Tobacco Carcinogens Research Articles

Abstract 3875: A novel STAT3 decoy inhibitor prevents lung cancer development

Abstract Exposure to tobacco carcinogens such as NNK leads to mutations in genes like K-RAS in premalignant lesions, which progress to adenocarcinoma. Early events in carcinogenesis are also characterized by inflammation. Central to these processes, STAT3 activation has pleotropic pro-tumor effects on proliferation, angiogenesis, and immune evasion. We previously showed that a “decoy” cyclic STAT3 oligonucleotide (CS3D) produced robust antitumor effects in human lung adenocarcinoma models. CS3D mimics the STAT3 DNA response element and acts as a “decoy” to which STAT3 binds, blocking its transcriptional activity. Effects of CS3D were compared to an inactive mutant construct unable to bind to STAT3 (CS3M). Therapeutic effects in human lung cancer xenografts suggested that CS3D might also impair lung cancer development. To test this, mice were exposed to NNK (3mg/kg) for 4 weeks (wks) followed by a 1-wk washout phase to model ex-smokers. Mice were then treated 3 times per wk with CS3D or CS3M (5mg/kg, IV) for 8 wks. Tumor burden and size were then assessed at 8 and 20 wks after the end of treatment. To evaluate effect of CS3D on the development of tumors with K-RAS activating mutations (since STAT3 knockout has been reported to promote KRAS mutant lung cancer), we isolated DNA from tumors by laser capture microdissection and assessed the incidence of G12D activating mutations. CS3D caused a reduction in preneoplasias (P<0.001), tumor size (P<.05), and tumor number (P<.05) by 30%, 50%, and 40% respectively, compared to CS3M. Immunohistochemistry (IHC) showed a tumor microenvironment (TME) that is less primed for tumor progression. Markers of angiogenesis (VEGF) and cell cycle progression (MYC) were downregulated by CS3D (P<.05). Strikingly, IHC analysis at 20 wks after the final treatment revealed a substantial decrease in STAT3 and NF-kB activation (P<.05) suggesting a long-lasting effect of CS3D. However, we detected higher levels of IL-6 at 8 wks and 20 wks after the end of treatment which could indicate a rescuing feedback loop. IHC also showed a 2.6 fold increase in macrophage infiltration at 8 wks. Sequencing of DNA from residual tumors collected at 20 wks (n = 10) showed no significant difference in tumors with K-RAS mutation between CS3D (33.3%) and CS3M (27.3%), showing that pharmacological inhibition of STAT3 with CS3D doesn’t select for K-RAS mutant tumors. To examine immunomodulation, mice were exposed to 4 wks of NNK with a 1-wk washout phase and 1 wk of CS3D or CS3M treatment. Lungs were collected for flow cytometry analysis to define the immune cell profile. CS3D favored an antitumor immune response over an immunosuppressive TME by increasing the number of lung M1 macrophages while decreasing M2 macrophages and MDSCs. Ratio of M1 to M2 was 4:1 with CS3D and 1:3 with CS3M. This is the first evidence that CS3D has chemopreventive properties in lung cancer without promoting KRAS-driven tumors, and modulating the TME is involved in the preventive effect. Citation Format: Christian Njatcha, Jiayi Wang, Huiyu Li, Mariya Farooqui, Jill M. Siegfried. A novel STAT3 decoy inhibitor prevents lung cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3875.

Abstract 2710: Depletion of CXCR2-dependent myeloid-derived suppressor cells (MDSCs) overcomes anti-PD-1 resistance in a murine model of LKB1-deficient non-small cell lung cancer (NSCLC) with high mutational load

