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1.  Implication of a chromosome 15q15.2 locus in regulating UBR1 and predisposing smokers to MGMT methylation in lung 
Cancer research  2015;75(15):3108-3117.
O6-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 (HBEC), 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.
doi:10.1158/0008-5472.CAN-15-0243
PMCID: PMC4526392  PMID: 26183928
Genome wide association study; promoter hypermethylation; MGMT; smoker; UBR1
2.  Epigenetic Repression of CCDC37 and MAP1B Links Chronic Obstructive Pulmonary Disease to Lung Cancer 
Introduction
Lung cancer and chronic obstructive pulmonary disease (COPD) share environmental risk factors. COPD also increases the risk of lung cancer; however, the molecular mechanisms are unclear.
Methods
An epigenome-wide association study of lung tumors and cancer-free lung tissue (CFLT) pairs from non-small cell lung cancer (NSCLC) cases with (n=18) or without (n=17) COPD was conducted using the HumanMethylation450 beadchip (HM450K). COPD-associated methylation of top-ranked genes was confirmed in a larger sample set, independently validated, and their potential as sputum-based biomarkers was investigated.
Results
Methylation of CCDC37 and MAP1B was more prevalent in lung tumors from COPD than non-COPD cases [54/71 (76%) vs. 20/46 (43%), p=0.0013] and [48/71 (68%) vs. 17/46 (37%), p=0.0035], respectively, after adjustment for age, sex, smoking status, and tumor histology. HM450K probes across CCDC37 and MAP1B promoters showed higher methylation in tumors than CFLT with the highest methylation seen in tumors from COPD cases (p<0.05). These results were independently validated using The Cancer Genome Atlas data. CCDC37 methylation was more prevalent in sputum from COPD than non-COPD smokers (p<0.005) from two cohorts. CCDC37 and MAP1B expression was dramatically repressed in tumors and CFLT from COPD than non-COPD cases, p<0.02.
Conclusions
The reduced expression of CCDC37 and MAP1B associated with COPD likely predisposes these genes to methylation that in turn, may contribute to lung cancer.
doi:10.1097/JTO.0000000000000592
PMCID: PMC4511946  PMID: 26200272
EWAS; Methylation; COPD; NSCLC; Sputum
3.  A prospective and retrospective analysis of smoking behavior changes in ever smokers with high risk for lung cancer from New Mexico and Pennsylvania 
Cigarette smoking is the leading preventable cause of death worldwide. The aim of this study is to conduct a prospective and retrospective analysis of smoking behavior changes in the Lovelace Smokers Cohort (LSC) and the Pittsburgh Lung Screening Study cohort (PLuSS). Area under the curve (AUC) for risk models predicting relapse based on demographic, smoking, and relevant clinical variables was 0.93 and 0.79 in LSC and PLuSS, respectively. The models for making a quit attempt had limited prediction ability in both cohorts (AUC≤0.62). We identified an ethnic disparity in adverse smoking behavior change that Hispanic smokers were less likely to make a quit attempt and were more likely to relapse after a quit attempt compared to non-Hispanic Whites. SNPs at 15q25 and 11p14 loci were associated with risk for smoking relapse in the LSC. Rs6495308 at 15q25 has a large difference in minor allele frequency between non-Hispanic Whites and Hispanics (0.46 versus 0.23, P<0.0001) and was associated with risk for ever relapse at same magnitude between the two ethnic groups (OR=1.36, 95% CI=1.10 to 1.67 versus 1.59, 95% CI=1.00 to 2.53, P=0.81). In summary, the risk prediction model established in LSC and PLuSS provided an excellent to outstanding distinguishing for abstainers who will or will not relapse. The ethnic disparity in adverse smoking behavior between Hispanics and non-Hispanic Whites may be at least partially explained by the sequence variants at 15q25 locus that contains multiple nicotine acetylcholine receptors.
PMCID: PMC4913225  PMID: 27335628
Smoking behavior; risk prediction model; ethnicity; single nucleotide polymorphism
4.  15q12 Variants, Sputum Gene Promoter Hypermethylation, and Lung Cancer Risk: A GWAS in Smokers 
Background:
Lung cancer is the leading cause of cancer-related mortality worldwide. Detection of promoter hypermethylation of tumor suppressor genes in exfoliated cells from the lung provides an assessment of field cancerization that in turn predicts lung cancer. The identification of genetic determinants for this validated cancer biomarker should provide novel insights into mechanisms underlying epigenetic reprogramming during lung carcinogenesis.
Methods:
A genome-wide association study using generalized estimating equations and logistic regression models was conducted in two geographically independent smoker cohorts to identify loci affecting the propensity for cancer-related gene methylation that was assessed by a 12-gene panel interrogated in sputum. All statistical tests were two-sided.
