Health Science Campus FINAL APPROVAL OF DISSERTATION Doctor of Philosophy in Biomedical Science (Cancer Biology) Aneuploidy: Using genetic instability to preserve a haploid genome? Submitted by: Ramona Ramdath In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Science Examination Committee Signature/Date Major Advisor: David Allison, M.D., Ph.D. Academic James Trempe, Ph.D. Advisory Committee: David Giovanucci, Ph.D. Randall Ruch, Ph.D. Ronald Mellgren, Ph.D. Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D. Date of Defense: April 10, 2009 Aneuploidy: Using genetic instability to preserve a haploid genome? Ramona Ramdath University of Toledo, Health Science Campus 2009 Dedication I dedicate this dissertation to my grandfather who died of lung cancer two years ago, but who always instilled in us the value and importance of education. And to my mom and sister, both of whom have been pillars of support and stimulating conversations. To my sister, Rehanna, especially- I hope this inspires you to achieve all that you want to in life, academically and otherwise. ii Acknowledgements As we go through these academic journeys, there are so many along the way that make an impact not only on our work, but on our lives as well, and I would like to say a heartfelt thank you to all of those people: My Committee members- Dr. James Trempe, Dr. David Giovanucchi, Dr. Ronald Mellgren and Dr. Randall Ruch for their guidance, suggestions, support and confidence in me. My major advisor- Dr. David Allison, for his constructive criticism and positive reinforcement. Kay Langenderfer, our secretary, who has always been there to help us in whatever we needed, regardless of the time of day or night, and who always advocated for me. Dr. Andrea Kalinoski, who taught me many of the techniques that I learnt, and who was a helped me understand the projects in the laboratory. Peter Bazeley, without whom this project would be impossible. His knowledge and expertise was invaluable to me, and all the long hours and countless times that he has explained concepts to me was far beyond his call of duty. Dr. Saad Sikhanderkhel who worked tirelessly on getting the breast cancer database up and running. Dr. David Weaver, for his help not only with managing our SNP data so efficiently, but also, for doing our expression arrays. iii Case Western Reserve Gene Expression and Genotyping core facility-Debora Poruban, Tom Merk and Dr. Martina Veigl, for doing all of our SNP arrays, and for working with us to help us achieve our goals. Dr. Seiichi Matsui and Jeffrey LaDuca for doing the SKY imaging for us. Dr. Amira Gohara, who spent many early morning hours patiently going through slides with me to identify cancerous and normal regions. Even with her exceptionally busy schedule, she always accommodated my requests for help, and did it with such pleasure. It was a pleasure working with you Dr. Gohara. University of Michigan, Microscopy and Imaging Centre for the use of their “devil child” Laser Capture Microdissection Microscope. All the friends I have made in the last few years, especially during my time in the Cicila laboratory. Kyle Robinson, my best friend, who always kept me on my toes, asking pertinent questions, and in other cases providing solid answers. He has supported me; been a shoulder to lean on when I was frustrated; been a listening ear when I needed one, or when I wanted to discuss ideas, and been patient with me, no matter what. My family- particularly my sister, and in many ways, most importantly, my mom, who has through the years picked me up when I fell, stayed up late with me working on projects, and been with me throughout all of my endeavors. Never have I known a stronger woman, thank you for being a superb role model. iv Table of Contents Introduction………………………………………………………..1 Literature Review………..………………………………………..5 Materials and Methods…………………………………………...38 Results……………………………………………………………..46 Discussion…………………………………………………………72 Conclusions………………………………………………………..85 Summary…………………………………………………………...86 References…………………………………………………………87 Appendix 1………………………………………………………….97 Appendix 2………………………………………………………….98 Appendix 3………………………………………………………….99 Appendix 4………………………………………………………….100 Appendix 5………………………………………………………….101 Appendix 6………………………………………………………….102 Appendix 7………………………………………………………….106 Appendix 8………………………………………………………….122 Appendix 9………………………………………………………….126 Appendix 10………………………………………………………...127 Abstract……………………………………………………………...128 v Introduction Although lifestyle and environment play a role in carcinogenesis, it is well-known that cancer is a genetic disease, in which the guardians of the genome have been mutated or lost, resulting in uncontrolled cell division and genetic instability, which favor