GENERATION AND ANALYSIS OF KRASV12 IN DRIVING LIVER TUMORIGENESIS USING TRANSGENIC ZEBRAFISH MODELS NGUYEN ANH TUAN (B.Sc., Vietnam National University; University of Natural Sciences, Ho Chi Minh City) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2011 ACKNOWLEDGEMENTS The work presented in this thesis was accomplished at the Department of Biological Sciences (DBS) and Temasek Life Sciences Labolatory (TLL), National University of Singapore, from August 2007 to August 2011. It gives me great pleasure to acknowledge all who made this thesis possible. First and foremost, I would like to express my deepest and most sincere gratitude to my supervisors, Prof. Gong Zhiyuan (DBS), Dr. Serguei Parinov (TLL) and Dr. Alexandre Emelyanov (TLL) for their innovative insights, valuable guidance, unending support, perpetual encouragements throughout my study. Their enormous scientific experience together with logical way of thinking have been of great value for me and also provided a good basis for the present thesis. I wish to appreciate Prof. Chou Loke Ming (DBS) for his coming to Vietnam to interview and offer me an invaluable opportunity to be NUS scholar. I owe my most sincere thanks to Dr. Koh Chor Hui Vivien (DBS) for her truthful friendship and great help that have remained consistent during my study. I warmly thank Dr. Jan Spitsbergen (University of Oregon) and Dr. Lam Siew Hong (DBS) for their professional advices and technical instructions in my research. I also appreciate the helps from many friends and colleges, including Yan Tie, Huiqing Zhan, Cecilia Winata, Lana Korzh, Zhen Li, Tiweng Chew, Bing Liang, Lili Sun, Helen Quach, Long Tran, Shimin Lim, Kumari Pooja, Thiet Vu and Lam Dang during my study. I would also like to thank DBS Graduate Officers, especially Ms. Reena and Ms. Priscilla, for their dedicated helps during the course of my study. Many thanks to all members in Prof. Gong laboratory (DBS), Dr. Karuna laboratory (TLL), Dr. Yue I labolatory (TLL), TLL fish facility, GIS microarray facility and Biopolis shared facilities for all their helps and support in this thesis. My most heartfelt gratitude goes to my dearest Family members. Thanks to my grandmother as my great mentor, together with my parents, aunts and sisters for their loving considerations and great believe in me all through these years. Most importantly, I feel so proud and lucky to have my most faithful partner, Quang Duc Dao, whose loving, understanding, supporting and accompanying me throughout all these years have given me strength to finish this study. Therefore, I wish to dedicate this thesis to my Family. Last but not least, I greatly acknowledge the National University of Singapore for awarding me the Graduate Research Scholarship. Singapore, 27th June 2011 II TABLE OF CONTENTS Acknowledgements………………………………………………………………. I Table of contents………………………………………………………………… III Summary………………………………………………………………………… .VIII List of tables………………………………………………………………………. XI List of figures …………………………………………………………………… . XII List of common abbreviations…………………………………………………… XIV Chapter 1: Introduction………………………………………………………… 1.1 1.2 Introduction to human liver cancer…………………………………… 1.1.1 Incidence, epidemiology and risk factors……………………… 1.1.2 Current trends in therapeutic strategies of human HCC…… … Zebrafish as a liver cancer model……………………………………… 1.2.1 Advantageous use of the zebrafish in research…………………… 1.2.2 Modeling human diseases using zebrafish…………………….…… 10 1.2.3 Zebrafish models of human liver cancer: Chemical carcinogenesis and transgenic approaches…………… . 13 1.2.4 Application of conditional expression systems in transgenic zebrafish .…… 14 1.3 1.4 Oncogenic Ras in human liver cancer…………………………………… . 17 1.3.1 Molecular perspective of Ras in cancer biology………………… 17 1.3.2 Association of Ras with HCC………………………………….…. 20 Objectives and significance of the study………………………………… . 22 III Chapter 2: Materials and Methods …………………………………………… . 24 2.1 General molecular biology techniques and plasmid construction… 25 2.1.1 Polymerase chain reaction (PCR) …………………………………. 25 2.1.2 Cloning…………………………………………………………… . 25 2.1.3 Isolation of zebrafish kras oncogene and construction of Tg(fabp10:EGFP-krasV12) plasmid…………… .……….…… 26 2.1.4 2.2 2.3 Construction of inducible transgenic systems…………………… . 29 Generation of transgenic zebrafish …………………………………… . 30 2.2.1 Zebrafish maintenance……………….…………………………… 30 2.2.2 RNA synthesis, microinjection and screening of transgenic fish… 30 Gross morphological and histopathological analyses of zebrafish tumor….….….….….….….….….….….….….….….….….… . 