Results:
Two single nucleotide polymorphisms (SNPs) at 15q12 (rs73371737 and rs7179575) that drove gene methylation were discovered and replicated with rs73371737 reaching genome-wide significance (P = 3.3×10–8). A haplotype carrying risk alleles from the two 15q12 SNPs conferred 57% increased risk for gene methylation (P = 2.5×10–9). Rs73371737 reduced GABRB3 expression in lung cells and increased risk for smoking-induced chronic mucous hypersecretion. Furthermore, subjects with variant homozygote of rs73371737 had a two-fold increase in risk for lung cancer (P = .0043). Pathway analysis identified DNA double-strand break repair by homologous recombination (DSBR-HR) as a major pathway affecting susceptibility for gene methylation that was validated by measuring chromatid breaks in lymphocytes challenged by bleomycin.
Conclusions:
A functional 15q12 variant was identified as a risk factor for gene methylation and lung cancer. The associations could be mediated by GABAergic signaling that drives the smoking-induced mucous cell metaplasia. Our findings also substantiate DSBR-HR as a critical pathway driving epigenetic gene silencing.
doi:10.1093/jnci/djv035
PMCID: PMC4555640  PMID: 25713168
5.  Radon Exposure, IL-6 Promoter Variants, and Lung Squamous Cell Carcinoma in Former Uranium Miners 
Environmental Health Perspectives  2015;124(4):445-451.
Background:
High radon exposure is a risk factor for squamous cell carcinoma, a major lung cancer histology observed in former uranium miners. Radon exposure can cause oxidative stress, leading to pulmonary inflammation. Interleukin-6 (IL-6) is a pro-carcinogenic inflammatory cytokine that plays a pivotal role in lung cancer development.
Objectives:
We assessed whether single nucleotide polymorphisms (SNPs) in the IL6 promoter are associated with lung cancer in former uranium miners with high occupational exposure to radon gas.
Methods:
Genetic associations were assessed in a case–control study of former uranium miners (242 cases and 336 controls). A replication study was performed using data from the Gene Environment Association Studies (GENEVA) Genome Wide Association Study (GWAS) of Lung Cancer and Smoking. Functional relevance of the SNPs was characterized using in vitro approaches.
Results:
We found that rs1800797 was associated with squamous cell carcinoma in miners and with a shorter time between the midpoint of the period of substantial exposure and diagnosis among the cases. Furthermore, rs1800797 was also associated with lung cancer among never smokers in the GENEVA dataset. Functional studies identified that the risk allele was associated with increased basal IL-6 mRNA level and greater promoter activity. Furthermore, fibroblasts with the risk allele showed greater induction of IL-6 secretion by hydrogen peroxide or benzo[a]pyrene diolepoxide treatments.
Conclusions:
An IL6 promoter variant was associated with lung cancer in uranium miners and never smokers in two external study populations. The associations are strongly supported by the functional relevance that the IL6 promoter SNP affects basal expression and carcinogen-induced IL-6 secretion.
Citation:
Leng S, Thomas CL, Snider AM, Picchi MA, Chen W, Willis DG, Carr TG, Krzeminski J, Desai D, Shantu A, Lin Y, Jacobson MR, Belinsky SA. 2016. Radon exposure, IL-6 promoter variants, and lung squamous cell carcinoma in former uranium miners. Environ Health Perspect 124:445–451; http://dx.doi.org/10.1289/ehp.1409437
doi:10.1289/ehp.1409437
PMCID: PMC4829998  PMID: 26372664
6.  Whole exome sequencing identifies novel candidate genes that modify chronic obstructive pulmonary disease susceptibility 
Human Genomics  2016;10:1.
Background
Chronic obstructive pulmonary disease (COPD) is characterized by an irreversible airflow limitation in response to inhalation of noxious stimuli, such as cigarette smoke. However, only 15–20 % smokers manifest COPD, suggesting a role for genetic predisposition. Although genome-wide association studies have identified common genetic variants that are associated with susceptibility to COPD, effect sizes of the identified variants are modest, as is the total heritability accounted for by these variants. In this study, an extreme phenotype exome sequencing study was combined with in vitro modeling to identify COPD candidate genes.
