Regulation of the TRIP BR1 proto oncoprotein a potential therapeutic target for human cutaneous and intracavitory proliferative lesions

REGULATION OF THE TRIP-BR1 PROTOONCOPROTEIN—A POTENTIAL THERAPEUTIC TARGET FOR HUMAN CUTANEOUS AND INTRACAVITARY PROLIFERATIVE LESIONS BY ZHIJIANG ZANG (MBBS, Kunming Medical College, China; MSc, National University of Singapore, Singapore) THESIS SUBMITTED FOR THE DEGREE OF PHILOSOPHICAL DOCTOR OF SCIENCE DEPARTMENT OF MEDICINE NATIONAL UNIVERISTY OF SINGAPORE 2007 Zhijiang Zang PhD. Thesis Medicine, NUS 2003-2007 ACKNOWLEDGEMENTS I wish to express my deep appreciation to the following individuals who made this work possible. First of all, my sincerest and deepest gratitude to my mentor Prof. Stephen I-Hong Hsu, for his guidance during my doctoral study. He provided a motivating, enthusiastic, and critical atmosphere during the many discussions we had. His constant support and encouragement, his inspiration and patience, his mentorship and friendship are the key for me to be able to submit this thesis. I feel very privileged to have worked with him and had a long journey with him together. With a deep sense of gratitude, I want to thank my co-supervisors, Prof. Manuel Salto-Tellez, who provided timely and valuable help at the crucial time. I like to express my deepest gratitude to Dr. Lakshman Gunaratnam, Brigham and Women's Hospital, for giving me excellent guidance, sharing valuable knowledge, asking challenging questions, reading and revising this thesis and providing good company. His experience and involvement is crucial for me to overcome many obstacles I met in this research project. I’d like to express my sincerest thanks to Prof. Joseph Vincent Bonventre, Robert H. Chief, Renal Division, Brigham and Women's Hospital, Harvard Medical School, for having me in his great lab during July 2005- July 2007. Thank him for allowing me to Zhijiang Zang PhD. Thesis Medicine, NUS 2003-2007 participate him lab meeting and present my data during this period of time. Thank him for his sound advice and constructive comments on my project. I wish to thank the lab members in the Renal Division, Brigham & Women Hospital of Harvard Medical School for their inspiring discussions and valuable advices. I am especially grateful to Dr. Jagesh Shah, Prof. Antonis S. Zervos, Dr. Tak ah aru Ic h i m u ra, Dr. Benjamin Humphreys, Dr. Li-Li Hsiao, Dr. Won Han, Dr. Alice Marie Sheridan, Dr. Dirk Hentschel, D r. Deguang Zhu and Dr. Vi s h al S . Vaid ya. I wish to thank Ms. Eileen O'Leary and Ms. Xiaoming Sun in Brigham & Women Hospital of Harvard Medical School for assisting me in many different ways. I would like to thank people in School of Medicine of National University of Singapore, especially Prof. Bay Boon Huat, Ms. Stacy Tan, Ms. Geetha Sreedhara Warrier and Ms. Malika Raguraman for their kind helps during the last four years in NUS. Without the assistance from them and others that I did not mention the names, my PhD candidature will not be smooth. Special thanks to fellow colleague Jit Kong Cheong for his good company, friendship and assistance during this long journey. I would like to also thank Susan Nasr who spent a lot of time in reading and editing this thesis although she is busy with preparing MCAT. I am grateful to all the members of the laboratory in Singapore: Dr. Sim Khe Guan, Dr.Yang Maolin Christopher, Chui Sun Yap, Hajjah Shahidah Bte Mohd Said who made the lab such a wonderful workplace and home. Zhijiang Zang PhD. Thesis Medicine, NUS 2003-2007 I am indebted to Dr. Tan Lai Yong & Lay Chin and Prof. Tan Kim Siang Luke for their invaluable support, encouragement and friendship during 2001-2005 when I was in Singapore. I’d like to thank my wonderful wife Liyun