Abstract Checkpoint inhibitors such as PD-L1/PD-1 blockade have rapidly integrated into the paradigm of NSCLC treatment. However, a majority of patients do not benefit from monotherapy with checkpoint inhibitors. High tumor mutational burden (TMB), along with pre-existing intratumoral T cell infiltration and baseline high PD-L1 expression, predicts response to checkpoint blockade. Furthermore, a recent retrospective study identified LKB1 alterations as the most prevalent genomic driver of resistance to PD-1 axis inhibitors in KRAS-mutant lung adenocarcinoma. In this study, we investigate the mechanisms underlying LKB1-mediated immunosuppression in NSCLC. We show that loss of LKB1 in human bronchial epithelial cells (HBECs) and NSCLC cells leads to increased secretion of CXCR2 ligands, including CXCL1, CXCL2, CXCL3, CXCL5 and CXCL8. These CXCR2 ligands are also elevated in LKB1-deficient tumors from patient-derived xenografts and genetically-engineered murine models. We find abundant tumor infiltrating MDSCs in murine Lkb1-deficient NSCLC, consistent with the capacity for CXCR2 ligands to recruit MDSCs. MDSCs mediate potent immune suppressive activities at multiple levels including release of immunosuppressive cytokines, recruitment of regulatory T cells (Tregs), inhibition of CD8 T cell tumor infiltration and upregulation of PD-L1 expression. Although MDSC depletion activates interferon gamma signaling and decreases systemic Tregs in murine KrasK12D;Tp53-/-;Lkb1-/- (KPL) tumors, it does not sensitize KPL tumors to anti-PD-1 therapy. One of the major challenges in the preclinical assessment of lung cancer immunotherapy is that the commonly utilized murine models lack the mutational burden of human NSCLC. To assess this combination therapy in the context of a mutational burden that more accurately reflects the clinical disease, we generated tumors with high TMB by exposing KPL cells in vitroto the tobacco carcinogen N-methyl-N-nitrosourea. In the context of high TMB, MDSC depletion demonstrates remarkable anti-tumor effects in combination with anti-PD-1 therapy. Finally, we delineate the regulation of CXCR2 ligands by LKB1 which is dependent on the MARK-mediated NF-κB pathway. In conclusion, we find that LKB1 deficiency leads to increased CXCR2 ligand production and tumor infiltrating MDSCs. MDSC depletion enhances the efficacy of anti-PD-1 blockade in LKB1-deficient tumors bearing high TMB. Citation Format: Rui Li, Ramin Salehi-Rad, Stephanie Ong, Milica Momcilovic, Bin Liu, Raymond Lim, Linh Tran, Ziling Huang, Zhe Jing, Manash Paul, Kostyantyn Krysan, Stacy Park, John Minna, Michael Teitell, David Shackelford, Steven Dubinett. Depletion of CXCR2-dependent myeloid-derived suppressor cells (MDSCs) overcomes anti-PD-1 resistance in a murine model of LKB1-deficient non-small cell lung cancer (NSCLC) with high mutational load [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2710.

Abstract 4952: Host lipocalin 2 protects against Kras mutant lung cancer development by maintaining an anti-tumor immune contexture

Abstract Relative to other lung adenocarcinomas (LUADs), Kras mutant LUAD displays dismal prognosis warranting the need for new strategies for early treatment of this lung cancer subtype. Limiting these advances is our poor understanding of events that drive Kras mutant LUAD development. In efforts to fill this void, we recently found that mice with knockout of G-protein coupled receptor 5A (Gprc5a-/-) and after tobacco carcinogen exposure (nicotine-specific nitrosamine ketone/NNK) developed LUADs harboring driver somatic Kras mutations. To understand early events preceding the onset of these Kras mutant LUADs, we performed RNA-sequencing (RNA-Seq) of normal airways in Gprc5a-/- mice and wild type (WT) littermates prior to NNK exposure. We found markedly elevated expression of the antimicrobial lipocalin 2 (Lcn2) gene in normal airways of Gprc5a-/- mice. Also, analysis of publicly available human datasets revealed elevated expression of LCN2 in human LUADs relative to normal lung tissues particularly in KRAS mutant tumors. We then sought to probe the role of Lcn2 in the pathogenesis of Kras mutant LUAD. Mice with knockout of host Lcn2 (Gprc5a-/-/Lcn2-/-) showed increased lung tumor development compared to Gprc5a-/- littermates. RNA-Seq of whole lungs at baseline and at seven months post-NNK (n = 9-10 mice each group) pinpointed differential expression profiles that denoted decreased anti-tumor immunity in Gprc5a-/-/Lcn2-/- mice. Computational estimation of immune infiltrates demonstrated increased levels of neutrophils post-NNK in Gprc5a-/-/Lcn2-/- mice but not in Gprc5a-/-littermates. By flow cytometry analysis of lung tissues, we found elevated numbers of various lymphoid and myeloid immune cells in both groups following NNK exposure and increased neutrophil counts in Gprc5a-/-/Lcn2-/- mice relative to Gprc5a-/- littermates. Histopathological assessment also revealed increased numbers of pro-inflammatory lesions in mice lacking Lcn2. Gprc5a-/-/Lcn2-/- mice at three months post-NNK were also found to exhibit increased production of IL-17A by lung-resident CD4+ and CD8+ T cells relative to Gprc5a-/- littermates concomitant with decreased expression of the anti-tumor Th1/cytolytic immune markers Ifnγ and Gzmb. Preliminary analysis also showed elevated expression of the major immune checkpoint PD-L1 on alveolar macrophages in Gprc5a-/-/Lcn2-/- mice relative to Gprc5a-/- mice with WT Lcn2. Lastly, we preliminarily found, by bacterial 16S rRNA sequencing of temporally collected fecal samples, globally altered microbiome profiles in mice lacking Lcn2. Our findings underscore novel cues in lung oncogenesis and suggest that Lcn2 decreases anti-tumor immunity, perturbs the host microbiome and promotes development of Kras mutant LUAD thereby offering new venues for early treatment of this fatal malignancy. Citation Format: Maya Hassane, Warapen Treekitkarnmongkol, Tina L. McDowell, Smruthy Sivakumar, Wenhua Lang, Joshua K. Ochieng, Sayuri Nunomura-Nakamura, Casey Finnicum, Christel Davis, Gareth E. Davies, Junya Fukuoka, Tina Cascone, Florencia McAllister, Ignacio I. Wistuba, Erik Ehli, Seyed J. Moghaddam, Paul Scheet, Junya Fujimoto, Humam Kadara. Host lipocalin 2 protects against Kras mutant lung cancer development by maintaining an anti-tumor immune contexture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4952.