Results
We performed whole exome sequencing of 62 highly susceptible smokers and 30 exceptionally resistant smokers to identify rare variants that may contribute to disease risk or resistance to COPD. This was a cross-sectional case-control study without therapeutic intervention or longitudinal follow-up information. We identified candidate genes based on rare variant analyses and evaluated exonic variants to pinpoint individual genes whose function was computationally established to be significantly different between susceptible and resistant smokers. Top scoring candidate genes from these analyses were further filtered by requiring that each gene be expressed in human bronchial epithelial cells (HBECs). A total of 81 candidate genes were thus selected for in vitro functional testing in cigarette smoke extract (CSE)-exposed HBECs. Using small interfering RNA (siRNA)-mediated gene silencing experiments, we showed that silencing of several candidate genes augmented CSE-induced cytotoxicity in vitro.
Conclusions
Our integrative analysis through both genetic and functional approaches identified two candidate genes (TACC2 and MYO1E) that augment cigarette smoke (CS)-induced cytotoxicity and, potentially, COPD susceptibility.
Electronic supplementary material
The online version of this article (doi:10.1186/s40246-015-0058-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s40246-015-0058-7
PMCID: PMC4705629  PMID: 26744305
Whole exome sequencing; Cigarette smoke; COPD; Susceptible smokers; Resistant smokers
7.  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, 1.3–33.6-fold (p=2.2×10−9), compared to corresponding normal lung. GATA2 promoter was unmethylated in normal lung (0/10) but frequently methylated in lung tumors (96%, 159/165) and NSCLC cell lines (97%, 30/31). This highly prevalent aberrant methylation was independently validated using TCGA data for 369 NSCLC tumor-normal pairs. In vitro studies using an established carcinogen-induced pre-malignancy 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 5-fold 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.
doi:10.1097/JTO.0000000000000165
PMCID: PMC4132640  PMID: 24807155
GATA-2; epigenetics; K-RAS; mutation; carcinogenesis
8.  Native American Ancestry Affects the Risk for Gene Methylation in the Lungs of Hispanic Smokers from New Mexico 
Rationale: Gene promoter methylation detected in sputum predicts lung cancer risk in smokers. Compared with non-Hispanic whites (NHW), Hispanics have a lower age-standardized incidence for lung cancer.
Objectives: This study compared the methylation prevalence in sputum of NHWs with Hispanics using the Lovelace Smokers cohort (n = 1998) and evaluated the effect of Native American ancestry (NAA) and diet on biomarkers for lung cancer risk.
Methods: Genetic ancestry was estimated using 48 ancestry markers. Diet was assessed by the Harvard University Dietary Assessment questionnaire. Methylation of 12 genes was measured in sputum using methylation-specific polymerase chain reaction. The association between NAA and risk for methylation was assessed using generalized estimating equations. The ethnic difference in the association between pack-years and risk for lung cancer was assessed in the New Mexico lung cancer study.
Measurements and Main Results: Overall Hispanics had a significantly increased risk for methylation across the 12 genes analyzed (odds ratio, 1.18; P = 0.007). However, the risk was reduced by 32% (P = 0.032) in Hispanics with high versus low NAA. In the New Mexico lung cancer study, Hispanic non–small cell lung cancer cases have significantly lower pack-years than NHW counterparts (P = 0.007). Furthermore, compared with NHW smokers, Hispanic smokers had a more rapidly increasing risk for lung cancer as a function of pack-years (P = 0.058).
Conclusions: NAA may be an important risk modifier for methylation in Hispanic smokers. Smoking intensity may have a greater impact on risk for lung cancer in Hispanics compared with NHWs.
doi:10.1164/rccm.201305-0925OC
PMCID: PMC3863742  PMID: 24032348
ethnicity; sputum; diet; risk; lung cancer
9.  Genetic variation in SIRT1 affects susceptibility of lung squamous cell carcinomas in former uranium miners from the Colorado plateau 
Carcinogenesis  2013;34(5):1044-1050.
Epidemiological studies of underground miners suggested that occupational exposure to radon causes lung cancer with squamous cell carcinoma (SCC) as the predominant histological type. However, the genetic determinants for susceptibility of radon-induced SCC in miners are unclear. Double-strand breaks induced by radioactive radon daughters are repaired primarily by non-homologous end joining (NHEJ) that is accompanied by the dynamic changes in surrounding chromatin, including nucleosome repositioning and histone modifications. Thus, a molecular epidemiological study was conducted to assess whether genetic variation in 16 genes involved in NHEJ and related histone modification affected susceptibility for SCC in radon-exposed former miners (267 SCC cases and 383 controls) from the Colorado plateau. A global association between genetic variation in the haplotype block where SIRT1 resides and the risk for SCC in miners (P = 0.003) was identified. Haplotype alleles tagged by the A allele of SIRT1 rs7097008 were associated with increased risk for SCC (odds ratio = 1.69, P = 8.2×10−5) and greater survival in SCC cases (hazard ratio = 0.79, P = 0.03) in miners. Functional validation of rs7097008 demonstrated that the A allele was associated with reduced gene expression in bronchial epithelial cells and compromised DNA repair capacity in peripheral lymphocytes. Together, these findings substantiate genetic variation in SIRT1 as a risk modifier for developing SCC in miners and suggest that SIRT1 may also play a tumor suppressor role in radon-induced cancer in miners.
doi:10.1093/carcin/bgt024
PMCID: PMC3643420  PMID: 23354305
10.  Increased methylation of lung cancer-associated genes in sputum DNA of former smokers with chronic mucous hypersecretion 
Respiratory Research  2014;15(1):2.
