well-known carcinogens. We analyzed the interaction of enzymes involved in the NER (nucleotide excision repair) pathway with ligands (NNK and NNAL). Binding was characterized for the enzymes sharing equivalent or better interaction as compared to +Ve control. The highest obtained docking energy between NNK and enzymes RAD23A, CCNH, CDK7, and CETN2 were -7.13 kcal/mol, -7.27 kcal/mol, -8.05 kcal/mol and -7.58 kcal/mol respectively. Similarly the highest obtained docking energy between NNAL and enzymes RAD23A, CCNH, CDK7, and CETN2 were -7.46 kcal/mol, -7.94 kcal/mol, -7.83 kcal/mol and -7.67 kcal/mol respectively. In order to find out the effect of NNK and NNAL on enzymes involved in the NER pathway applying protein-protein interaction and protein-complex (i.e. enzymes docked with NNK/NNAL) interaction analysis. It was found that carcinogens are well capable to reduce the normal functioning of genes like RAD23A (HR23A), CCNH, CDK7 and CETN2. In silico analysis indicated loss of functions of these genes and their corresponding enzymes, which possibly might be a cause for alteration of DNA repair pathways leading to damage buildup and finally contributing to cancer formation.

Abstract 4563: Muscadine grape extract reduces lung carcinogenesis in female mice exposed to 3-methylcholanthrene in utero

Abstract The developing fetus is highly sensitive to the carcinogenic effects of dietary and environmental carcinogens in cigarette smoke, charbroiled foods and air pollution following transplacental exposure. Oxidative metabolism of toxicants by both maternal and fetal tissue causes DNA damage which can ultimately result in the initiation of childhood cancers. Due to the latency of cancer formation, in utero exposure to carcinogens may even predispose the individual to cancers developing later in life. The goal of this study is to determine whether a proprietary muscadine grape extract (MGE) effectively reduces lung tumor formation in the fetus following transplacental exposure to a polycyclic hydrocarbon. The muscadine grape (Vitis rotundifolia) has a distinct phytochemical composition compared to other grape varieties and limited reports indicate that the extract has potential anti-tumor activity. BALB/c male mice were mated to C57BL/6 female mice to serve as a model for in utero exposure to environmental toxicants. The pregnant mice were treated on the 17th day of gestation by intraperitoneal injection with a 45 mg/kg dose of 3-methylcholanthrene dissolved in olive oil, to induce lung tumors in the fetus. An injection of the olive oil vehicle (0.5 mL/0.35 kg) served as the control. Day 0 was considered as the first day when the vaginal plug was detected. After weaning, cohorts of mice received drinking water alone or water with MGE (approximately 1.0 mg phenolics/25 g mouse) and were sacrificed at 12 months of age. Although all of the mice developed lung tumors, MGE significantly reduced tumor burden in female mice by approximately 48% as compared to the tumor tissue found in control mice drinking regular water (12.4 ± 1.9 g versus 6.5 ± 1.2 g, n = 26-32, p = 0.014). In addition, tumor multiplicity was higher in control mice as compared to MGE-treated animals (6.8 ± 0.4 observed tumors versus 4.2 ± 0.5 observed tumors, n = 26-32, p = 0.0001). Tumor tissue sections from mice administered MGE had a significant decrease in proliferating cell nuclear antigen as compared to tumors from control animals (22.4 ± 1.8 versus 14.2 ± 1.3, p = 0.0009), suggesting that in part MGE reduces tumor burden by decreasing tumor cell proliferation. Lung tumor sections from female mice drinking regular water or MGE were incubated with an antibody to the endothelial cell marker, CD34, and vessels were identified by a combination of morphology and positive CD34 immunoreactivity. MGE significantly reduced blood vessel density in lung tumor tissue as compared to female mice drinking regular water (20.5 ± 2.2 versus 11.3 ± 1.3, p = 0.0012), suggesting that the grape extract inhibits angiogenesis. These results indicate that MGE reduces both tumor proliferation and angiogenesis, leading to a decrease in tumor burden and multiplicity. Thus, MGE may represent a novel nutraceutical for the prevention of lung tumors induced by in utero exposure to environmental toxicants. Citation Format: Brooke E. Porter, Joseph E. Moore, Mark S. Miller, E. Ann Tallant, Patricia E. Gallagher. Muscadine grape extract reduces lung carcinogenesis in female mice exposed to 3-methylcholanthrene in utero. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4563. doi:10.1158/1538-7445.AM2015-4563