tumor progression. Genetic instability is defined as subtle changes in DNA sequence such as point mutations, that can subsequently result in chromosomal losses, gains, translocations or copy number changes. The changes from genetic instability can, in turn, lead to the loss of genomic information from one homologous chromosome (parent), known as the loss of heterozygosity (Olshen et al.) (Argos et al., 2008; Dutt & Beroukhim, 2007; Zheng, Peng, Li, & He, 2005). Thus, LOH can potentially be used as a gauge of genomic instability. LOH can be measured using single nucleotide polymorphism (SNP) assays, which are common genetic variations of one base pair, widely distributed throughout the human genome. SNPs are widely used as markers for genetic diseases, and can detect LOHs in relatively small regions of the genome. As a result of genetic instability, many tumors are aneuploid and no longer have normal diploid genomes (Zheng et al., 2005). By definition, aneuploidy is a change in chromosome number that is not the exact multiple of the normal haploid karyotype (Torres, Williams, & Amon, 2008). Due to the genetic instability characteristic of aneuploid cancers, these cancers frequently contain structurally abnormal chromosomes such as translocations and deletions. Another subtle type of genetic instability, copy number alterations (CNAs), can lead to genome wide deregulation of gene expression, even in diploid cancers and contribute to the development or progression of several cancers (Pollack et al., 2002). Spectral karyotyping (SKY) is an accurate technique of staining each chromosome to visualize chromosomal aberrations, including CNAs (Schrock et al., 2006). 1 Another commonly employed method to look at genetic instability of chromosomes is to investigate the genome wide alterations of gene expression. Gene expression arrays can be used to explore the expression of genes that are essential for survival and also determine which genes are involved in carcinogenesis and tumor progression (Christensen, McCoy, & Ford, 2008). Our first hypothesis is that aneuploidy occurs in cells as a system of retaining at least a haploid genome in cancer cells. We will use genome- wide, Single Nucleotide Polymorphism (SNPs) patterns to test this hypothesis. The novel method developed in this project, which we call Probability Stripes, will aid the detection of LOHs in the SNP patterns to look at aneuploidy, chromosomal instability and LOH. This method also eliminates noise in the SNP data. The probability stripes model will be applied to: 1) normal samples obtained from healthy, twin sisters, to obtain a baseline level of LOH (if any), and to illustrate how the model works; and 2) prostate cancer cell line DU-145 to visualize the LOH patterns in cancer cell lines. This data together with copy number analysis done using Spectral Karyotyping will allow us to see if aneuploid chromosomal abnormalities and SNP- detected LOHs interact. A potential clinical application of this investigation into the molecular mechanisms of aneuploidy could potentially be an important prognostic indicator for cancer patients. Our second hypothesis is that there will be genes expressed throughout the genome, both in tissues and in long-term cell lines, which may be responsible for the retention of structurally abnormal chromosomes found in aneuploid cancers. To test this hypothesis, we first take a genome wide look at the expression of all human genes for which gene expression arrays are available, in a large variety of normal and cancerous cell lines and tissues, to define a set of universally expressed genes. We 2 will then determine whether or not the universally expressed cell survival genes (hypothesis 2) are preserved in the haploid genome that is retained by the cancer cell lines after LOH formation (hypothesis 1). The motivation behind our first long term goal of improving the SNP-LOH assay is to obtain a better screening method for deciding optimal, individualized treatment regimens for breast cancer patients. We currently do not have a method to effectively and individually screen breast cancer patients to determine the best patient treatment regimen from the staging criteria of individual breast cancers. This results in many women receiving treatments that may not be needed or beneficial. Our work may aid in determining whether the observed genetic events can be used as a screen which may be part of a diagnostic panel. It is possible that the use of a whole genome SNP technique to obtain a LOH pattern may distinguish different tumor grades and thereby help predict better treatment regimens for individual patients. The motivation behind our second hypothesis is to explain the reason for selectively retained chromosomal