Background
Chronic mucous hypersecretion (CMH) contributes to COPD exacerbations and increased risk for lung cancer. Because methylation of gene promoters in sputum has been shown to be associated with lung cancer risk, we tested whether such methylation was more common in persons with CMH.
Methods
Eleven genes commonly silenced by promoter methylation in lung cancer and associated with cancer risk were selected. Methylation specific PCR (MSP) was used to profile the sputum of 900 individuals in the Lovelace Smokers Cohort (LSC). Replication was performed in 490 individuals from the Pittsburgh Lung Screening Study (PLuSS).
Results
CMH was significantly associated with an overall increased number of methylated genes, with SULF2 methylation demonstrating the most consistent association. The association between SULF2 methylation and CMH was significantly increased in males but not in females both in the LSC and PLuSS (OR = 2.72, 95% CI = 1.51-4.91, p = 0.001 and OR = 2.97, 95% CI = 1.48-5.95, p = 0.002, respectively). Further, the association between methylation and CMH was more pronounced among 139 male former smokers with persistent CMH compared to current smokers (SULF2; OR = 3.65, 95% CI = 1.59-8.37, p = 0.002).
Conclusions
These findings demonstrate that especially male former smokers with persistent CMH have markedly increased promoter methylation of lung cancer risk genes and potentially could be at increased risk for lung cancer.
doi:10.1186/1465-9921-15-2
PMCID: PMC3893562  PMID: 24405663
Methylation of gene promoters; Persistent cough and phlegm; Sputum DNA; Former smoker; Lung cancer genes
11.  Sex-specific association of sequence variants in CBS and MTRR with risk for promoter hypermethylation in the lung epithelium of smokers 
Carcinogenesis  2012;33(8):1542-1547.
Gene promoter hypermethylation is now regarded as a promising biomarker for the risk and progression of lung cancer. The one-carbon metabolism pathway is postulated to affect deoxyribonucleic acid (DNA) methylation because it is responsible for the generation of S-adenosylmethionine (SAM), the methyl donor for cellular methylation reactions. This study investigated the association of single nucleotide polymorphisms (SNPs) in six one-carbon metabolism-related genes with promoter hypermethylation in sputum DNA from non-Hispanic white smokers in the Lovelace Smokers Cohort (LSC) (n = 907). Logistic regression was used to assess the association of SNPs with hypermethylation using a high/low methylation cutoff. SNPs in the cystathionine beta synthase (CBS) and 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR) genes were significantly associated with high methylation in males [CBS rs2850146 (-8283G > C), 
OR = 4.9; 95% CI: 1.98, 12.2, P = 0.0006] and low methylation in females [MTRR rs3776467 (7068A > G), OR = 0.57, 95% CI: 0.42, 0.77, P = 0.0003]. The variant allele of rs2850146 was associated with reduced gene expression and increased plasma homocysteine (Hcy) concentrations. Three plasma metabolites, Hcy, methionine and dimethylglycine, were associated with increased risk for gene methylation. These studies suggest that SNPs in CBS and MTRR have sex-specific associations with aberrant methylation in the lung epithelium of smokers that could be mediated by the affected one-carbon metabolism and transsulfuration in the cells.
Abbreviations:CBScystathionine beta synthaseDNAdeoxyribonucleic acidHBEChuman bronchial epithelial cellHcyhomocysteineLD, linkage disequilibrium; LSClovelace Smokers CohortMAFminor allele frequencyMTHFRmethylenetetrahydrofolate reductaseMTRRmethyltransferase reductaseSNPsingle nucleotide polymorphismsSAHS-adenosylhomocysteineSAMS-adenosylmethionine
doi:10.1093/carcin/bgs194
PMCID: PMC3499054  PMID: 22665368
12.  Defining a Gene Promoter Methylation Signature in Sputum for Lung Cancer Risk Assessment 
Purpose
To evaluate the methylation state of 31 genes in sputum as biomarkers in an expanded nested, case-control study from the Colorado Cohort and to assess the replication of results from the most promising genes in an independent case-control study of asymptomatic Stage I lung cancer patients from New Mexico.
