Epigenetic Mechanisms in Lung Cancer Development

Abstract

Epigenetic control of gene expression involves covalent reversible modifications of DNA, RNA, and histones which lead to changes in chromatin structure and accessibility. This ability to maintain precise control over gene expression in cells and tissues is critical for cellular development and differentiation, as well as for normal cellular processes. The most important epigenetic mark in DNA is cytosine methylation, an epigenetic mark created by the addition of a methyl (CH3) group to the C5 position of cytosine. 5-methylcytosines (mC) are stable epigenetic marks introduced by DNA methyltransferases (DNMTs). 5-methylcytosines can be iteratively oxidized by ten-eleven translocation (TET) dioxygenases to form 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. Epigenetic deregulation is increasingly recognized as a critical event in the development of cancer and many other human diseases. 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 also covers the role that epigenetic enzymes such as DNMTs and TETs play in human disease. We next describe how global levels of epigenetic DNA modifications can be quantified using antibodies and mass spectrometry-based approaches. We then discuss methods available to site-specifically map 5-methylcytosine and 5-hydroxymethylcytosine along the genome. Finally, Chapter I covers the role of epigenetic dysregulation in cancer and other human diseases; specifically, we focus on the roles epigenetics and inflammation play in the development of lung cancer. Chapter II of this thesis examines how oxidized and extended forms of mC influence the activity of maintenance DNA methyltransferase (DNMT1). DNMT1 specifically recognizes mC bases at hemimethylated CG sites in DNA and conducts maintenance methylation. Maintenance methyltransferase activity was the highest towards DNA containing the natural DNMT1 substrate, mC. 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. We also found that methyl transfer rates were reduced when mC was oxidized to hmC, fC, and caC, consistent with the model that TET-mediated oxidation contributes to passive DNA demethylation. We employed molecular modeling simulations of the enzyme with modified DNA to understand the interactions of oxidized and extended forms of mC with the DNMT1 enzyme. We found that modified forms of mC cause disruptions in its interactions with the target recognition domain (TRD) of DNMT1, causing the enzyme to adopt an unproductive mode of binding. In Chapter III, we investigated inflammation-mediated epigenetic changes in type II alveolar epithelial cells (AECII) using A/J mouse model of smoking-induced lung cancer. We characterized changes in methylation, hydroxymethylation, and gene expression in the AECII of A/J mice after exposure to LPS or cigarette smoke. We found that exposure to both cigarette smoke and LPS leads to significant changes in the epigenome of murine AECII. These results provide evidence that epigenetic deregulation, along with genetic changes, likely contributes to the development of smoking-induced lung cancer. The final chapter of this thesis (Chapter IV) aimed to identify potent and specific small molecule inhibitors of TET proteins. TET proteins play a central role in epigenetic regulation by removing repressive DNA methylation marks and thus allowing for gene expression; however, TET expression and activity are deregulated in many diseases including cancer. Specific inhibitors of TET proteins are needed to elucidate their roles in human disease. We utilized several different techniques in order to identify small molecule inhibitors of TET enzymes including virtual screening, high-throughput screening approaches, and structure-based design. Overall, during the course of the studies described in this thesis, we have investigated how oxidized forms of mC alter DNMT1 activity and affinity for DNA as well as provided a structural explanation for how these modifications can participate in passive demethylation. We have characterized epigenetic changes associated with inflammation and cigarette smoke exposure in order to better understand the role that epigenetic deregulation plays in smoking-induced lung cancer. Finally, we sought to identify specific inhibitors of TET proteins as a possible therapeutic target for treatment of cancer and other human diseases. Overall, this work contributes to current understanding of epigenetic regulation in normal cells and epigenetic deregulation in cancer.