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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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

Results 1-13 (13)