31 2.4 Tumor screening for the inducible systems….….….….….….….….… . 33 2.5 Transplantation of liver tumors into wild-type zebrafish 2.6 Isolation of total RNA/genomic DNA and ….….….….…33 reverse transcriptase/quantitative real-time/genotyping PCR….….……… 34 2.6.1 Isolation of total mRNA….….….….….….….….….….….….…… 34 2.6.2 Reverse transcriptase PCR….….….…….….….…….….….……… 34 2.6.3 Quantitative real-time PCR….….….…….….….…….….….…… . 35 2.6.4 Isolation of genomic DNA and genotyping PCR….….….……… . 38 2.7 Western blot analysis….….….…….….….…….….….…….….….…… . 39 2.8 Immunohistochemistry….….….…….….….…….….….…….….….…… 40 2.9 Cellular senescence and cell death analyses….….….…….….….………… 40 IV 2.10 Zebrafish oligonucleotide microarray construction and hybridization…… 41 2.11 Transcriptomic analyses….….….…….….….…….….….…….….….…….42 2.12 Inhibitor treatment….….….…….….….…….….….…….….….…………. 44 2.13 Statistical analyses….….….…….….….…….….….…….….….……… . 45 Chapter 3: Results….….….…….….….…….….….…….….….…….….….…….46 3.1 Analysis of constitutive liver-specific expression of oncogenic krasV12 in driving liver tumorigenesis in transgenic zebrafish….………… 47 3.1.1 Generation of Tg(fabp10:EGFP-krasV12) transgenic zebrafish……. 47 3.1.2 High level of krasV12 expression led to early lethality and induced HCC….….….…….….….…….….….…….….….……… 51 3.1.3 Transplantability of krasV12 liver tumors in WT recipients………… 56 3.1.4 Differential activation of ERK, JNK and p38 mitogen-activated protein kinase pathways during krasV12 liver tumorigenesis…… . 58 3.1.5 Activation of the Wnt/β-catenin pathway during krasV12 liver tumorigenesis….….….…….….….…….….….…….….….…. 62 3.1.6 Acceleration of Liver tumor onset by loss of p53-mediated senescence….….….…….….….…….….….…….….….…….……. 64 3.1.7 Transcriptomic analyses of krasV12 liver tumorigenesis…… …… 68 3.1.8 Identification of a HCC-specific signature and a liver cancer progression signature….….….…….….….… …… . 71 3.2 Development and analysis of mifepristone-inducible and -reversible krasV12 liver tumorigenesis in transgenic zebrafish……… 79 V 3.2.1 System design ….….….…….….….…….….….…….….….…… . 79 3.2.2 Control of liver tumor progression and regression in krasV12 transgenic zebrafish by mifepristone administration….….….…… 82 3.2.3 Activation of ERK and AKT pathways required for krasV12-driven liver tumorigenesis and tumor maintenance………. 88 3.2.4 Prevention of krasV12 liver tumorigenesis by inhibiting ERK and/or AKT pathways ….….….…….….….…….….….… …… . 91 3.3 Development and analysis of mifepristone-inducible Cre/loxP recombination to conditionally control krasV12 liver tumorigenesis in transgenic zebrafish….….….…….….….…….….….…….….….…… . 94 3.3.1 System design ….….….…….….….…….….….…….….….…… 94 3.3.2 Determination of concentration- and time-dependent mifepristone induction of Cre expression….….….…….….….…… 97 3.3.3 Mosaicism of EGFP-krasV12 expression in Triple-Tg fish causing hepatocellular carcinoma and other types of liver tumor……… .100 3.3.4 Deregulation of ERK and Wnt/β-catenin pathways during krasV12-induced liver tumor progression….….….…….….….…… 106 Chapter 4: Discussions….….….…….….….…….….….…….….… …….….…. 108 4.1 A high level of krasV12 expression leading to HCC in transgenic zebrafish….….….…….….….…….….….…….….….…….….….……… 109 4.2 Conserved gene expression signatures underlying liver tumorigenesis in humans and krasV12 transgenic zebrafish….….….…….….….…….…… 113 VI 4.3 Mifepristone-inducible and -reversible krasV12system potential for high throughput anti-cancer drug screens….….….…….….….…….…. 114 4.4 Mifepristone-inducible Cre/loxP regulating krasV12 system induces various liver tumors and closely mimics spontaneous cancer development….….….…….….….…….….….…….….….…….….….…… 118 4.5 Summary and conclusions….….….…….….….…….….….…….….….…. 120 Bibliography.….….…….….….…….….….…….….….…….….….…….….… 125 Appendices VII SUMMARY Human liver cancer is one of the deadliest cancers worldwide, with hepatocellular carcinoma (HCC) being the most common type. The neoplastic development of human HCCs is a complex multistage process, with heterogeneity in morphology and genetics that makes its ultimate clinical benefit negligible. Despite the relevance of HCC malignancy, a fundamental understanding of the molecular mechanisms of hepatocarcinogenesis is currently rather limited. As a potent proto-oncogene and bona fide central regulator of signal transduction pathways in many human cancers, Ras is at the leading edge of most tumorigenic events and is activated in nearly all HCC cases. Thus, targeting Ras signaling has emerged as a potential strategy to treat advanced HCC. However, the