IDENTIFICATION OF BAP1 AS A PREDISPOSING GENE FOR MALIGNANT MESOTHELIOMA A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI’I AT MANOA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN MOLECULAR BIOSCIENCES AND BIOENGINEERING DECEMBER 2012 By MASAKI NASU Dissertation Committee: Michele Carbone (Chairperson) Giovanni Gaudino David Ward Toshihiko Kawamori Haining Yang 0 ACKNOWLEDGMENTS I would like to express my gratitude to my mentor Dr. Michele Carbone, and my dissertation committee members, Dr. David Ward, Dr. Giovanni Gaudino, Dr. Toshihiko Kawamori, and Dr. Haining Yang for their support, suggestion, and encouragement. I also thank to Dr. Patricia S. Lorenzo, who gave me kind guidance until my comprehensive exams. This work was done by the collaboration with Dr. Testa Lab (Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA). This project is supported by US National Institutes of Health (NIH) grants P01CA-114047, P30CA-06927 and P30CA-71789, by the AACR-Landon Award for International Collaboration in Cancer, and by the Local No. 14 Mesothelioma Fund of the International Association of Heat and Frost Insulators & Allied Workers. Many thanks go to my lab mates Andrea Napolitano, Brian Kendrick, Cormac Jennings, Erin Flores, Fang Qi, Laura Pellegrini, Lauren Gardner, Mika Tanji, Sabahattin Comertpay, Sandro Jube, and Vishal Singh Neigi. I would like to express my special gratitude to my wife, Naoko Nasu, son, Misato Nasu, daughters, Fua Nasu, and Solala Nasu, and my mother Akiko Nasu for their support, patience, and encouragement. i ABSTRACT Malignant mesothelioma (MM) is a very aggressive tumor which arises from mesothelial cells found in the lining of the pleural, pericardial and peritoneal cavities. An estimated 3,000 people are diagnosed with MM each year in the United States and the prognosis is very poor with a median survival of about 12 to 18 months from diagnosis. The relationship between asbestos exposure and mesothelioma is now widely accepted and approximately 80% of patients with MM in the United States have been exposed to asbestos. However, because only a small fraction of asbestos-exposed individuals develop MM (about 5%) and clustering of this disease was observed in some families, we hypothesized the existence of a genetic predisposing factor. We have been searched for mutated genes in germline cells of individuals from “mesothelioma village” in Cappadocia, Turkey, and in two American families, which have history of mesothelioma. We discovered mutations in the gene encoding BRCA1 associated protein-1 (BAP1) from the two American families with a high incidence of MM. We also found germline BAP1 mutations in 2 of 26 sporadic MM; both individuals with mutant BAP1 were previously diagnosed with uveal melanoma, too. We also observed somatic truncating BAP1 mutations and aberrant BAP1 expression in sporadic MM with no germline mutations. This is the first project which proved that BAP1 as a predisposing gene for malignant mesothelioma. ii TABLE OF CONTENTS ACKNOWLEDGMENT ……………………………………….……………… i ABSTRACT ……………………………………………………….……………….ii LIST OF TABLES …………………………………………...………………iv LIST OF FIGURES ………………………………………………………….v SPECIFIC AIMS …………………………………………………….………….vii CHAPTER 1 INTRODUCTION 1.1 Malignant mesothelioma …………………………………………………1 1.2. Genetic Susceptibility of Malignant Mesothelioma …………………….3 1.3. Genetic alterations in malignant mesothelioma …………………….…6 1.4. Malignant Mesothelioma and Chromosome 3p21.1 ………..……..….12 1.5. Ubiquitination, Deubiquitination and BAP1 …………………….…..19 1.6. Polycomb and Histone ubiquitination …………………………..……..35 1.7. BAP1 mutations in cancer. ………………………………………………39 CHAPTER 2 RESULTS 2.1. Identification of Mesothelioma pedigree in two American families. ..41 2.2. BAP1 mutation predicted by array-CGH analysis. …………………..43 2.3. Germline DNA sequencing of BAP1 in the two families. …………...45 2.4. Germline DNA sequencing of BAP1 in sporadic mesothelioma. …….51 2.5. BAP1 mutations were found in 22% of sporadic tumor DNA samples. …………………………………….………54 2.6. BAP1 mutations were found in one tumor DNA sample from Cappadocia, Turkey. ………………………….………………61 2.7. BAP1 mutations were found in 31% of MM cell lines. ………………….62 CHAPTER 3 DISCUSSION AND FUTURE PROSPECTS …………….……… 67 CHAPTER 4 MATERIALS & METHODS …………………….…………..…….76 REFERENCES …… ……………………………………………….……………..…80 iii LIST OF TABLES Table Page Table 1. List of genes surrounding the BAP1 and located in 3p21.1. ……….….18 Table 2. Summary of BAP1 mutations. ……………………………………….…….75 Table 3. Primers used for the amplification of genomic DNA for sequencing …79 iv LIST OF FIGURES Figure Page Figure 1. Pedigrees of families from the “mesothelioma village” Cappadocia, Turkey. ………………………………………………….