Abstract LB-091: Characterization of molecular changes occurring during long-term treatment of human bronchial epithelial cells with cigarette smoke total particulate matter

Abstract Objectives: Cigarette smoking is the leading cause of lung cancer worldwide. Carcinogens in cigarette smoke are responsible for airway epithelial changes, although our knowledge about the molecular events underlying lung tumorigenesis are still not very detailed. Our aim was first of all to establish an in vitro model which mimics chronic exposure conditions found in the airways of smokers and to more comprehensively characterize the chronological changes that occur in bronchial epithelial cells under those conditions. Materials and methods: Total particulate matter (TPM) was generated from the 3R4F reference cigarette according to the ISO smoking regimen. Human bronchial epithelial cells (BEAS-2B; ATCC) were repeatedly exposed to TPM for 4 weeks. Gene regulation and expression were investigated using Affymetrix GeneChip® twice weekly during exposure, and data were analyzed using R scripts and Ingenuity Pathway Analysis® (IPA®). Phenotypic alterations due to exposure including, for example, cell cycle changes and epithelial-to-mesenchymal transition (EMT) were studied on a weekly basis and at the end of the exposure period, respectively, using high content imaging. The imaging data were subjected to statistical analysis using linear models (SAS 9.2). Results: Treatment of BEAS-2B cells with TPM resulted in increased DNA damage after 1 week, continuously rising until the end of the 4-week exposure, although this trend was not statistically significant. We also observed a shift of the cell cycle to a G2/M-phase arrest, with a growing number of cells accumulating in this cell cycle phase over the treatment period. In addition, gradual activation of canonical pathways (e.g. PI3K/AKT, p53 and IL6 signaling) and enrichment in biological functions related to tumorigenesis over time was evidenced by overexpression of tumor promoting genes such as TP63, COX-2, GDF15, CCND1 and tumor suppressive genes like SERPINB5, THBS1, FAS and CDKN1A from as early as days 3-7. Furthermore, overexpression of miR-200 and miR-205 was identified first following 17 days of treatment, remaining significantly higher than controls until day 28. However, no visible morphological changes could be detected at the time when gene and miRNA expression changes first became significant. In contrast, those events typically occurred later. Conclusion: Our experiments indicate that repeated exposure of bronchial epithelial cells to TPM induces ongoing alterations in gene expression as well as phenotypic changes related to tumorigenesis. The data here may lead to a better mechanistic understanding of the stepwise transformation of normal airway epithelial cells to full malignancy. Citation Format: Marco van der Toorn, Niklas Kuehn, Stefan Frentzel, Diego Marescotti, Emmanuel Guedj, Nikolai Ivanov, Patrice Leroy, Manuel C. Peitsch, Julia Hoeng, Karsta Luettich. Characterization of molecular changes occurring during long-term treatment of human bronchial epithelial cells with cigarette smoke total particulate matter. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-091. doi:10.1158/1538-7445.AM2015-LB-091

Genetic polymorphisms in UDP-glucuronosyltransferase 1A6 and 1A7 and the risk for benign Warthin's tumors of the parotid gland.

Warthin's tumors of the parotid gland are associated with smoking, whereas pleomorphic adenomas are not. Genetic polymorphisms in biotransformation enzymes, involved in detoxification of toxins and carcinogens in cigarette smoke, might modify the corresponding enzyme activity and influence detoxifying capacity. We hypothesize that these genetic polymorphisms may influence the individual risk for Warthin's tumor, but not for pleomorphic adenomas. Blood from 146 patients with benign parotid gland tumors and 437 controls were investigated for polymorphisms in several biotransformation enzymes. Based on these polymorphisms, patients and controls were divided according to predicted enzyme activity (low, intermediate, and high). Prevalence of predicted intermediate and high activity UGT1A7 and UGT1A6 genotypes was significantly higher in the patients with Warthin's tumors, but not in patients with pleomorphic adenomas, compared with healthy controls. Predicted intermediate and high activity UGT1A7 and UGT1A6 genotypes are associated with an increased risk for Warthin's tumor. © 2015 Wiley Periodicals, Inc. Head Neck 38: E717-E723, 2016.

Repression of CC16 by cigarette smoke (CS) exposure.