Experimental Design
Cases and controls from Colorado and New Mexico were interrogated for methylation of up to 31 genes using nested, methylation specific PCR. Individual genes and methylation indices were used to assess the association between methylation and lung cancer with logistic regression modeling.
Results
Seventeen genes with odds ratios of 1.4 – 3.6 were identified and selected for replication in the New Mexico study. Overall, the direction of effects seen in New Mexico was similar to Colorado with the largest increase in case discrimination (odds ratios, 3.2 – 4.2) seen for the PAX5α, GATA5, and SULF2 genes. ROC curves generated from seven gene panels from Colorado and New Mexico studies showed prediction accuracy of 71% and 77%, respectively. A 22-fold increase in lung cancer risk was seen for a subset of New Mexico cases with five or more genes methylated. Sequence variants associated with lung cancer did not improve the accuracy of this gene methylation panel.
Conclusions
These studies have identified and replicated a panel of methylated genes whose integration with other promising biomarkers could initially identify the highest risk smokers for computed tomography screening for early detection of lung cancer.
doi:10.1158/1078-0432.CCR-11-3049
PMCID: PMC3483793  PMID: 22510351
gene methylation; sputum; lung cancer; biomarker
13.  Difference in airflow obstruction between Hispanic and non-Hispanic white female smokers 
COPD  2008;5(5):274-281.
Rationale
Smoking-related respiratory diseases are a major cause of morbidity and mortality. However, the relationship between smoking and respiratory disease has not been well-studied among ethnic minorities in general and among women in particular.
Objective
The objective of this cross-sectional study was to evaluate the risk of airflow obstruction and to assess lung function among Hispanic and non-Hispanic white (NHW) female smokers in a New Mexico cohort.
Methods
Participants completed a questionnaire detailing smoking history and underwent spirometry testing. Outcomes studied included airflow obstruction, selected lung function parameters, and chronic mucus hyper-secretion. Chi square, logistic, and linear regression techniques were utilized.
Main findings
Of the 1,433 eligible women participants, 248 (17.3%) were Hispanic; and 319 had airflow obstruction (22.3%). Hispanic smokers were more likely to be current smokers, and report lower pack-years of smoking, compared to NHW smokers (p < 0.05 for all analyses). Further, Hispanic smokers were at a reduced risk of airflow obstruction compared to NHW smokers, with an O.R. of 0.51, 95% C.I. 0.34, 0.78 (p = 0.002) after adjustment for age, BMI, pack-years and duration of smoking, and current smoking status. Following adjustment for covariates, Hispanic smokers also had a higher mean absolute and percent predicted post-bronchodilator FEV1/FVC ratio, as well as higher mean percent predicted FEV1 (p < 0.05 for all analyses).
Principal conclusions
Hispanic female smokers in this New Mexico-based cohort had lower risk of airflow obstruction and better lung function than NHW female smokers. Further, smoking history did not completely explain these associations.
doi:10.1080/15412550802363345
PMCID: PMC3616889  PMID: 18972275
Hispanic ethnicity; Smokers; Airflow obstruction; Pulmonary function; Chronic mucus hyper-secretion; Women
14.  Genetic Determinants for Promoter Hypermethylation in the Lungs of Smokers: A Candidate Gene-Based Study 
Cancer Research  2011;72(3):707-715.
The detection of tumor suppressor gene promoter methylation in sputum-derived exfoliated cells predicts early lung cancer. Here we identified genetic determinants for this epigenetic process and examined their biological effects on gene regulation. A two-stage approach involving discovery and replication was employed to assess the association between promoter hypermethylation of a 12-gene panel and common variation in 40 genes involved in carcinogen metabolism, regulation of methylation, and DNA damage response in members of the Lovelace Smokers Cohort (n=1434). Molecular validation of three identified variants was conducted using primary bronchial epithelial cells. Association of study-wide significance (P<8.2×10−5) was identified for rs1641511, rs3730859, and rs1883264 in TP53, LIG1, and BIK, respectively. These SNPs were significantly associated with altered expression of the corresponding genes in primary bronchial epithelial cells. In addition, rs3730859 in LIG1 was also moderately associated with increased risk for lung cancer among Caucasian smokers. Together, our findings suggest that genetic variation in DNA replication and apoptosis pathways impacts the propensity for gene promoter hypermethylation in the aerodigestive tract of smokers. The incorporation of genetic biomarkers for gene promoter hypermethylation with clinical and somatic markers may improve risk assessment models for lung cancer.
doi:10.1158/0008-5472.CAN-11-3194
PMCID: PMC3271143  PMID: 22139380
DNA damage response; promoter hypermethylation; single nucleotide polymorphism; sputum; smoker
15.  The A/G Allele of Rs16906252 Predicts for MGMT Methylation and Is Selectively Silenced in Premalignant Lesions from Smokers and in Lung Adenocarcinomas 
Purpose
To address the association between sequence variants within the MGMT promoter-enhancer region and methylation of MGMT in premalignant lesions from smokers and lung adenocarcinomas, their biological effects on gene regulation, and targeting MGMT for therapy.