….5 Figure 2. Mechanism of ubiquitination and deubiquitination in proteasome pathway. ……………………………………………….…21 Figure 3. Schematic diagram of UCH family proteins. ……………………………. 24 Figure 4. Schematic diagram of wild type BAP1. ……….………………….…..27 Figure 5. Possible involvement of BAP1 in DNA damage response. …….…... 30 Figure 6. HCF-1/YY1/E2F1/BAP1 complex activate gene transcription for S phase transition… ………………………………………….…………32 Figure 7. Histone ubiquitination and deubiquitination by Polycomb complex...34 Figure 8. Pedigrees of two US families with high incidence of mesothelioma…41 Figure 9. Asbestos in the L family and W family homes. ………………….……...42 Figure 10. Array-CGH analysis of members of two American families ………...44 Figure 11. Sequencing results of germline mutations in the American families..48 Figure 12. Immunohistochemistry on mesotheliomas from the L and W. families………….…….49 Figure 13. The individuals in which BAP1 mutations were identified in the two families. …………………………………………………………….50 Figure 14. BAP1 mutations in SP-002, and SP-008. ……………….………….....52, 53 Figure 15. BAP1 mutations in SP-001 SP-015 SP-013 and SP-018. ………....55 - 59 Figure 16. BAP1 truncating mutations and aberrant protein expression in sporadic mesothelioma tumor biopsies …………………..…….60 Figure 17. Mutation analysis of BAP1 in tumor DNA sample from Turkey …….62 v Figure 18. Expression analysis of BAP1 in mesothelioma (MM) cell lines…..….64 Figure 19. Homozygous deletion of BAP1 in ROB ………………………………64 Figure 20. Sequencing alignment of Phi ………………….…………………………65 Figure 21. Sequencing alignment of Hmeso …..…………….………………………66 vi SPECIFIC AIMS Background and Hypothesis Malignant mesothelioma (MM) is a rare, but an aggressive tumor that originates from the mesothelial cells. There is a strong association between MM and asbestos exposure, and it is known that about 80% of MM cases occur in individuals who have been exposed to asbestos. SV40 infection is also known as a co-factor of asbestos-related MM. However, MM was diagnosed in only about 5% of asbestos miners, and there are some families with high incidence of MM without the occupational asbestos exposure and without the SV40 infection. For example, we found that an epidemic of MM in Cappadocia, Turkey, was due to erionite exposure. Erionite is a fibrous zeolite mineral with asbestos-like characteristics. Pedigree analysis showed that MM was inherited in prevalent in certain families. When an individual from a high-risk MM family married into a family with no history of MM, MM started to appear in the descendants. Our data from Cappadocia and published data suggest that the possibility of other co-factor, besides SV40 infection, is involved in MM carcinogenicity. Therefore, we hypothesized that predisposing gene(s) for MM might exist. Specific Aim 1- Determine the possible predisposing gene for MM. In order to identify the predisposing gene, we collected DNA samples not only from families in Cappadocia, but also from two American families with high incidence of MM. One of the American families has 5 MM patients, and the other family has 7 patients. By CGH array analysis, we tried to find a common deleted region shared by the two American families. Literature review on this region (on chromosome 3p) leads us to identify particular gene, BAP1, which we needed to check germline mutations by DNA sequencing. We checked if the gene was mutated only in the MM patients in these families, but not mutated in healthy spouse in the families, nor in DNA from other healthy individuals. vii Specific Aim 2 – Determine whether BAP1 mutations are involved in MM tumorigenesis. In this aim, we checked BAP1 mutations in sporadic MM samples, and MM cell lines to determine the activity of BAP1 in MM tumorigenesis. We checked BAP1 mutations by DNA sequencing on tumor samples, and then checked if mutated proteins were functional or not by western blotting. Significance of this project. Identification of predisposing genes can prove that MM is a cancer caused by gene-environment interaction and suggest that a novel cell signaling pathway can be involved in MM tumorigenesis. Further investigation into the function of BAP1 will lead to a novel preventive and therapeutic strategy for families with inherited BAP1 mutations. Results of this project will contribute to the early diagnosis and prevention of MM. viii CHAPTER 1. INTRODUCTION 1.1 Malignant mesothelioma Mesothelioma (MM) is a very aggressive tumor that arises from mesothelial cells of the lining of pleural, pericardial, and peritoneal cavities [1]. In the US, an estimated 3,000 people are diagnosed with MM each year, with nearly 100,000 new cases expected to occur over the next 40 years [2]. The relationship between asbestos