Club (Clara) Cell Secretory Protein (CCSP, or CC16) is produced mainly by non-ciliated airway epithelial cells including bronchiolar club cells and the change of its expression has been shown to associate with the progress and severity of Chronic Obstructive Pulmonary Disease (COPD). In an animal model, the lack of CC16 renders the animal susceptible to the tumorigenic effect of a major CS carcinogen. A recent population-based Tucson Epidemiological Study of Airway Obstructive Diseases (TESAOD) has indicated that the low serum CC16 concentration is closely linked with the smoke-related mortality, particularly that driven by the lung cancer. However, the study of CC16 expression in well-defined smoke exposure models has been lacking, and there is no experimental support for the potential causal link between CC16 and CS-induced pathophysiological changes in the lung. In the present study, we have found that airway CC16 expression was significantly repressed in COPD patients, in monkey CS exposure model, and in CS-induced mouse model of COPD. Additionally, the lack of CC16 exacerbated airway inflammation and alveolar loss in the mouse model. Therefore, CC16 may play an important protective role in CS-related diseases.

A Call for a Greater Emphasis upon Smoking Cessation in Urology

During a Capitol Hill summit in 2013 on cancer prevention, a scientific panel discussed the link between lung, esophageal, bladder and other human cancers with smoking. A United States Senator on the panel was puzzled and asked “Why does tobacco cause bladder cancer if cigarette smoke doesn’t come in contact with the bladder? The Senator’s unfamiliarity of the role of the renal system in the metabolism of carcinogens in cigarette smoke, the subsequent exposure to the uroepithelial lining, and concentration within the bladder reflect an opportunity for the field of urology to educate not only our Elected Officials but also the general public about the link between smoking with renal, ureteral, and bladder cancer.

Abstract 1525: Exome sequencing to identify permissive mutations representing acquired vulnerabilities in lung cancer

Abstract When a lung epithelial cell acquires a driver mutation (e.g. in KRAS) it often needs to acquire additional ‘permissive’ genetic alterations to enable the cell to tolerate the oncogenic change. These ‘permissive' alterations represent an ‘acquired vulnerability' and consequently a novel therapeutic target in lung cancers driven by known, but currently un-targetable, driver mutations. Immortalized Human Bronchial Epithelial Cells (HBECs) represent a cell appropriate in vitro model to study the origin and pathogenesis of lung cancer following introduction of defined oncogenic changes. We have previously transformed HBECs to full malignancy by introducing five genetic manipulations (to CDK4, TERT, TP53, KRAS, MYC, BCL2, PTEN and/or STK11). While malignant transformation will be largely driven by the exogenous oncogenotype -i.e. the introduced oncogenic alterations- we hypothesize the cells also must undergo additional somatic ‘permissive' changes to bypass anti-tumor mechanisms. While lung cancers are often highly mutated (mainly due to carcinogens in cigarette smoke), manipulated HBECs should exhibit fewer somatic mutations, providing greater power to identify important somatically acquired mutations that facilitate tumorigenic progression of lung epithelial cells. We have sequenced the exomes of a series of 22 genetically manipulated HBECs derived from two individuals and representing 15 unique oncogenotypes (combinations of 3-5 introduced manipulations). Paired-end sequencing was performed with an Illumina HiSeq 2000 using a VCRome 2.1 liquid capture design ‘exome chip’. The median fold coverage was 114X, resulting in at least 90% of targeted bases are covered at &amp;gt;20-fold. Somatic mutations were called by comparing manipulated HBECs to their immortalized parental cell line. Manipulated HBECs had a median somatic mutation rate of 0.92 mutations/Mb, 7-9-fold lower than observed in lung cancers and tumor cell lines. The median mutation rate increased with the number of introduced manipulations (HBECs with three, four and five manipulations had 0.47, 0.97 and 0.92 mutations/Mb, respectively). Non-synonymous somatic mutations were identified in 470 genes. Thirty-nine genes were mutated in ≥3 HBECs (recurrent mutations) and of these, 35 genes (none currently regarded as ‘driver’ oncogenes and 5 potentially ‘druggable' [DGIdb]) were also mutated in primary lung adenocarcinoma and squamous cell carcinomas (TCGA data). We found the introduction of defined oncogenic alterations in HBECs is accompanied by the acquisition of somatic mutations. Leveraging against public somatic mutation data for lung tumors has allowed us to prioritize mutations for follow up to better identify potential acquired vulnerabilities in lung cancer cells and therefore, novel therapeutic targets. Citation Format: Jill E. Larsen, Caleb F. Davis, Kenneth Huffman, Luc Girard, David A. Wheeler, Richard A. Gibbs, John D. Minna. Exome sequencing to identify permissive mutations representing acquired vulnerabilities in 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 1525. doi:10.1158/1538-7445.AM2014-1525