Experimental Design
SNPs identified through sequencing a 1.9kb fragment 5' of MGMT were examined in relation to MGMT methylation in 169 lung adenocarcinomas and 1731 sputum samples from smokers. The effect of promoter haplotypes on MGMT expression was tested using a luciferase reporter assay and cDNA expression analysis along with allele-specific sequencing for methylation. The response of MGMT methylated lung cancer cell lines to the alkylating agent temozolomide was assessed.
Results
The A allele of rs16906252 and the haplotype containing this SNP were strongly associated with increased risk for MGMT methylation in adenocarcinomas (ORs ≥ 94). This association was observed to a lesser extent in sputum samples in both smoker cohorts. The A allele was selectively methylated in primary lung tumors and cell lines heterozygous for rs16906252. With the most common haplotype as the reference, a 20–41% reduction in promoter activity was seen for the haplotype carrying the A allele that correlated with lower MGMT expression. The sensitivity of lung cancer cell lines to temozolamide was strongly correlated with levels of MGMT methylation and expression.
Conclusions
These studies provide strong evidence that the A allele of a MGMT promoter-enhancer SNP is a key determinant for MGMT methylation in lung carcinogenesis. Moreover, temozolamide treatment may benefit a subset of lung cancer patients methylated for MGMT.
doi:10.1158/1078-0432.CCR-10-3026
PMCID: PMC3070839  PMID: 21355081
MGMT; allele specific methylation; single nucleotide polymorphism; sputum; lung cancer
16.  Combination Therapy with Vidaza and Entinostat Suppresses Tumor Growth and Reprograms the Epigenome in an Orthotopic Lung Cancer Model 
Cancer research  2011;71(2):454-462.
Epigenetic therapy for solid tumors could benefit from an in vivo model that defines tumor characteristics of responsiveness and resistance to facilitate patient selection. Here we report that combining the histone deacetylase inhibitor entinostat with the demethylating agent vidaza profoundly affected growth of K-ras/p53 mutant lung adenocarcinomas engrafted orthotopically in immunocompromised nude rats by targeting and ablating pleomorphic cells that occupied up to 75% of the tumor masses. A similar reduction in tumor burden was seen with epigenetic therapy in K-ras or EGFR mutant tumors growing orthotopically. Increased expression of pro-apoptotic genes and the cyclin dependent kinase inhibitor p21 was seen. Hundreds of genes were demethylated highlighted by the re-expression of polycomb-regulated genes coding for transcription factor binding proteins and the p16 gene, a key regulator of the cell cycle. Highly significant gene expression changes were seen in key regulatory pathways involved in cell cycle, DNA damage, apoptosis, and tissue remodeling. These findings demonstrate the promise for epigenetic therapy in cancer management and provide an orthotopic lung cancer model that can assess therapeutic efficacy and reprogramming of the epigenome in tumors harboring different genetic and epigenetic profiles to guide use of these drugs.
doi:10.1158/0008-5472.CAN-10-3184
PMCID: PMC3075424  PMID: 21224363
lung cancer; DNA methylation; polycomb; epigenetic therapy; nude rat
17.  Wood Smoke Exposure and Gene Promoter Methylation Are Associated with Increased Risk for COPD in Smokers 
Rationale: Wood smoke–associated chronic obstructive pulmonary disease (COPD) is common in women in developing countries but has not been adequately described in developed countries.
Objectives: Our objective was to determine whether wood smoke exposure was a risk factor for COPD in a population of smokers in the United States and whether aberrant gene promoter methylation in sputum may modify this association.
Methods: For this cross-sectional study, 1,827 subjects were drawn from the Lovelace Smokers' Cohort, a predominantly female cohort of smokers. Wood smoke exposure was self-reported. Postbronchodilator spirometry was obtained, and COPD outcomes studied included percent predicted FEV1, airflow obstruction, and chronic bronchitis. Effect modification of wood smoke exposure with current cigarette smoke, ethnicity, sex, and promoter methylation of lung cancer-related genes in sputum on COPD outcomes were separately explored. Multivariable logistic and poisson regression models were used for binary and rate-based outcomes, respectively.