Modulation of Reverse Cholesterol Transport by Lycopene Is Associated with Its Protective Role Against Cigarette Smoke Induced COPD and Lung Carcinogenesis in Ferrets (OR05-02-19)

ObjectivesReverse cholesterol transport (RCT), which mediates removal of excess cholesterol from peripheral tissues, is a key player in persistent lung inflammation, a common feature of chronic obstructive pulmonary disease (COPD) and lung cancer, after cigarette smoke (CS) exposure. We have previously shown that lycopene, a carotenoid naturally occurring in tomatoes and tomato products, inhibits tobacco carcinogen (NNK)/CS-induced COPD and preneoplastic lesions in lung of ferrets, but the mechanism is unknown. We hypothesize that the protective role of lycopene against NNK/CS-induced COPD and lung preneoplastic lesions is associated with restoring the RCT in ferrets. MethodsMale ferrets (4 groups, n = 12–16/group) were exposed to combination of NNK and CS with or without lycopene feeding at low (LL) and high (HL) doses (equivalent to ∼30 mg and ∼90 mg lycopene/day in humans, respectively) for 22 weeks. Ferrets in NNK/CS groups were given NNK (50 mg/kg body weight, i.p. injection) once a month for 4 consecutive months, and were exposed to CS for 30 min/day for 22 weeks. The animals were fed lycopene or placebo starting three weeks prior to the NNK injections and continued until the end of the study. ResultsNNK/CS exposure significantly increased total cholesterol levels in both plasma and lungs of ferrets (P < 0.05), which was associated with COPD and lung preneoplastic lesion development. In lungs, HL and LL doses lowered NNK/CS-induced total cholesterol, with HL dose reaching significance (P < 0.05); this was accompanied with the decreased lung lesions. HL group had significantly higher mRNA expression of critical genes involved in RCT (LXRα, PPARα, ABCA1 and ABCG1) as compared to NNK/CS group (P < 0.05). Additionally, HL feeding significantly increased protein levels of both nuclear hormone receptors, LXRα and PPARα, known regulators of ABCA1/G1 transporters (P < 0.05). In plasma, lycopene restored total cholesterol and HDL-C concentrations to normal. Plasma triglyceride, LDL-C and VLDL-C concentrations were not significantly different between groups. ConclusionsThese data demonstrate an important association between lycopene protection against NNK/CS induced lung lesions and pulmonary cholesterol homeostasis through the restoration of the impaired RCT. Funding SourcesNIH/NCI, USDA/ARS grants.

Club cells form lung adenocarcinomas and maintain the alveoli of adult mice.

Lung cancer and chronic lung diseases impose major disease burdens worldwide and are caused by inhaled noxious agents including tobacco smoke. The cellular origins of environmental-induced lung tumors and of the dysfunctional airway and alveolar epithelial turnover observed with chronic lung diseases are unknown. To address this, we combined mouse models of genetic labeling and ablation of airway (club) and alveolar cells with exposure to environmental noxious and carcinogenic agents. Club cells are shown to survive KRAS mutations and to form lung tumors after tobacco carcinogen exposure. Increasing numbers of club cells are found in the alveoli with aging and after lung injury, but go undetected since they express alveolar proteins. Ablation of club cells prevents chemical lung tumors and causes alveolar destruction in adult mice. Hence club cells are important in alveolar maintenance and carcinogenesis and may be a therapeutic target against premalignancy and chronic lung disease.

Detoxification effects of aloe polysaccharide and propolis on the urinary excretion of metabolites in smokers

The aim of the present study was to investigate the detoxifying effects of aloe polysaccharide (APS), propolis, and the mixture of APS and propolis on the urinary excretion of major human tobacco carcinogens, BaP and an addictive stimulant alkaloid, nicotine. Smokers (≥20 cigarettes/day) were randomly classified into four subgroups (10 people/group) and were given 600 mg/day of APS, 600 mg/day of propolis, or 600 mg/day of the mixture of APS (420 mg/day) and propolis (180 mg/day) for four weeks. Urinary excretion of BaP and cotinine (a metabolite of nicotine) increased in a time-dependent manner increased after supplementation with APS (BaP, 2.23-fold; cotinine, 2.64-fold), propolis (BaP, 1.30-fold; cotinine, 2.08-fold), and the mixture (BaP, 2.33-fold; cotinine, 2.28-fold) compared with smoker control. Creatinine, glucose, and total bilirubin levels significantly decreased in a time-dependent manner after supplementation with APS (creatinine, 15.24%; glucose, 40.22%; total bilirubin, 48.82%), propolis (creatinine, 16.83%; glucose, 36.25%; total bilirubin, 52.59%), and the mixture (creatinine, 16.36%; glucose, 46.37%; total bilirubin, 39.20%) (p < 0.05). These results suggest that supplementation with APS, propolis, or the mixture could reduce the risk of cancer or other diseases associated with tobacco smoking by enhancing urinary excretion of BaP and nicotine.