A knockdown with smoke model reveals FHIT as a repressor of Heme oxygenase 1

Fragile histidine triad (FHIT) gene deletions are among the earliest and most frequent events in carcinogenesis, particularly in carcinogen-exposed tissues. Though FHIT has been established as an authentic tumor suppressor, the mechanism underlying tumor suppression remains opaque. Most experiments designed to clarify FHIT function have analyzed the consequence of re-expressing FHIT in FHIT-negative cells. However, carcinogenesis occurs in cells that transition from FHIT-positive to FHIT-negative. To better understand cancer development, we induced FHIT loss in human bronchial epithelial cells with RNA interference. Because FHIT is a demonstrated target of carcinogens in cigarette smoke, we combined FHIT silencing with cigarette smoke extract (CSE) exposure and measured gene expression consequences by RNA microarray. The data indicate that FHIT loss enhances the expression of a set of oxidative stress response genes after exposure to CSE, including the cytoprotective enzyme heme oxygenase 1 (HMOX1) at the RNA and protein levels. Data are consistent with a mechanism in which Fhit protein is required for accumulation of the transcriptional repressor of HMOX1, Bach1 protein. We posit that by allowing superinduction of oxidative stress response genes, loss of FHIT creates a survival advantage that promotes carcinogenesis.

Increased NQO1 but not c-MET and survivin expression in non-small cell lung carcinoma with KRAS mutations.

Cigarette smoking is one of the most significant public health issues and the most common environmental cause of preventable cancer deaths worldwide. EGFR (Epidermal Growth Factor Receptor)-targeted therapy has been used in the treatment of LC (lung cancer), mainly caused by the carcinogens in cigarette smoke, with variable success. Presence of mutations in the KRAS (Kirsten rat sarcoma viral oncogene homolog) driver oncogene may confer worse prognosis and resistance to treatment for reasons not fully understood. NQO1 (NAD(P)H:quinone oxidoreductase), also known as DT-diaphorase, is a major regulator of oxidative stress and activator of mitomycins, compounds that have been targeted in over 600 pre-clinical trials for treatment of LC. We sequenced KRAS and investigated expression of NQO1 and five clinically relevant proteins (DNMT1, DNMT3a, ERK1/2, c-MET, and survivin) in 108 patients with non-small cell lung carcinoma (NSCLC). NQO1, ERK1/2, DNMT1, and DNMT3a but not c-MET and survivin expression was significantly more frequent in patients with KRAS mutations than those without, suggesting the following: (1) oxidative stress may play an important role in the pathogenesis, worse prognosis, and resistance to treatment reported in NSCLC patients with KRAS mutations, (2) selecting patients based on their KRAS mutational status for future clinical trials may increase success rate, and (3) since oxidation of nucleotides also specifically induces transversion mutations, the high rate of KRAS transversions in lung cancer patients may partly be due to the increased oxidative stress in addition to the known carcinogens in cigarette smoke.

Clinical and metabolic parameters in non-small cell lung carcinoma and colorectal cancer patients with and without KRAS mutations.