Measurements and Main Results: Self-reported wood smoke exposure was independently associated with a lower percent predicted FEV1 (point estimate [± SE] −0.03 ± 0.01) and a higher prevalence of airflow obstruction and chronic bronchitis (odds ratio, 1.96; 95% confidence interval, 1.52–2.52 and 1.64 (95% confidence interval, 1.31–2.06, respectively). These associations were stronger among current cigarette smokers, non-Hispanic whites, and men. Wood smoke exposure interacted in a multiplicative manner with aberrant promoter methylation of the p16 or GATA4 genes on lower percent predicted FEV1.
Conclusions: These studies identify a novel link between wood smoke exposure and gene promoter methylation that synergistically increases the risk for reduced lung function in cigarette smokers.
doi:10.1164/rccm.201002-0222OC
PMCID: PMC3001253  PMID: 20595226
wood smoke; cigarette smokers; airflow obstruction; gene promoter methylation in sputum DNA
18.  Multi-Vitamins, Folate, and Green Vegetables Protect Against Gene Promoter Methylation in the Aerodigestive Tract of Smokers 
Cancer research  2010;70(2):568-574.
The detection of gene promoter hypermethylation in sputum is a promising molecular marker for early lung cancer detection. Epidemiologic studies suggest that dietary fruits and vegetables and the micronutrients they contain may reduce risk of lung cancer. This investigation evaluated whether diet and multi-vitamin use influence the prevalence for gene methylation in the cells exfoliated from the aerodigestive tract of current and former smokers. Members (n = 1101) of the Lovelace Smokers Cohort completed the Harvard Food Frequency Questionnaire and provided a sputum sample that was assessed for promoter methylation of eight genes commonly silenced in lung cancer and associated with risk for this disease. Methylation status was categorized as low (< 2 genes methylated) or high (≥2 genes methylated). Logistic regression models were used to identify associations between methylation status and 21 dietary variables hypothesized to affect the acquisition of gene methylation. Significant protection against methylation was observed for leafy green vegetables (OR = 0.83 per 12 monthly servings, CI: 0.74, 0.93) and folate (OR = 0.84 per 750 mcg/day, CI: 0.72, 0.99). Protection against gene methylation was also seen with current use of multi-vitamins (OR = 0.57, CI: 0.40, 0.83). This is the first cohort-based study to identify dietary factors associated with reduced promoter methylation in cells exfoliated from the airway epithelium of smokers. Novel interventions to prevent lung cancer should be developed based on the ability of diet and dietary supplements to affect reprogramming of the epigenome.
doi:10.1158/0008-5472.CAN-09-3410
PMCID: PMC3076796  PMID: 20068159
gene methylation; folate; multi-vitamins; green vegetables; smokers
19.  Discovery of common SNPs in the miR-205/200 family-regulated epithelial to mesenchymal transition pathway and their association with risk for non-small cell lung cancer 
The activation of the epithelial-to-mesenchymal transition (EMT) program is an important step for tumor initiation, invasion, and metastasis in solid tumors, including lung cancer. The purpose of this study was to identify the sequence variants in the miR-205/200 family-regulated EMT pathway and test their association with risk for lung cancer. Fifty samples were resequenced to identify sequence variants in the miR-205/200 family-regulated EMT pathway. The association between tagSNPs and risk for non-small cell lung cancer was discovered and validated in New Mexico (386 cases and 514 controls) and Massachusetts (2453 cases and 1555 controls) case-control studies, respectively. The function of SNPs on miR-200b-a-429 promoter activity was tested using luciferase reporter and expression assays. Forty-one sequence variants with minor allele frequency ≥ 0.03 were identified, and 16 variants were selected as tagSNPs. Genetic association analysis identified that the G allele of rs61768479 was associated with a 50% reduced risk for lung cancer (OR=0.50, 95%CI=0.30-0.85, uncorr-P=0.01); however, this association was not validated (OR=0.90, 95%CI=0.72-1.13, uncorr-P=0.35). The G allele of rs61768479 was associated with lower promoter activity and miR expression by disrupting the binding of NKX2.5. In summary, no association was identified between sequence variants in the miR-205/200 family-regulated EMT pathway and risk for lung cancer. However, this study identified a comprehensive panel of tagSNPs (n=16) in the miR-205/200 family-regulated EMT pathway that can be applied to other EMT-related phenotypes such as cancer chemoresistence and prognosis.
PMCID: PMC3110389  PMID: 21686129
miR-200 family; miR-205; sequence variant; risk; lung cancer
20.  Haplotypes of DNMT1 and DNMT3B are associated with mutagen sensitivity induced by benzo[a]pyrene diol epoxide among smokers 
Carcinogenesis  2008;29(7):1380-1385.
