Browsing by Subject "lung cancer"

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    Epigenetic Mechanisms in Lung Cancer
    (2018-09) Seiler, Christopher
    Epigenetic control of gene expression involves covalent reversible modifications of DNA, RNA, and histones which lead to changes in chromatin structure and accessibility. The ability to maintain precise control over gene expression in cells and tissues is critical for ensuring normal cellular development and homeostasis. The most important epigenetic mark of DNA is methylation of cytosine at the C5 position (MeC). This stable epigenetic mark is introduced by de novo methyltransferases DNMT3a/b and maintained through cell division by maintenance methyltransferase DNMT1. Ten Eleven Translocation (TET) dioxygenases oxidize 5-methylcytosine (MeC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC), a process known to induce DNA demethylation and gene reactivation. A precise balance of DNA methylation and demethylation is important for establishing tissue specific gene expression patterns, maintaining cell identity, and guiding development. However, inflammation and exposure to exogenous agents can lead to changes in DNA methylation patterns or “epimutations” which together with genetic mutations can lead to the development of cancer. Chapter I of this Thesis provides an overview of the major mechanisms of epigenetic regulation including epigenetic marks of DNA, non-coding RNAs, and histone modifications. Chapter I then describes epigenetic dysregulation in cancer and other human diseases. We then go on to describe how epigenetic changes in DNA can be detected and quantified using antibodies and mass spectrometry-based approaches. After considering global quantitation of epigenetic DNA modifications, we discuss the methods available for mapping epigenetic modifications along the genome. In Chapter II of this thesis, the effects of C5-cytosine substituents with increased steric bulk on catalytic activity of maintenance DNA methyltransferase (DNMT1) were examined. This protein specifically recognizes 5-methylcytosine (MeC) bases at hemimethylated CG sites in DNA and conducts maintenance methylation. Maintenance methyltransferase activity was the highest towards DNA containing the natural DNMMT1 substrate, MeC. The enzyme was capable of performing maintenance methylation when 5-ethyl-dC was the substrate, while the more rigid and bulky C5-alkyl substituents such as 5-vinyl- dC, and 5-propyl-dC could not direct maintenance methylation. In Chapter III, we investigated the kinetics of maintenance DNA methylation towards DNA duplexes containing oxidized forms of MeC (hmC, fC, and caC). We also employed a molecular dynamics simulation of the enzyme with the DNA to understand the interactions of oxidized forms of MeC with the DNMT1 enzyme. We found that methyl transfer rates were reduced when MeC was oxidized to hmC, fC, and caC, consistent with the model that Tet mediated oxidation contributes to passive DNA demethylation. In Chapters IV and V, we investigated inflammation-mediated epigenetic changes in the lung using A/J mouse model of smoking induced lung cancer. In collaboration with NuGEN (Santa Carlos, CA), we developed a novel reduced representation bisulfite sequencing (RRBS) methodology to map both MeC and hmC genome-wide. Our studies provide evidence that inflammation of the lung induces both global and site-specific epigenetic changes in DNA methylation and hydroxymethylation, alters global histone acetylation, and deregulates gene expression. These studies also provide evidence that exposure to cigarette smoke can alter site-specific DNA methylation and hydroxymethylation of genes that are associated with the cancer phenotype. The final chapter of this dissertation (Chapter VI) employs affinity proteomics to identify protein readers of epigenetic marks of DNA in the lung. DNA duplexes functionalized with C, MeC, hmC, fC, and caC were attached to solid support and incubated with nuclear protein extracts from human bronchial epithelial cells (HBEC). Proteins specifically recognizing DNA epigenetic marks were identified using Orbitrap Velos mass spectrometer and quantified using 8-plex TMT tags. This chapter details the development of a method for carrying out the affinity proteomics experiments, including solid phase synthesis of DNA targets, peptide tagging, sample clean-up, fractionation, and nanoHPLC-ESI+-MS2 based methodology for protein identification and quantification. Overall, during the course of the studies described in this thesis, we have investigated the specificity and kinetics of human maintenance DNA methyltransferase (DNMT1), employed animal models to characterize epigenetic changes in the lung caused by inflammation and exposure to cigarette smoke, and examined novel mechanisms of epigenetic regulation at oxidized forms of MeC. Overall, this work contributes to current understanding of epigenetic regulation in normal cells and epigenetic deregulation in cancer.
