Overexpression of hMTH1 mRNA: a molecular marker of oxidative stress in lung cancer cells

AbstractHuman MutT homologue (hMTH1) mRNA was overexpressed in SV-40-transformed non-tumorigenic human bronchial epithelial cells (BEAS-2B cells) and in 11 out of 12 human lung cancer cell lines relative to normal human bronchial epithelial cells. Expression levels of hMTH1 mRNA were inversely proportional to cellular levels of 8-oxo-deoxyguanosine. Together, these results suggest that hMTH1 gene expression may represent a molecular marker of oxidative stress that could ultimately be used to elucidate the temporal relationships between oxidative stress, genomic instability and the development of lung cancer

LOW-DOSE GAMMA-RADIATION INHIBITS BENZO[A]PYRENE-INDUCED LUNG ADENOMA DEVELOPMENT IN A/J MICE

Low-dose ionizing radiation (LDR) may lead to suppression of smoking-related lung cancer. We examined the effects of a known cigarette smoke carcinogen Benzo[a]pyrene (B[a]P) alone or in combination with fractionated low-dose gamma radiation (60 – 600 mGy total dose) on the induction of lung neoplasms in the A/J mouse. Our results show that 600 mGy of gamma radiation delivered in six biweekly fractions of 100 mGy starting 1 month after B[a]P injection significantly inhibits the development of lung adenomas per animal induced by B[a]P. Our data also indicated that the six biweekly doses suppressed the occurrence of spontaneous hyperplastic foci in the lung, although this suppression failed to reach statistical significance when analyzed as average foci per lung possibly related to the small sample sizes used for the control and test groups

Increased methylation of lung cancer-associated genes in sputum DNA of former smokers with chronic mucous hypersecretion

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

Differential Epigenetic Regulation of TOX Subfamily High Mobility Group Box Genes in Lung and Breast Cancers

Aberrant cytosine methylation affects regulation of hundreds of genes during cancer development. In this study, a novel aberrantly hypermethylated CpG island in cancer was discovered within the TOX2 promoter. TOX2 was unmethylated in normal cells but 28% lung (n = 190) and 23% breast (n = 80) tumors were methylated. Expression of two novel TOX2 transcripts identified was significantly reduced in primary lung tumors than distant normal lung (p<0.05). These transcripts were silenced in methylated lung and breast cancer cells and 5-Aza-2-deoxycytidine treatment re-expressed both. Extension of these assays to TOX, TOX3, and TOX4 genes that share similar genomic structure and protein homology with TOX2 revealed distinct methylation profiles by smoking status, histology, and cancer type. TOX was almost exclusively methylated in breast (43%) than lung (5%) cancer, whereas TOX3 was frequently methylated in lung (58%) than breast (30%) tumors. TOX4 was unmethylated in all samples and showed the highest expression in normal lung. Compared to TOX4, expression of TOX, TOX2 and TOX3 in normal lung was 25, 44, and 88% lower, respectively, supporting the premise that reduced promoter activity confers increased susceptibility to methylation during lung carcinogenesis. Genome-wide assays revealed that siRNA-mediated TOX2 knockdown modulated multiple pathways while TOX3 inactivation targeted neuronal development and function. Although these knockdowns did not result in further phenotypic changes of lung cancer cells in vitro, the impact on tissue remodeling, inflammatory response, and cell differentiation pathways suggest a potential role for TOX2 in modulating tumor microenvironment