Black Light Smokers: How Nicotine Intake and Carcinogen Exposure Differ Across Various Biobehavioral Factors

ObjectiveThe study objective was to identify biobehavioral variables associated with greater intake of nicotine and a tobacco carcinogen among Black light smokers who smoke 1 to 10 cigarettes per day (CPD). MethodsWe analyzed baseline data collected from 426 Black light smokers enrolled in Kick It at Swope III (KIS III), a smoking cessation trial for Black smokers. We examined differences in concentrations of tobacco biomarkers, including urinary total nicotine equivalents (TNE) and total 4-(methylnitrosamino)-1-(3)pyridyl-1-butanonol (NNAL; a human carcinogen), across gender, age, plasma nicotine metabolite ratio (NMR), CPD, and measures of tobacco dependence, including time to first cigarette (TFC), using ANOVA. ResultsTobacco biomarker levels were significantly higher among those who smoked more CPD (6–10 vs 1–5 CPD) and those with greater reported physical dependence on tobacco. Concurrently, those who smoked 1–5 CPD smoked each cigarette more intensely than those who smoked 6–10 CPD. While we found no gender differences overall, among those who smoked 1–5 CPD, women had higher NNAL levels compared to men. The rate of nicotine metabolism, measured by the nicotine metabolite ratio, was not significantly related to TNE or NNAL levels. ConclusionAmong Black Light smokers, higher cigarette consumption and greater physical dependence—but not rate of nicotine metabolism, menthol use, or socioeconomic status—were associated with greater toxicant exposure and thus a likely increased risk of tobacco-related diseases. The lack of data on light smokers, and specifically on Blacks, make this observation important given the disproportionate burden of lung cancer in this population.

Detection and quantification of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) from smoker albumin and its potential as a surrogate biomarker of tobacco-specific nitrosamines exposure and bioactivation

4-(Methylnitrosamino)-l-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), two tobacco specific nitrosamine carcinogens, can form adducts with DNA and proteins via pyridyloxobutylation upon phase I enzyme-mediated bioactivation. Such DNA modifications have been proposed as the root cause to initiate carcinogenesis. Upon hydrolysis, both DNA and protein modifications would release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). The released HPB, being tobacco carcinogen specific, has the potential to serve as a surrogate biomarker for both tobacco exposure and carcinogen bioactivation. Because of its easy access, blood is a great source of such investigations with the potential in epidemiological application. HPB quantification from haemoglobin (Hb), however, has been demonstrated with limited success. To further explore this potentially paradigm-shift opportunity, we reported, for the first time, the detection and quantification of HPB from albumin (Alb) adducts formed by the tobacco-specific nitrosamines in mice and in human smokers. The time-course quantitative analysis of HPB from mouse Alb upon NNK exposure suggests that such an Alb adduct is stable. The amounts of HPB from Alb adducts in smoker plasma averaged 1.82 ± 0.19 pg/mg Alb (0.42 to 3.11 pg/mg Alb), which was 36 times the value in nonsmokers (0.05 ± 0.01 pg/mg Alb). Importantly, HPB level from Alb correlated positively with the level of human tobacco exposure estimated by urinary total nicotine equivalent (TNE) (R2 = 0.6170). For comparison, HPB level from Alb was 16.5 times that of Hb (0.12 ± 0.02 pg/mg Hb) in the plasma and red blood cell (RBC) samples of the same smokers. In addition, there was no significant correlation between HPB levels from Hb and TNE (R2 = 0.0719). These data overall suggest that HPB from Alb adducts can serve as a surrogate biomarker to monitor the level of tobacco exposure and carcinogenic nitrosamine bioactivation.