mechanism of Ras-induced liver cancer remains elusive and in vivo models that enable investigations of the important role of Ras in liver tumorigenesis are lacking. To address these problems, a constitutive transgenic zebrafish liver cancer model was first generated using a hepatocyte-specific promoter (fabp10) to target oncogenic krasV12 expression to the liver. Fusion with EGFP allowed visualization of the process of tumor development from early stages. Only high level of krasV12 expression initiated liver tumorigenesis. The krasV12 tumors showed progressive features from hyperplasia to invasive HCC which was accompanied by a loss of p53-dependent senescence response. HCC cells derived from this line also displayed transplantability. Transcriptomic analyses delineated several pathways and identified two conserved gene signatures accounting for HCC specificity and HCC progression in both zebrafish and human. These findings validated the potential of krasV12 transgenic fish in modeling human liver cancer. However, several limitations were found in this model such as low HCC penetrance and VIII premature lethality due to early Ras activation. Motivated by previous findings, another model allowing for liver-specific and inducible EGFP-krasV12 expression was generated using mifepristone-inducible strategy, which allowed to induce oncogene expression at any desirable time and to accelerate tumor onset. Robust and homogeneous HCC growth was achieved in 100% transgenics after month induction. HCC was found to be “addicted” to Ras signaling for tumor maintenance as mifepristone withdrawal led to tumor regression via cell death. Targeting KrasV12 liver tumorigeneis via its downstream effectors, Raf/MEK/ERK and PI3K/AKT/mTOR, by chemical inhibitors significantly suppressed the over-growth of hyperplastic liver in EGFP-krasV12 larvae. Collectively, this model offered an effective and predictable liver cancer model for large-scale studies. It is well known that human cancer is usually initiated by a sporadic event of mutations occurring in a single or group of cells. Therefore, a third krasV12 liver cancer model was established using the mifepristone-inducible Cre/loxP approach. By exposure to mifepristone, Cre recombination was induced to permanently activate the liver-specific EGFP-krasV12 expression. Due to incomplete Cre-mediated recombination, a mosaic pattern of krasV12 expression resulted in broad liver tumor spectrum. Clonal proliferation of neoplastic cells expressing EGFP-krasV12 in normal-appearing liver can be observed in transgenic fish, offering a unique model to study spontaneous oncogenic mutations in humans. In summary, the krasV12 transgenic zebrafish is the first in vivo model unveilling molecular mechanisms underlying Ras-induced liver tumorigenesis that recapitulates typical hallmarks of human HCC. The two conserved HCC gene signatures identified in IX Discussions to some rare human liver cancer cases, suggesting for the first time the ubiquitous and important role of Ras signaling in the initiation and development of a wide range of hepatocarcinogenesis. In this aspect, this system would be useful for mining novel genetic variants as well as evolution mechanism between different liver tumor types. Experimentally, this system requires only a short pulse of inducer treatment and it is more convenient and less laborious than the mifepristone-inducible system which needs the continuous presence of inducer to maintain the expression of krasV12. In conclusion, this project comprehensively demonstrates the generation and analyses of krasV12 driving liver tumorigenesis in different transgenic zebrafish model systems and also suggests a practical step-by-step experimental route in studying effect of a certain oncogene in cancer. The tight conservation of liver cancer-related pathways between fish and human underscoring that krasV12 transgenics are valuable liver cancer models in which studies of different transgenic systems describe their unique characteristics and utilizations as well as provide further insights into krasV12 liver tumorigenesis. High incidence and consistent pattern of cancer progression coupled with low maintenance costs allowed systematic study of tumor biology in transgenic zebrafish. Furthermore, the fluorescence-tagged liver tumors would enable live imaging of tumor progression and tumor-host interaction during transplantation. 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Drug Discov. 4, 35-44. 138 [...]... 