Gene methylation biomarkers in sputum as a classifier for lung cancer risk

CT screening for lung cancer reduces mortality, but will cost Medicare ∼2 billion dollars due in part to high false positive rates. Molecular biomarkers could augment current risk stratification used to select smokers for screening. Gene methylation in sputum reflects lung field cancerization that remains in lung cancer patients post-resection. This population was used in conjunction with cancer-free smokers to evaluate classification accuracy of a validated eight-gene methylation panel in sputum for cancer risk. Sputum from resected lung cancer patients (n=487) and smokers from Lovelace (n=1380) and PLuSS (n=718) cohorts was studied for methylation of an 8-gene panel. Area under a receiver operating characteristic curve was calculated to assess the prediction performance in logistic regressions with different sets of variables. The prevalence for methylation of all genes was significantly increased in the ECOG-ACRIN patients compared to cancer-free smokers as evident by elevated odds ratios that ranged from 1.6 to 8.9. The gene methylation panel showed lung cancer prediction accuracy of 82–86% and with addition of clinical variables improved to 87–90%. With sensitivity at 95%, specificity increased from 25% to 54% comparing clinical variables alone to their inclusion with methylation. The addition of methylation biomarkers to clinical variables would reduce false positive screens by ruling out one-third of smokers eligible for CT screening and could increase cancer detection rates through expanding risk assessment criteria

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

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

MUC1 Contributes to BPDE-Induced Human Bronchial Epithelial Cell Transformation through Facilitating EGFR Activation

Although it is well known that epidermal growth factor receptor (EGFR) is involved in lung cancer progression, whether EGFR contributes to lung epithelial cell transformation is less clear. Mucin 1 (MUC1 in human and Muc1 in animals), a glycoprotein component of airway mucus, is overexpressed in lung tumors; however, its role and underlying mechanisms in early stage lung carcinogenesis is still elusive. This study provides strong evidence demonstrating that EGFR and MUC1 are involved in bronchial epithelial cell transformation. Knockdown of MUC1 expression significantly reduced transformation of immortalized human bronchial epithelial cells induced by benzo[a]pyrene diol epoxide (BPDE), the active form of the cigarette smoke (CS) carcinogen benzo(a)pyrene (BaP)s. BPDE exposure robustly activated a pathway consisting of EGFR, Akt and ERK, and blocking this pathway significantly increased BPDE-induced cell death and inhibited cell transformation. Suppression of MUC1 expression resulted in EGFR destabilization and inhibition of the BPDE-induced activation of Akt and ERK and increase of cytotoxicity. These results strongly suggest an important role for EGFR in BPDE-induced transformation, and substantiate that MUC1 is involved in lung cancer development, at least partly through mediating carcinogen-induced activation of the EGFR-mediated cell survival pathway that facilitates cell transformation

Genetic Associations and Architecture of Asthma-COPD Overlap

BACKGROUND: Some people have characteristics of both asthma and COPD (asthma-COPD overlap), and evidence suggests they experience worse outcomes than those with either condition alone. RESEARCH QUESTION: What is the genetic architecture of asthma-COPD overlap, and do the determinants of risk for asthma-COPD overlap differ from those for COPD or asthma? STUDY DESIGN AND METHODS: We conducted a genome-wide association study in 8,068 asthma-COPD overlap case subjects and 40,360 control subjects without asthma or COPD of European ancestry in UK Biobank (stage 1). We followed up promising signals (P < 5 x 10(-6)) that remained associated in analyses comparing (1) asthma-COPD overlap vs asthma-only control subjects, and (2) asthma-COPD overlap vs COPD-only control subjects. These variants were analyzed in 12 independent cohorts (stage 2). RESULTS: We selected 31 independent variants for further investigation in stage 2, and discovered eight novel signals (P < 5 x 10(-8)) for asthma-COPD overlap (meta-analysis of stage 1 and 2 studies). These signals suggest a spectrum of shared genetic influences, some predominantly influencing asthma (FAM105A, GLB1, PHB, TSLP), others predominantly influencing fixed airflow obstruction (IL17RD, C5orf56, HLA-DQB1). One intergenic signal on chromosome 5 had not been previously associated with asthma, COPD, or lung function. Subgroup analyses suggested that associations at these eight signals were not driven by smoking or age at asthma diagnosis, and in phenome-wide scans, eosinophil counts, atopy, and asthma traits were prominent. INTERPRETATION: We identified eight signals for asthma-COPD overlap, which may represent loci that predispose to type 2 inflammation, and serious long-term consequences of asthma.Peer reviewe