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    Urinary cadmium and lung cancer risk in smokers from the Multiethnic Cohort Study
    (2019-08) Cigan, Shannon
    Abstract Objective: The overall objective of this research is to investigate the effects of smoking, occupational exposures, demographics (e.g. race/ethnicity and education) and common genetic variants on the levels of urinary cadmium (Cd), a validated biomarker of long-term Cd exposure, in current smokers from the Multiethnic Cohort (MEC) Study. In addition, test whether this biomarker in smokers is associated with lung cancer risk. The overall objective of this research was accomplished in three separate dissertation chapters (chapter 2, 3 and 4) and set up as three sequential papers. Specific Aim 1: Aim: To investigate the relationship between self-reported occupation and urinary Cd levels in 1,956 current smokers at time of urine collection from five race/ethnicity groups from the MEC Study with complete covariate data. Methods: Urinary Cd was measured by inductively coupled plasma mass spectrometry (ICP-MS). Internal smoking dose was estimated by the biomarker urinary total nicotine equivalents (TNE). A censored multiple linear regression model (tobit regression) was used to estimate the percent adjusted change in the association between occupational exposure categories and levels of urinary Cd, while adjusting for confounding variables, with estimated 95% confidence intervals (CI) to characterize precision. Occupational exposure categories were based on the combined response to questions regarding the industry and occupation the participant worked the longest. Results: Participants categorized as ‘Likely exposed’ to Cd based on their occupations had 12.1% (95%CI: 1.2%, 24.3%) higher levels of urinary Cd as compared to those ‘Not likely exposed’ to Cd in the workplace, after adjusting for age, sex, race/ethnicity, creatinine (natural-log), education, smoking dose (TNE), and duration (years of smoking). Similarly, participants categorized as ‘Possibly exposed’ to Cd in the workplace had 7.7% (95%CI: 0.1%, 16.0%) higher levels of urinary Cd as compared to workers ‘Not likely exposed’ to Cd in the workplace. Specific Aim 2: Aim: To conduct a genome-wide association study to identify common genetic variants that may be associated with urinary Cd in smokers (N=1,977). In addition, evaluate the association between urinary Cd and single nucleotide polymorphisms (SNPs) previously reported in literature to be associated with Cd and located within genes that have biological plausibility to be associated with Cd absorption, distribution, metabolism and elimination. Methods: Linear regression was used to test the association of each genetic variant with urinary Cd levels, adjusted for age at urine collection, sex, self-reported race/ethnicity, creatinine (natural log), smoking dose (urinary TNE), and the top 10 leading PCs. Allele dosage was used as the explanatory variable of most interest in the analysis. Genome-wide significance was based on a Bonferroni-corrected 5% significant threshold of p < 8.4 x 10-9 (0.05/5,944,091 SNPs for analyses). In addition, a candidate SNP approach was used to identify associations in 1,169 single nucleotide polymorphisms (SNPs) comprised from 15 SNPs reported in literature to be associated with Cd biomarkers and SNPs within 29 candidate genes identified a priori that have biological plausibility to be involved in transport of Cd; Metallothionein (MT) gene family, metal-regulatory factor 1 (MTF1), and Glutathione S-transferase (GST) gene family. Results: No single SNP was associated with urinary Cd in the genome-wide association study. The SNP with the lowest p-value for the association with urinary Cd was rs673456 on chromosome 11, an intron variant close to the TENM4 gene (p=3.47 x 10-7). The candidate SNP analyses also did not reach statistically significance (lowest p-values 3.89 x 10-4). After adjusting for age at urine collection, sex, creatinine (natural log), and total nicotine equivalents, geometric mean levels of urinary Cd were significantly different across the five race/ethnicity groups (p<0.001). Latinos and Native Hawaiians had the highest geometric mean urinary Cd (0.87 and 0.84 ng/mL) followed by Japanese Americans (0.81 ng/mL), African Americans (0.80 ng/mL), and Whites (0.74 ng/mL). Geometric means were also higher in females than males across all race/ethnicity groups. Specific Aim 3: Aim: To investigate the association of urinary Cd and lung cancer risk in a subset of 1,956 MEC current smokers at the time of urine collection. Methods: Cd was analyzed by ICP-MS in urine samples collected between 1997 and 2006. Lung cancer incidence was identified by linkages with the statewide Surveillance, Epidemiology and End Results (SEER) registries of Hawaii and California through December 31, 2016. Cox proportional hazards regression was used to estimate multivariable-adjusted hazard ratios (HR) and 95% confidence intervals (95% CI) for lung cancer. Urinary Cd was modelled as (1) continuous urinary Cd levels (natural log) and (2) quartiles of urinary Cd levels. Results: A total of 89 lung cancer cases were diagnosed in our subset of 1,956 current smokers with a median follow-up time of 12.4 years. Higher urinary Cd was associated with increased lung cancer risk after adjustment for age, sex, race/ethnicity, creatinine (natural log), education, smoking dose (urinary total nicotine equivalents), smoking duration (years of smoking), and occupational Cd exposure (HR: 1.69; 95% CI: 1.26, 2.26). Categorical analysis by quartiles of urinary Cd levels showed the multivariable-adjusted HR for lung cancer increased by increasing quartile of urinary Cd. Relative to the lowest quartile of urinary Cd levels, the adjusted HR for the fourth (highest) quartile was 3.45 (95% CI: 1.73, 6.89), third quartile was 2.02 (95% CI: 1.05, 3.89) and second quartile was 1.21 (95% CI: 0.62, 2.36). Overall Conclusions: Overall, our results indicate that urinary Cd is an important determinant of lung cancer risk in current smokers at the time of urine collection from the MEC Study and future studies investigating lung cancer risk will benefit from these findings. Specific Aim 1 demonstrated that at similar levels of smoking, smokers in occupations that have a potential for Cd exposure had higher levels of urinary Cd. This suggest workers in these types of occupations who also smoke may be at an increased risk for lung cancer and should be targeted for intervention measures. In Specific Aim 2, no common genetic variants were associated with urinary Cd at a genome-wide level. In addition, the candidate SNP analyses also did not demonstrate a statistically significant association with urinary Cd levels. Specific Aim 3 demonstrated that levels of urinary Cd were associated with lung cancer incidence in smokers from the MEC study and this association persisted after adjustment for occupational Cd exposure, smoking dose (TNE), duration (years of smoking) and other potential risk factors. Replication of findings in a larger sample size and/or studies with well-characterized measures of occupational Cd exposure, dietary and environmental exposures are warranted. The findings of this study were consistent with literature, confirm Cd is a risk factor for lung cancer and this relationship can be detected in current smokers at the time of urine collection. Public health efforts to reduce Cd exposure including tobacco cessation programs, reducing the environmental and industrial impact of Cd, and the implementation of educating smokers in occupations that pose a high risk for Cd exposure, may contribute to the reduction of lung cancer in the future.