ABERRANT DNA METHYLATION IN HUMAN NON-SMALL CELL LUNG CANCER DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Romulo Martin Brena, B.A. ***** The Ohio State University 2007 Dissertation Committee: Approved by: Dr. Christoph Plass, Adviser Dr. Thomas J. Rosol ____________________________________ Dr. Michael C. Ostrowski Adviser Graduate Program in Molecular Genetics Dr. Gregory A. Otterson Dr. Laura J. Rush ABSTRACT Lung cancer is the leading cause of cancer-related death worldwide. Given its impact on human health, extensive research is being conducted in an effort to reduce the global lung cancer death toll. Specifically, much interest has been placed on the development of biomarkers and the discovery of novel prognostic factors. Over the past 2 decades it has become evident that the cancer genome is not only affected by genetic abnormalities, such as mutations, deletions and chromosomal rearrangements, but also by epigenetic changes which, together, contribute to the deregulation of transcription profiles. Epigenetic changes are defined as heritable lesions to the DNA affecting gene expression without altering the primary DNA sequence. These lesions typically involve a genomewide reduction in 5-methylcytosine, increased DNA methylation in gene promoter sequences and substitutions in histone tail modifications. Epigenetic changes have been shown to interact with one another, resulting in genomic instability, silencing of tumor suppressor genes, activation of oncogenes and derepression of transposable elements. As opposed to the irreversible nature of genetic lesions, epigenetic lesions can be reversed. Because of their reversibility, epigenetic alterations have become an attractive target for new therapies, which has resulted in the development of new anticancer compounds, several of which are currently in clinical trials. ii The field of epigenetics has expanded considerably since its inception. Integrative research approaches aimed at elucidating the contribution of genetic and epigenetic alterations to the tumorigenic process are being undertaken worldwide, generating promising results. In hopes of furthering the body of knowledge currently afforded by the epigenetics field, the work presented in this thesis is focus primarily on unraveling the role of DNA methylation in the diagnosis, etiology and potential treatment of lung cancer. Early detection would play a major role in reducing lung cancer-related death. Since standardized early detection methods for lung cancer are currently lacking, extensive efforts have been devoted in the lung cancer field to the identification of molecular markers which might be useful for disease detection or which might afford improvement in prognostic parameters. In recent months, several studies have reported important advances in these two areas. Lu and colleagues were able to identify a gene expression signature that helps predict survival of patients with stage I non-small cell lung cancer. Following a similar investigative approach, Chen and colleagues reported a 5 gene expression signature that correlates with clinical outcome in non-small cell lung cancer patients, regardless of stage. Other investigators, such as Guo et al and Raponi et al have focus primarily on specific lung cancer subtypes and have reported molecular classifiers that help refine the clinical prognosis of adenocarcinomas and squamous cell carcinomas, respectively. Other prominent investigators in the lung cancer field have focused their efforts on the discovery of markers which could be utilized for early detection. A landmark study on this line of research has recently been published by Shames and colleagues. These investigators reported a number of molecular markers that could help detect not only lung cancer, but also other types of common neoplasias, such as those of the breast and colon. The importance of this study resides in that it is iii one of the few research endeavors geared towards the discovery of pan-cancer markers. Furthermore, Shames and colleagues decided to focus on aberrant DNA methylation as a marker for neoplasias, an emerging investigative approach of great potential that still remains to be fully explored. Following this line of investigation, we hypothesize that epigenetic abnormalities, in particular aberrant DNA methylation, is involved in the etiology of lung cancer. Furthermore, we hypothesized that different non-small cell lung cancer subtypes can be distinguished by their aberrant DNA methylation profiles. We tested our hypotheses by analyzing human primary lung tumors via Restriction Landmark Genomic Scanning (RLGS) in an effort to identify not only novel DNA methylation targets, but also targets specific to lung cancer. In Chapter 1, we introduce DNA methylation as biological process intimately involved in the regulation of key cellular functions, from early development to adulthood. We continue to expand on how DNA methylation is involved in human cancer and particularly discuss the identification of key genes silenced by DNA methylation in lung cancer. We conclude the chapter by discussing how DNA methylation could be used as a biomarker and a prognostic marker in lung cancer. Chapter 2 provides a synopsis of the techniques currently available for the assessment of DNA methylation, coupled with a discussion on the suitability of each of these techniques for their application in disease diagnosis and classification. We put special emphasis on addressing the applicability of these techniques in clinical settings, in an effort to highlight the current disconnect between bench work achievements and their translation into novel approaches which could directly impact patient lives. Chapter 3 describes a novel technique, Bio-COBRA, for the quantitative analysis of DNA methylation. Specifically, we discuss how the determination of DNA methylation iv levels in a quantitative and reproducible manner could aid in increasing the efficacy of treatments involving DNA demethylating agents. In Chapter 4, we describe a genomewide DNA methylation scan of primary human lung adenocarcinomas and squamous cell carcinomas. Our scan revealed that these two lung cancer subtypes can in fact be distinguished based on their aberrant DNA methylation profiles. Also, we report the discovery a novel prognostic factor, oligodendrocyte transcription factor 1 (OLIG1), whose expression at the protein level was strongly correlated with survival in patients suffering from non-small cell lung cancer. Chapter 5 presents a survey of lung-specific DNA methylation events, as determined by comparing NotI RLGS profiles from human cancers derived from 12 different organs. Our survey showed that lung cancer is the neoplasia with the highest number of tumor-specific aberrant DNA methylation events. We performed extensive mRNA expression and DNA methylation analyses in an effort to provide a comprehensive report of the genes most frequently silenced by lung-specific DNA methylation. Finally, we conclude with Chapter 6, where we discuss the future steps that need to be taken in order to further the current understanding of how aberrant DNA methylation impacts the etiology of lung cancer. v Dedicated to my family and to all those whose constant support has helped me made a small contribution towards the advancement of science vi ACKNOWLEDGMENTS The past 6 years have been a memorable journey. This journey, like many others, seemed, at times, of uncertain destination. However, now that I find myself at the end of its road, I can clearly see the destination had always been there. Knowing this about the journey might enable me to impart some words of wisdom to future travelers, to let them know that although it may seem unattainable at times, they will also get to the journey’s end. Past travelers did impart those same words to me, but they did not resonate as loudly as they would today. Nevertheless, their intention to pass on their experience and encouragement was unmistakable, for which I will always be grateful. Many people have helped me get to the point where I can finally be writing these words. There are so many in fact that, I will certainly forget to mention some of them, for which I apologize in advance. First, I would like to thank Dr. Christoph Plass, whose incredible patience has made me realize that the best attribute of a great mentor is being able to make his students feel they are always welcome and that no question or concern is too trite for his attention. Dr. Thomas Rosol and Dr. Laura Rush have been instrumental in my learning to look at scientific problems not just from a molecular standpoint, but as a puzzle that affects an entire being, whether it be human or animal, and that the answer to solving the puzzle most likely entails thinking about that being as a whole. I would also like to thank Dr. Rush for her constant support and for saying the right words at the right time to help me stay on track. vii A very special thank you goes to Dr. Sandya Liyanarachchi, who not only played a central role in the statistical analysis of the data for almost all my research projects, but who also permanently took time out of her busy schedule to teach me about the theoretical background involved in her work. Dr. Liyanarachchi’s involvement in my graduate career has been fundamental in my training to be able to critically assess the validity of statistical analyses presented in biomedical research publications.