Lai, who has been extremely understanding and supportive of my studies. She has never complained although we had to experience a lot of hardship when we are alone in overseas. Without her sacrifice, it is totally impossible for me to focus on research. I thank my daughter, Yi Zang, for giving me so much joy. Sorry for the countless weekends and holidays that I could not accompany you and your mom during these four years. I love your two! I thank my brother, Zhixin Hu and his wife, my parents-in-law, my aunty and her family for their moral support. Lastly, and the most importantly, I wish to thank my mother, Xixiu Zang and my father Shufan Fu. They bore me, raised me, taught me, and loved me. Although my mother is no longer with us, she is forever remembered. Her love is always in my heart. I am sure she shares my joy and happiness in the heaven. To my mother and father I dedicate this thesis. Zhijiang Zang PhD. Thesis Medicine, NUS 2003-2007 TABLE OF CONTENTS Page TITLE ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF PUBLICATIONS 11 LIST OF FIGURES & TABLES 12 LIST OF ABBREVIATIONS 16 ABSTRACT 19 CHAPTER ONE---GENERAL INTRODUCTION 1.1 TRIP-Br proteins 1.1.1 TRIP-Br protein family 21 1.1.2 Domain structure of the TRIP-Br Proteins 25 1.1.3 TRIP-Br proteins co-regulate transcriptional activity of E2F-1/DP-1 24 1.1.4 TRIP-Br proteins interact with PHD zinc fingerand /or the bromodomain- containing proteins 1.1.4.1 PHD zinc finger domain and proteins 26 1.1.4.2 Bromodomain and bromodomain-containing proteins 31 1.1.4.3 Interaction between PHD zinc finger/bromodomain -containing transcription factors and TRIP-Br proteins 34 Zhijiang Zang PhD. Thesis 1.1.4.4 Biological significance of the interaction between PHD zinc finger/bromodomain proteins and TRIP-Br proteins Medicine, NUS 2003-2007 37 1.1.5 TRIP-Br1 is a CDK interacting protein 38 1.1.6 TRIP-Br proteins interact with cyclin A 39 1.1.7 TRIP-Br proteins are novel cell cycle regulators 40 1.1.8 The integrator model of TRIP-Br protein function in cell cycle regulation 41 1.2 TRIP-Br proteins and cancer 1.2.1 TRIP-Br1 and RBT1 locate at 19q13, a common amplicon in human cancer 44 1.2.2 TRIP-Br proteins promote cell growth 45 1.2.3 TRIP-Br proteins are a family of oncogenes 46 1.2.4 The integrator function of TRIP-Br proteins and cancer 47 CHAPTER TWO---OBJECTIVES 49 CHAPTER THREE---EVALUATION OF THE TRIP-BR PROTEINS AS CHEMOTHERAPEUTIC DRUG TARGET IN HUMAN CUTANEOUS AND INTRACAVITARY HYPERPROLIFERATIVE LESIONS 2.1 Introduction 51 2.2 Material and methods 2.2.1 Materials 54 2.2.1.1 Decoy peptide 54 2.2.1.2 Cell culture 54 2.2.2 Methods 2.2.2.1 Generation and characterization of monoclonal and polyclonal antibodies against hTRIP-Br proteins 55 2.2.2.2 Western blot for detection of TRIP-Br expression 55 2.2.2.3 In vitro and in vivo decoy peptide uptake assay by flow cytometric analysis and confocal microscopy 55 Zhijiang Zang PhD. Thesis Medicine, NUS 2003-2007 2.2.2.4 Cell cycle analysis 55 2.2.2.5 BrdU incorporation assay 56 2.2.2.6 Colony formation assay 57 2.2.2.7 Generation of nude mouse tumor xenograft models 57 2.2.2.8 Chick embryo chorioallantoic membrane (CAM) tumor xenograft model establishment and validation 57 2.2.2.9 In vivo effect of peptide treatment on tumor growth using the chick CAM model 58 2.2.2.10 Statistical analysis 59 2. Results 2.3.1 TRIP-Br decoy peptides inhibit the proliferation of CNE2, Ca Ski and MeWo cells in vitro 60 2.3.2 Tumors xenografts in the chick embryo CAM model are accessible to the topically applied decoy peptide treatment 60 2.3.3 TRIP-Br decoy peptides inhibit the growth of CNE2-, Ca Ski- and Me Wo-derived tumors in the chick embryo CAM model 64 2.4 Discussion 66 CHAPTER FOUR---REGULATION OF THREONINE