The mutagen sensitivity assay is an in vitro measure of DNA repair capacity used to evaluate intrinsic susceptibility for cancer. The high heritability of mutagen sensitivity to different mutagens validates the use of this phenotype to predict cancer susceptibility. However, genetic determinants of mutagen sensitivity have not been fully characterized. Recently, several studies found that three major cytosine DNA methyltransferases (DNMTs), especially DNMT1, have a direct role in the DNA damage response, independent of their methyltransferase activity. This study evaluated the hypothesis that sequence variants in DNMT1, DNMT3A and DNMT3B are associated with mutagen sensitivity induced by the tobacco carcinogen benzo[a]pyrene diol epoxide (BPDE) in 278 cancer-free smokers. Single-nucleotide polymorphisms (n = 134) dispersed over the entire gene and regulatory regions of these DNMTs were genotyped by the Illumina Golden Gate Assay. DNA sequence variation in the DNMT1 and DNMT3B loci was globally associated with breaks per cell (P < 0.04 for both). No global association between DNMT3A and breaks per cell was seen (P = 0.09). Two haplotypes in block1 of DNMT1 (H284) and 3B (H70) were associated with 16 and 24% increase in breaks per cell, respectively. Subjects with three or four adverse haplotypes of both DNMT1 and 3B had a 50% elevation in mean level of breaks per cell compared with persons without adverse alleles (P = 0.004). The association between sequence variants of DNMT1 and 3B and mutagen sensitivity induced by BPDE supports the involvement of these DNMTs in protecting the cell from DNA damage.
doi:10.1093/carcin/bgn121
PMCID: PMC2899849  PMID: 18499700
21.  Double-strand break damage and associated DNA repair genes predispose smokers to gene methylation 
Cancer research  2008;68(8):3049-3056.
Gene promoter hypermethylation in sputum is a promising biomarker for predicting lung cancer. Identifying factors that predispose smokers to methylation of multiple gene promoters in the lung could impact strategies for early detection and chemoprevention. This study evaluated the hypothesis that double-strand break repair capacity and sequence variation in genes in this pathway are associated with a high methylation index in a cohort of current and former cancer-free smokers. A 50% reduction in the mean level of double-strand break repair capacity was seen in lymphocytes from smokers with a high methylation index, defined as ≥ 3 of 8 genes methylated in sputum, compared to smokers with no genes methylated. The classification accuracy for predicting risk for methylation was 88%. Single nucleotide polymorphisms within the MRE11A, CHEK2, XRCC3, DNA-Pkc, and NBN DNA repair genes were highly associated with the methylation index. A 14.5-fold increased odds for high methylation was seen for persons with ≥ 7 risk alleles of these genes. Promoter activity of the MRE11A gene that plays a critical role in recognition of DNA damage and activation of ATM was reduced in persons with the risk allele. Collectively, ours is the first population-based study to identify double-strand break DNA repair capacity and specific genes within this pathway as critical determinants for gene methylation in sputum, that is, in turn, associated with elevated risk for lung cancer.
doi:10.1158/0008-5472.CAN-07-6344
PMCID: PMC2483467  PMID: 18413776
promoter methylation; DNA double strand break; single nucleotide polymorphism; DNA repair capacity; association study
22.  Genotypes in matrix metalloproteinase 9 are a risk factor for COPD 
A growing body of evidence indicates that matrix metalloproteinases (MMPs) play a role in the pathogenesis of COPD. Therefore, we conducted a candidate gene association study of 4 promoter polymorphisms that are known to modify expression levels of the MMP-1, MMP-2, and MMP-9 genes and a Gln279Arg polymorphism in exon 6 of MMP-9 that modifies the substrate-binding region. We examined the association of each variant and haplotypes in 385 male veterans with greater than 20 pack-years of cigarette smoking whose COPD status was characterized using spirometry. The association of these polymorphisms was also examined with decline of pulmonary function in a subset of participants. Only the 279Arg variant was more common in participants with COPD and the homozygous variant was associated with a 3-fold increased risk for COPD. In the haplotype analysis, the haplotype comprising the 249Arg and the CA promoter polymorphism within the MMP-9 gene was associated with risk, suggesting that either 279Arg or a linked variant on this haplotype underlies the association. No association of this polymorphism was found with decline in pulmonary function. These studies show that variants of the MMP-9 gene are associated with COPD in this cohort of veterans.
PMCID: PMC2707156  PMID: 18046864
metalloproteinase; smoking; pulmonary function; single nucleotide polymorphism; molecular epidemiology

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