Lung cancer (LC) and colorectal cancer (CRC) are the first and second deadliest types of cancer worldwide. EGFR-based therapy has been used in the treatment of these cancers with variable success. Presence of mutations in the KRAS driver oncogene, possibly induced by environmental factors such as carcinogens in diet and cigarette smoke, may confer worse prognosis and resistance to treatment for reasons not fully understood. Data on possible associations between KRAS mutational status and clinical and metabolic parameters, which may help in clinical management, as well as in identifying risk factors for developing these cancers, are limited in the current literature. We sequenced the KRAS gene and investigated the associations of variations in 108 patients with non-small cell lung carcinoma (NSCLC), the most common form of LC, and in 116 patients with CRC. All of the mutations originated from the guanosine nucleotide and over half of all transversions in NSCLC and CRC were c.34 G>T and c.35 G>T, respectively. c.35 G>A was the most frequent type of transition in both cancers. Excluding smoking, the clinical and metabolic parameters in patients carrying mutant and wild type KRAS were similar except that the CRC patients with transversion mutations were 8.6 years younger than those carrying the transitions (P < 0.01). Dyslipidemia, hypertension, family cancer history, and age of diagnosis older than 60 years were more frequent in NSCLC than CRC (P ≤ 0.04). These results suggest that most of the clinical and metabolic parameters investigated in this study are probably not associated with the more aggressive phenotype and differences in response to EGFR-based treatment previously reported in patients with KRAS mutations. However, the increased rates of abnormal metabolic parameters in patients with NSCLC in comparison to CRC indicate that these parameters may be more important in the management of NSCLC. CRC patients carrying transition mutations are older than those carrying transversions, suggesting that age may determine the type of KRAS mutation in CRC patients.

Combined effects of asbestos and cigarette smoke on the development of lung adenocarcinoma: different carcinogens may cause different genomic changes.

The carcinogens in cigarette smoke are distinct from asbestos. However, an understanding of their differential effects on lung adenocarcinoma development remains elusive. We investigated loss of heterozygosity (LOH) and the p53 mutation in 132 lung adenocarcinomas, for which asbestos body burden (AB; in numbers per gram of dry lung) was measured using adjacent normal lung. All cases were classified into 9 groups based on a matrix of cumulative smoking (CS in pack-years; CS=0, 0<CS<25, ≥25 CS) and AB (AB=0, 0<AB<1,000, ≥1,000 AB). AB=0 indicates a lower level than the detection limit of ~100. LOH frequency increased only slightly with the elevation of CS in the AB=0 groups. In the AB>0 groups, LOH frequency increased as AB and/or CS was elevated and was significantly higher in the ≥1,000 AB, ≥25 CS group (p=0.032). p53 mutation frequency was the lowest in the AB=0, CS=0 group, increased as AB and/or CS rose, and was significantly higher in the ≥1,000 AB, ≥25 CS group (p=0.039). p53 mutations characteristic of smoking were frequently observed in the CS>0 groups contrary to non-specific mutations in the CS=0, AB>0 groups. Combined effects of asbestos and smoking were suggested by LOH and p53 analyses. Sole exposure to asbestos did not increase LOH frequency but increased non-specific p53 mutations. These findings indicate that the major carcinogenic mechanism of asbestos may be tumor promotion, acting in an additive or synergistic manner, contributing to the genotoxic effect of smoking. Since this study was based on a general cancer center’s experience, the limited sample size did not permit the consideration that the result was conclusive. Further investigation with a large sample size is needed to establish the mechanism of asbestos-induced lung carcinogenesis.

Benzo(a)pyrene promotes A549 cell migration and invasion through up-regulating Twist.

Benzo(a)pyrene (BaP) is one of the strongest carcinogens in cigarette smoke, which is an established human carcinogen. Twist, a transcription factor of the basic helix-loop-helix class, is reported to regulate lung cancer metastasis. Evidence has shown that BaP could induce Twist mRNA expression in non-small cell lung cancer (NSCLC) cell line A549 and promote lung adenocarcinoma cell invasion. However, it is unclear whether BaP promotes the migration and invasion of NSCLC cells by Twist modulation. A549 cell was exposed to BaP for different time. MTT assay was applied to assess cell proliferation. Silencing of Twist was done by small interfering RNA. Wound-healing assay was used to evaluate the capability of cell migration. Transwell assay was used to detect the capability of cell invasion. Western blotting and quantitative polymerase chain reaction were used to detect Twist expression. The levels of Twist protein expression and mRNA expression were increased with the treatment of BaP, compared with solvent control. The capability of wound healing of A549 cells was increased in BaP-treated group, compared with solvent control. BaP enhanced the capability of invasion of A549 cells. Twist knockdown could block the migration and invasion of A549 cells induced by BaP treatment. The mRNA levels of Twist were higher in metastatic NSCLC tissue samples than in primary NSCLC tissue samples, and higher levels of Twist mRNA were observed in metastatic NSCLC tissue samples with smoking history than in those with nonsmoking history. BaP treatment could promote the migration and invasion of NSCLC cells by up-regulating Twist expression.