PROTEIN PHOSPHATASE 2A TRIP-BR1 BY SERINE/ 3.1 Introduction 3.1.1 Protein kinases and protein phosphatases 75 3.1.2 Protein phosphatase 2A (PP2A) 3.1.2.1 Subunit composition, localization and distribution 76 3.1.2.2 Biological role of PP2A 3.1.2.2.1 Regulation by PP2A via protein-protein interaction 81 Zhijiang Zang PhD. Thesis Medicine, NUS 2003-2007 3.1.2.2.2 PP2A and cell cycle 83 3.1.2.2.3 PP2A and tumorigenesis 84 3.2 Materials and methods 3.2.1 Materials 3.2.1.1 Plasmid DNA 87 3.2.1.2 Biochemical reagents 87 3.2.2 Methods 3.2.2.1 Cell culture and maintenance 88 3.2.2.2 Expression and purification of GST fusion proteins 89 3.2.2.3 GST pull-down assay 90 3.2.2.4 Silver staining 90 3.2.2.5 Coomassie Blue staining 91 3.2.2.6 Liquid chromatography/mass spectrometry 91 3.2.2.7 Immunoprecipitation assay 92 3.2.2.8 Serine/threonine protein phosphatase activity assays 92 3.2.2.9 Indirect immunofluorescence staining of cells 93 3.2.2.10 Subcellular fractionation 93 3.2.2.11 Co-localization study of TRIP-Br1 and PP2A-Bα 94 3.2.2.12 DNA and siRNA transfection procedures 94 3.2.2.13 Preparation of proteins from tissue culture 94 3.2.2.14 Protein quantitation 95 3.2.2.15 Western blot for protein immunodetection 95 3.2.2.16 Extraction of total cellular RNA 96 3.2.2.17 Reverse transcription of RNA 96 3.2.2.18 Gel electrophoresis of DNA products 97 Zhijiang Zang PhD. Thesis Medicine, NUS 2003-2007 3.2.2.19 Real time-RCR 97 3.2.2.20 Luciferase assays 98 3.3 Results 3.3.1 Identification of the Bα and Bδ subunits of serine/threonine protein phosphatase 2A as potential interactors of TRIP-Br1 3.3.1.1 Expression and purification of glutathione S-transferase mouse TRIP-Br1 fusion protein 99 3.3.1.2 Probing mammalian cellular protein extracts with GSTmTRIP-Br1 3.3.1.3 Mass spectrometric analysis 3.3.2 3.3.4 3.3.5 3.3.8 110 3.3.2.2 Catalytically active PP2A holoenzyme associates with GST-mTRIP-Br1 114 Endogenous human TRIP-Br1 associates with PP2A holoenzyme in vivo 116 Endogenous TRIP-Br1, a cytoplasmic dominant protein, co-localizes with the Bα subunit of PP2A 120 TRIP-Br1 is a serine-phosphorylated protein 128 3.3.6 Okadaic acid treatment alters the level of serine-phosphorylated and total TRIP-Br1 protein 3.3.7 105 TRIP-Br1 associates with catalytically active PP2A holoenzyme in vitro 3.3.2.1 GST-mTRIP-Br1 binds to the PP2A holoenzyme comprising the A/Bα/C subunits 3.3.3 102 132 Transcriptional silencing of the PP2A catalytic subunit decreases the level of TRIP-Br1 protein 137 Overexpression of the PP2A-C subunit increases the level of TRIP-Br1 protein and TRIP-Br1 co-activated E2F1/DP1transcription 141 3.4 Discussion 150 Zhijiang Zang PhD. 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Biochemistry 33, 11858-11867. 180 [...]... cervical (Ca Ski) and melanoma (MeWo) cancer cell lines in vitro as well as in corresponding chick embryo chorioallantoic membrane (CAM) tumor xenografts in vivo, suggesting that TRIP Br1 may represent a novel therapeutic target for the treatment of human cutaneous and intracavitary hyperproliferative lesions To further understand the regulation of this potential therapeutic target, GST-mTRIP -Br1 fusion... function of TRIP- Br proto- oncoprotein may represent important chemotherapeutic drug target for superficial cutaneous and intracavitary hyperproliferative lesions The protein level of TRIP- Br1 is positively regulates by serine/threonine protein phosphatase 2A, via dephoshrlation of serine residue(s) on TRIP- Br1 Uncovering the mechanism of TRIP- Br protein regulation will facilitate the application of therapeutic. .. Progression and in the Maintenance of Genomic Stability Cell Cycle 3:1296-304 3 Zang ZJ, et al Regulation of TRIP- Br1 by serine/threonine protein phosphatase 