Menthol Inhibits UGT2B10‐catalyzed Detoxification of the Potent Tobacco‐carcinogen, NNAL

Menthol may be added to tobacco products as either naturally occurring L‐menthol or synthesized racemic DL‐menthol and is excreted as a menthol glucuronide (MG). The most potent tobacco carcinogen is 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK). NNK is extensively metabolized to enantiomers [(R) or (S)] of the equally carcinogenic chiral 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL). Both (R)‐ and (S)‐NNAL have been shown to be carcinogenic. Detoxification of (R)‐ and (S)‐NNAL through glucuronidation on NNAL's pyridine ring nitrogen (NNAL‐N‐gluc) is primarily mediated by UGT2B10. Menthol has been shown to inhibit NNAL‐N‐gluc formation in human liver microsomes (HLM). The goal of the present study was to elucidate the impact of menthol on NNAL‐N‐gluc formation in vivo. In vitro inhibition assays using HLM or microsomes from UGT2B10 over‐expressing HEK293 cells were performed with racemic (R/S)‐NNAL as substrate with each menthol enantiomer added separately as inhibitor. In vivo data were gathered from analysis of MG, NNAL, and NNAL metabolites from 50 menthol smokers and 50 non‐menthol smokers. Samples were obtained from the Southern Community Cohort Study. Both L‐ and D‐menthol enantiomers were inhibitors of UGT2B10 in vitro with IC50 values of 236 and 202 μM, respectively. In vivo D‐MG levels, when present, were far below the levels of L‐MG observed in smokers of both menthol or non‐menthol cigarettes, with D‐MG comprising an average of 1.3% of total MG. Urinary L‐MG was detectable in 98 subjects and levels were not significantly different between menthol and non‐menthol smokers (p=0.50). When stratified by tertiles of urinary MG levels, NNAL‐N‐gluc levels were highest in smokers with the lowest MG levels, with NNAL‐N‐gluc levels inversely associated with MG levels in smokers (p&lt;0.05). These data suggest that menthol inhibits a major detoxification pathway of the tobacco carcinogen, NNK. The relatively high levels of urinary menthol in smokers of both menthol and non‐menthol cigarettes raise the possibility that menthol branding may not be the best approach to studying the impact of menthol as a tobacco additive.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluation of tobacco‐specific nitrosamine (TSNA) content in smokeless tobacco products in Bangladesh

Head and neck cancers are among the most common cancers in Southeast Asia and are likely to face sharp increases in the coming years due to the widespread use of smokeless tobacco (SLT) products. Bangladesh has the second highest oral cancer mortality rate as well as the second highest rate of SLT product usage worldwide. SLT products contain high levels of the highly carcinogenic tobacco‐specific nitrosamines (TSNAs); they include N‐nitrosonornicotine (NNN), 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK), N‐nitrosoanatabine (NAT), and N‐nitrosoanabasine (NAB). To date, the amount of TSNAs present in Bangladeshi SLT products has not been reported. The goal of the present study was to develop an efficient TSNA and nicotine extraction method to quantify the levels of TSNAs and nicotine present in 31 Bangladeshi SLT products. The concentration of NNN, NNK, NAT, and NAB in Bangladeshi SLT brands ranged from 1.07–59.18 μg/g SLT, 0.15–33.70 μg/g SLT, 0.79–44.64 μg/g SLT, and 0.03–12.85 μg/g SLT, respectively. Pan Masala, which is labeled as a non‐tobacco product, also contains low levels of TSNAs. Betel nut, a common flavor additive in SLT products in Bangladesh, contained no detectible TSNA levels. Bangladeshi SLT products contained significantly higher (p&lt;0.05) levels of TSNAs as compared to SLT brands from the United States (U.S.), and were higher on average than the levels observed in brands in other regions of southern Asia. NNN and NNK levels were 16 and 19, 2 and 3.5, and 62 and 85 fold higher than U.S., Indian and Pakistani SLT brands, respectively. Additionally, the levels of NNN and NNK measured in U.S. brands in this study were 1.2‐fold higher than previously reported values from 2013. This study demonstrates that the levels of TSNAs, including the major tobacco carcinogens NNN and NNK, are far higher in Bangladeshi SLT brands than they are in other regions of the world. Exposure to such high levels of carcinogens may be the most important contributing factor to the rate of head and neck as well as other tobacco‐related cancers observed in Bangladesh.Support or Funding InformationThis work is supported in part by grants from NIH, National Cancer Institute (grant R01‐ES025460, to P. Lazarus) and the Health Sciences and Services Authority of Spokane, Washington (grant WSU002292, to College of Pharmacy and Pharmaceutical Sciences, Washington State University)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glycosylation of polycyclic aromatic hydrocarbons by UDP‐glycosyltransferase 3A2 (UGT3A2) and aerodigestive tract tissues