3.4 (p.54) Liver tumors progression in krasV12 transgenic zebrafish Figure 3.5 (p.57) Growth of transplanted krasV12 liver tumors in WT recipients Figure 3.6 (p.60) Hyperactivation of the mitogen-activated protein kinase (MAPK) signaling pathway in krasV1 2transgenic zebrafish Figure 3.7 (p.63) Activation of the Wnt/β-catenin pathway during krasV1 2liver tumorigenesis Figure 3.8 (p.66) KrasV12-induced p53-dependent... frequency in human cancers Figure 2.1 (p.28) Alignment of human and zebrafish KrasV12 amino acid sequences Figure 3.1 (p.49) Generation and characterization of Tg(fabp10:EGFP-krasV12) transgenic zebrafish Figure 3.2 (p.50) Morphological development of liver in transgenic fish expressing EGFP-krasV12 Figure 3.3 (p.53) Premature lethality caused by high level of krasV12 expression in transgenic zebrafish. .. use of zebrafish Recently, there is increased generation and analysis of zebrafish models of human diseases (Amatruda and Patton, 2008; Liu and Leach, 2011) Owing to the ease of housing maintenance, short generation time and fecundity, zebrafish studies are cost-effective and provide advantages over other models in high-throughput small molecule screening All these key attributes underpin the use of zebrafish. .. double transgenic zebrafish harboring the Liver- driver and Creeffector constructs Driver/Ras-effector double transgenic zebrafish harboring the Liver- driver and Ras-effector constructs dpi day(s) post-injection dpf day(s) post-fertilization EGFP enhanced green fluorescent protein EGFP-KrasV12 fusion protein of N-terminal EGFP and C-terminal zebrafish KrasV12 ENU ethylnitrosourea fabp10 fatty-acid binding... Advanced liver cancer in krasV12 transgenic fish XII Figure 3.14 (p.90) Roles of Raf/MEK/ERK and PI3K/AKT/mTOR pathways during krasV12 tumor progression and regression Figure 3.15 (p.93) Suppression of liver tumorigenesis by inhibition of Raf/MEK/ERK and PI3K/AKT/mTOR pathways Figure 3.16 (p.96) Strategies for the mifepristone-induced Cre-mediated conditional expression of krasV12 in transgenic zebrafish. .. in Triple-Tg zebrafish overexpressing krasV12 since 1-month-old Table 4.1 (p.124) Comparison of the three krasV12-induced liver cancer models using transgenic zebrafish in this project XI LIST OF FIGURES Figure 1.1 (p.4) Multi-stage process of hepatocarcinogenesis Figure 1.2 (p.9) Advantages of zebrafish as a powerful model organism for cancer research Figure 1.3 (p.19) Distribution of KRAS somatic... senescence in the pre-neoplastic liver Figure 3.9 (p.69) Flowchart of microarray data analysis Figure 3.10 (p.72) GSEA identification of conserved gene signatures common between zebrafish and human HCC Figure 3.11 (p.81) Mifepristone-inducible liver- specific oncogenic krasV12expression in transgenic zebrafish Figure 3.12 (p.85) Dosage-dependent induction of krasV12 expression and liver tumor induction and. .. Wnt/β-catenin pathways during krasV12induced liver tumor progression Figure 4.1 (p.112) Proposed mechanism of Ras-induced liver tumorigenesis in transgenic zebrafish model Figure 4.2 (p.117) Tumorigenesis and tumor regression in the mifepristone-inducible krasV12 liver tumor model XIII LIST OF COMMON ABBREVIATIONS Ac/Ds Activator/Dissociation transposon system bp base pair cryB crystallin beta B DMSO dimethylsulphoxide... markers and potential therapeutic targets in human liver cancer Adopting these krasV12 transgenic zebrafish model systems in which high incidence and consistent pattern of cancer progression are coupled with low maintenance costs of zebrafish would allow systematic study of liver cancer progression and regression as well as provide novel platforms for high-throughput screening of anticancer drugs X LIST OF. .. highthroughput screening 1.2 Zebrafish as a liver cancer model Animal models have been widely used in biomedical research to define the pathogenesis of cancer and as in vivo systems for developing and testing new therapies Indeed, drug discovery involves a complex process of biomedical and cellular assays, with final validation in mammalian models before ultimate test in humans (Zon and Peterson, 2005) . 3.1 Analysis of constitutive liver- specific expression of oncogenic kras V12 in driving liver tumorigenesis in transgenic zebrafish .………… 47 3.1.1 Generation of Tg(fabp10:EGFP -kras V12 ) transgenic. Histopathologic findings in Triple-Tg zebrafish overexpressing kras V12 since 1-month-old Table 4.1 (p.124) Comparison of the three kras V12 -induced liver cancer models using transgenic zebrafish in. GENERATION AND ANALYSIS OF KRAS V12 IN DRIVING LIVER TUMORIGENESIS USING TRANSGENIC ZEBRAFISH MODELS NGUYEN ANH TUAN (B.Sc., Vietnam National University; University of Natural