2A (In preparation) CONFERENCE PAPERS Zang Z, et al Proof -of- principal studies of peptides that antagonize the integrator function of TRIP- Br transcription factors for the treatment of cutaneous and intracavitary lesion (Poster presentation), ASN 2006 Annual... (containing a putative nuclear localization signal), a novel uncharacterized motif termed SERTA (SEI-1, RBT1 and TARA), a PHD zinc finger- and/ or bromodomain-containing protein binding motif, and a conserved acidic C-terminal domain (shown to mediate a transactivation function for TRIP- Br1 and TRIP- Br2) (Calgaro et al., 2002) All four domains are highly evolutionarily conserved across mammalian species The. .. level of TRIP- Br1 protein Furthermore, overexpression of the PP 2A- C subunit increased the TRIP- Br1 protein level and TRIP- Br1 co-activated of E2F1/DP1 transcription These results suggest that PP 2A holoenzyme ABαC associates with TRIP- Br1 in vitro and in vivo in mammalian cells The level of TRIP- Br1 protein is positively regulated by PP 2A In summary, our data indicates that antagonizing the integrator... transcriptional activity It was first noted that GAL4 -TRIP- Br1 and GAL4 -TRIP- Br2 fusion protein stimulates both basal and enhancer-activated transcription when recruited to a heterologous promoter bearing GAL4 DNA binding site (Hsu et al., 2001) Thereafter, potent transactivation activity was demonstrated for the RBT-1 and CDCA4 proteins (Cho et al., 2000; Hayashi et al., 2006) Truncation analysis suggests that the. .. structurally and functionally related mammalian proteins (TRIP- Br1, TRIP- Br2, RBT1 and CDCA4) and a Drosophila homologue, TARA Murine TRIP- Br1 (p34SEI-1/SEI-1/SERTAD1) was originally identified from a mouse whole embryo cDNA library based on its unique ability to interact with the composite PHD-bromodomain of the transcription factor KRIP-1 (KRAB associated protein, also known as TIF1β or KAP 1) in a yeast... CBP, p300, PCAF and STAF65γ have been shown to interact with multiple members of the TRIP- Br protein family (TRIP- Br1, TRIP- Br2 and CDCA4) in vitro and in vivo to regulate transcription (Watanabe-Fukunaga et al.,2005; Lai et al., 2007; Hirose et al., 2003; Hsu et al., 2001) The features and proposed functions of these proteins are summarized in Table 1.3 Deletion analysis reveals that amino acid residues... Deletions, translocations, or point mutations in the CBP gene which alter the histone 28 Zhijiang Zang PhD Thesis Medicine, NUS 2003-2007 acetyltransferase activity (HAT) of the encoded mutant protein lead to RubinsteinTaybi Syndrome (RTS), a human developmental disorder comprised by mental retardation, an unusual facial appearance, broad thumbs, and broad big toes (Murata et al., 2001; Rubinstein and Taybi,... et al., 2001) The magnitude of the co-activation by TRIP- Br proteins was similar to that observed for MDM2, a known E2F1 interactor that stimulates transcriptional activity and DNA synthesis (Hsu et al., 2001) Further investigations determined that TRIP- Br1 and TRIP- Br2 bind DP1 but not E2F1, which may constitute the basis of TRIP- Br protein- mediated E2F1/DP1 transcriptional coactivation (Hsu et al., . that TRIP Br1 may represent a novel therapeutic target for the treatment of human cutaneous and intracavitary hyperproliferative lesions. To further understand the regulation of this potential. REGULATION OF THE TRIP- BR1 PROTO- ONCOPROTEIN A POTENTIAL THERAPEUTIC TARGET FOR HUMAN CUTANEOUS AND INTRACAVITARY PROLIFERATIVE LESIONS BY ZHIJIANG ZANG (MBBS, Kunming Medical College,. School of Medicine of National University of Singapore, especially Prof. Bay Boon Huat, Ms. Stacy Tan, Ms. Geetha Sreedhara Warrier and Ms. Malika Raguraman for their kind helps during the last