Smoking is a primary risk factor for the development of lung and tobacco‐related cancers. There are over 70 carcinogens in tobacco smoke, and they belong to multiple chemical classes including polycyclic aromatic hydrocarbons (PAHs). The UDP‐glycosyltransferase (UGT) family of enzymes play a key role in the detoxification of several major tobacco carcinogens by glucuronidation, using UDP‐glucuronic acid as cosubstrate. While the UGT 1As, 2As, and 2Bs are well known to detoxify simple and complex PAHs, the UGT3A subfamily has been understudied in the detoxification of PAHs. UGT3A2 uses UDP‐glucose and UDP‐xylose as cosubstrates and is active against 1‐hydroxypyrene, a biomarker for PAH exposure. Our previous research has shown that UGT3A2 is expressed in the aerodigestive tract and is active against simple hydroxylated PAHs. The purpose of the present study was to determine the kinetic parameters of UGT3A2 against complex PAHs and determine glycosylation in aerodigestive tract tissues using UDP‐glucose and UDP‐xylose. Microsomes prepared from UGT3A2 overexpressing HEK293 cells exhibited glycosylation activity against complex PAHs including benzo[a]pyrene (B[a]P)‐7,8‐diol, B[a]P‐9,10‐diol, DB[a, l]P‐11,12‐diol, and 5‐methylchrysene‐1,2‐diol, with Km's ranging from 96 to 1250 μM using UDP‐glucose. The Km's were significantly lower when using UDP‐xylose, with Km's ranging from 120 to 168 μM for the same substrates. The Vmax/Km ratio of UDP‐xylose/UDP‐glucose ranged from 0.71 to 4.0. To determine the physiological significance and relevance of UDP‐xylose‐ and UDP‐glucose‐dependent glycosylation, activity assays were performed in tissues where UGT3A2 is expressed using 1‐hydroxypyrene as substrate. Using UDP‐glucose as the cosubstrate, glycosylation rates of 3.5, 5.2, and 13 pmol·min−1·mg−1 were observed for liver, intestine and kidney microsomes, respectively. The rates were higher using UDP‐xylose, with rates of 22, 9.3, and 20 pmol·min−1·mg−1 for liver, intestine and kidney, respectively. The glycosylation rate ratio for UDP‐xylose/UDP‐glucose ranged from 1.6 to 6.3 in the three tissues, with liver exhibiting the highest ratio. In addition, detectable glycosylation activity was observed in various aerodigestive tract tissues including esophagus, tonsil, bronchus, floor of mouth, tongue, trachea, and larynx, with tonsil exhibiting the highest relative activity with both UDP‐sugars. Incubations with UDP‐xylose resulted in higher levels of glycosylation activity than that observed with UDP‐glucose in esophagus, tonsil, floor of mouth, and larynx. These data suggest that UGT3A2 is playing an important role in the detoxification of PAHs in tobacco target tissues, with UDP‐xylose being the preferred cosubstrate in most tissues.Support or Funding InformationThis study is supported by the National Institute of Environmental Health Sciences (NIEHS) of the National Institutes of Health to P Lazarus (Grant R01‐ES025460), a NIEHS Research Supplement to Promote Diversity in Health‐Related Research to AG Vergara (Grant R01‐ES025460‐02S1), and a grant from the Health Sciences and Services Authority of Spokane, WA (Grant WSU002292).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mutational Signatures in Mandibular Ameloblastoma Correlate with Smoking

Ameloblastoma is a rare tumor of odontogenic epithelium, the low incidence rate of which precludes statistical determination of its molecular characterizations. Despite recent genomic and transcriptomic profiling, the etiology of ameloblastomas remains poorly understood. Risk factors of ameloblastoma development are also largely unknown. Whole exome sequencing was performed on 11 mandibular ameloblastoma samples. We identified 2 convergent mutational signatures in ameloblastoma: 1) a signature found in multiple types of lung cancers with probable etiology of tobacco carcinogens (COSMIC signature 4) and 2) a signature present in gingivobuccal oral squamous cell carcinoma and correlated with tobacco-chewing habits (COSMIC signature 29). These mutational signatures highlight tobacco usage or related mutagens as one possible risk factor of ameloblastoma, since the association of BRAF mutations and smoking was demonstrated in multiple studies. In addition to BRAF hotspot mutations (V600E), we observed clear inter- and intratumor heterogeneities. Interestingly, prior to BRAF mutation, important genes regulating odontogenesis mutated (e.g., corepressor BCOR), possibly playing important roles in tumorigenesis. Furthermore, recurrent mutations in the CDC73 gene, the germline mutations of which predispose patients to the development of jaw tumors, were found in 2 patients, which may lead to recurrence if not targeted by therapeutic drugs. Our unbiased profiling of coding regions of ameloblastoma genomes provides insights to the possible etiology of mandibular ameloblastoma and highlights potential disease risk factors for screening and prevention, especially for Asian patients. Because of the limited sample size and incomplete habitual, dietary, and occupational data, a causal link between tobacco usage and ameloblastoma still requires further investigations.

The Aryl hydrocarbon receptor mediates tobacco-induced PD-L1 expression and is associated with response to immunotherapy

Whether tobacco carcinogens enable exposed cells immune escape resulting in carcinogenesis, and why patients who smoke respond better to immunotherapies than non-smokers, remains poorly understood. Here we report that cigarette smoke and the carcinogen benzo(a)pyrene (BaP) induce PD-L1 expression on lung epithelial cells in vitro and in vivo, which is mediated by aryl hydrocarbon receptor (AhR). Anti-PD-L1 antibody or deficiency in AhR significantly suppresses BaP-induced lung cancer. In 37 patients treated with anti-PD-1 antibody pembrolizumab, 13/16 (81.3%) patients who achieve partial response or stable disease express high levels of AhR, whereas 12/16 (75%) patients with progression disease exhibit low levels of AhR in tumor tissues. AhR inhibitors exert significant antitumor activity and synergize with anti-PD-L1 antibody in lung cancer mouse models. These results demonstrate that tobacco smoke enables lung epithelial cells to escape from adaptive immunity to promote tumorigenesis, and AhR predicts the response to immunotherapy and represents an attractive therapeutic target.

Tobacco chemical-induced mouse lung adenocarcinoma cell lines pin the prolactin orthologue proliferin as a lung tumour promoter.

Lung adenocarcinoma (LADC) is the leading cause of cancer death worldwide. Nevertheless, syngeneic mouse models of the disease are sparse, and cell lines suitable for transplantable and immunocompetent mouse models of LADC remain unmet needs. We established multiple mouse LADC cell lines by repeatedly exposing two mouse strains (FVB, Balb/c) to the tobacco carcinogens urethane or diethylnitrosamine and by culturing out the resulting lung tumours for prolonged periods of time. Characterization of the resulting cell lines (n = 7) showed that they were immortal and phenotypically stable in vitro, and oncogenic, metastatic and lethal in vivo. The primary tumours that gave rise to the cell lines, as well as secondary tumours generated by transplantation of the cell lines, displayed typical LADC features, such as glandular architecture and mucin and thyroid transcription factor 1 expression. Moreover, these cells exhibited marked molecular similarity with human smokers' LADC, including carcinogen-specific Kras point mutations (KrasQ61R in urethane- and KrasQ61H in diethylnitrosamine-triggered cell lines) and Trp53 deletions and displayed stemness features. Interestingly, all cell lines overexpressed proliferin, a murine prolactin orthologue, which functioned as a lung tumour promoter. Furthermore, prolactin was overexpressed and portended poor prognosis in human LADC. In conclusion, we report the first LADC cell lines derived from mice exposed to tobacco carcinogens. These cells closely resemble human LADC and provide a valuable tool for the functional investigation of the pathobiology of the disease.

Disparity in age at lung cancer diagnosis between current and former smokers.

In a previous study of smoking cessation in veterans with lung cancer, we noted as an incidental finding that current smokers were much younger than former smokers at diagnosis. To confirm and extend this observation, we analyzed the association of smoking status with age at diagnosis and survival of lung cancer patients. The Jefferson Cancer Registry collects information on all cancer patients registered at this hospital. Information on smoking status has been recorded since 1995. We determined age at diagnosis and survival of current and former smokers with lung cancer. 5111 lung cancer cases were identified in the registry from 1995 to 2011 inclusive. Smoking status was recorded in 4687 cases (91.7%). Of these, 1859 (39.7%) were current, 2423 (51.7%) were former, and 405 (8.6%) were never smokers. There was a 6-year difference in median age at lung cancer diagnosis between the current (63years) and former smokers (69years) (P < 0.0001). The median survival was 12.1months for current versus 14.5months for former smokers (P < 0.0001). These results confirm and extend our observation that among patients diagnosed with lung cancer, current smokers are younger than former smokers. The possible explanations include higher competing causes of death and increased risk of lung cancer among current smokers as well as increasing proportions of former smokers in older populations. Ongoing exposure to tobacco carcinogens may accelerate the development of lung cancer in continuing smokers. This provides more incentive for smokers to quit at the earliest age possible.

Are there any theranostic biomarkers in small cell lung carcinoma?

Small cell lung cancer (SCLC), an aggressive lung tumour with a poor prognosis, has a high load of somatic mutations, mainly induced by tobacco carcinogens given the strong association with smoking. Advances in genomic, epigenetic and proteomic profiling have significantly improved our understanding of the molecular and cellular biology of SCLC. Given the high mutational burden of SCLC the immune microenvironment is another exciting area under investigation even if it seems to be quite distinct from that of other solid tumours. In this review we will outline the current progress in molecular etiology of SCLC mentioning some key markers considered promising theranostic biomarkers.

Lung-Enriched Mutations in the p53 Tumor Suppressor: A Paradigm for Tissue-Specific Gain of Oncogenic Function.

Lung cancer, the leading cause of cancer-related mortality in the United States, occurs primarily due to prolonged exposure to an array of carcinogenic compounds in cigarette smoke. These carcinogens create bulky DNA adducts, inducing alterations including missense mutations in the tumor suppressor gene TP53 TP53 is the most commonly mutated gene in many human cancers, and a specific set of these variants are enriched in lung cancer (at amino acid residues V157, R158, and A159). This perspective postulates that lung-enriched mutations can be explained, in part, by biological selection for oncogenic gain-of-function (GOF) mutant p53 alleles at V157, R158, and A159. This hypothesis explaining tissue-specific TP53 mutations is further supported by mouse model studies of the canonical TP53 hotspots showing that tumor spectra and GOF activities are altered with mutation type. Therefore, although smoking-related lung cancer unequivocally arises due to the mutagenic environment induced by tobacco carcinogens, this perspective provides a rationale for the preferential selection of lung-enriched V157, R158, and A159 mutant p53.

Modulation of Vascular Endothelial Growth Factor and Annexin A2 in Response to 4-(Methylnitrosamino)-1-(3-pyridyl)-1-Butanone -Induced Inflammation via Swimming Training

The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK; nicotine derived nitrosamine ketone) is one of the strongest carcinogens in tobacco which is involved in induction of lung cancer by changing the stimulation of vascular endothelial growth factor (VEGF) and annexin A2 expression. The aim of this study was to investigate the changes in resting levels of annexin A2 and VEGF in lung tissues of rats exposed NNK after 12 weeks of aerobic submaximal swimming training. For this purpose, 46 Wistar rats were randomly divided into five groups consist of training, training + NNK, NNK, saline and control. NNK-induced groups received NNK subcutaneously one day per week at a rate of 12/5 mg per kg body weight and the training groups performed submaximal swimming training for 12 weeks. The levels of VEGF and annexin A2 in lung tissue were measured respectively by ELISA and immunohistochemistry. To analyze the data; ANOVA and Tukey's test were used at a significance level of p<0.05. Findings indicated that 12 weeks submaximal swimming training decreased the levels of VEGF and annexin A2 in lung tissue significantly when compared to NNK group (p<0.001). There was no significant correlation between VEGF and annexin A2 levels in all study groups (p≥0.05). Generally, it could be confirmed that regular submaximal aerobic training plays an important role in inhibition of the effects of lung inflammation induced by NNK via decreased levels of VEGF and annexin A2.

Highly Efficient Degradation of Tobacco Specific N‐Nitrosamines by TiO2 Mesocrystals with Robust and Tailored Microporous Structure

AbstractIt is essential to control tobacco specific N‐nitrosamines (TSNAs) pollution for the protection of public health, which were identified as the most abundant carcinogens in tobacco and tobacco smoke. Herein, we provide microporous TiO2 mesocrystals as a new material for the remove of TSNAs with high degradation efficiency. The anatase TiO2 mesocrystals with tunable microporous structure were synthesized by a facile method under the acid solution. The TiO2 mesocrystals were firstly used to the remove of TSNAs through photocatalytic degradation. It's exciting that TSNAs of tobacco extraction solution can be almost entirely reduced (up to 97%) by anatase TiO2 mesocrystals after 30 mins through photocatalytic degradation. Moreover, it's found that the microporous size of mesocrystals play a key role on the degradation capability. The TiO2 mesocrystals with microporous size comparable to the size of TSNAs molecule exhibit the best degradation performance. This finding offers a robust microporous material and efficient method for removing the carcinogen like TSNAs as well as other pollutants in environment.

Prostacyclin and EMT Pathway Markers for Monitoring Response to Lung Cancer Chemoprevention.

Lung cancer is the leading cause of cancer death worldwide and global burden could be reduced through targeted application of chemoprevention. The development of squamous lung carcinoma has been linked with persistent, high-grade bronchial dysplasia. Bronchial histology improved in former smokers in a chemoprevention trial with the prostacyclin analogue iloprost. Prostacyclin acts through peroxisome proliferator-activated receptor gamma (PPARγ) to reverse epithelial to mesenchymal transition and promote anticancer signaling. We hypothesized that the prostacyclin signaling pathway and EMT could provide response markers for prostacyclin chemoprevention of lung cancer. Human bronchial epithelial cells were treated with cigarette smoke condensate (CSC) or iloprost for 2 weeks, CSC for 16 weeks, or CSC for 4 weeks followed by 4 weeks of CSC and/or iloprost, and RNA was extracted. Wild-type or prostacyclin synthase transgenic mice were exposed to 1 week of cigarette smoke or one injection of urethane, and RNA was extracted from the lungs. We measured potential markers of prostacyclin and iloprost efficacy in these models. We identified a panel of markers altered by tobacco carcinogens and inversely affected by prostacyclin, including PPARγ, 15PGDH, CES1, COX-2, ECADHERIN, SNAIL, VIMENTIN, CRB3, MIR34c, and MIR221 These data introduce a panel of potential markers for monitoring interception of bronchial dysplasia progression during chemoprevention with prostacyclin. Chemoprevention is a promising approach to reduce lung cancer mortality in a high-risk population. Identifying markers for targeted use is critical for success in future clinical trials of prostacyclin for lung cancer chemoprevention. Cancer Prev Res; 11(10); 643-54. ©2018 AACR.