Roles of RUNX3 as a tumor supressor protein

ROLES OF RUNX3 AS A TUMOUR SUPPRESSOR PROTEIN LEE WEI LIN BSc (Hons), NUS NATIONAL UNIVERSITY OF SINGAPORE 2008 ROLES OF RUNX3 AS A TUMOUR SUPPRESSOR PROTEIN LEE WEI LIN BSc (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS Graduate School for Integrative Sciences and Engineering NATIONAL UNIVERSITY OF SINGAPORE 2008 Acknowledgements “Science can only determine what is, but not what shall be, and beyond its realm, value judgements remain indispensable” Albert Einstein Research is a constant pursuit of knowledge, of trying to unravel answers to a never-ending stream of questions. In this pursuit of knowledge, there are no easy answers and the importance of good supervisors to learn from and a proper conducive research environment cannot be undermined. None of the work described in this thesis would have been possible without the help and support of a large number of people who have been a part of my graduate education, as friends, mentors and colleagues. Although many have come and gone, they have each played a significant role at various stages of my graduate pursuit. First and foremost, I would like to express my deepest gratitude to Professor Yoshiaki Ito for being my PhD supervisor. I am very thankful for his guidance and honoured to have had the opportunity to be part of his laboratory the past four years. I am grateful to have been able to learn from him. His depth of knowledge and vast wisdom is a constant inspiration and motivation. In addition, I am also appreciative of his continued encouragement and helpful suggestions. I am deeply grateful to Dr Kosei Ito, for guiding me through my thesis project the past two and a half years, and for his invaluable guidance, supervision, understanding and patience throughout the course of the project. I am grateful to him for imparting his knowledge and wisdom to me, guiding me along this difficult journey. I would also like i to say a big thank you to Dr Yasuko Yamamura, who taught me the basic research skills and techniques, which laid the foundation for my thesis project. A special word of thanks to Dr Hiroshi Ida, who taught me the basics of real-time PCR, which eventually became my favourite technique. In addition, I would also like to thank all the members of the RUNX research group, past and present, both from IMCB and ORI. It has been wonderful working in this lab, and it wouldn’t have been the same without each and every one of you. In particular, I am grateful to Dr Anthony Lim , Dr Linda Chuang, Dr Dominic Voon and Dr Hiroyuki Kato for providing timely and constructive comments and suggestions. I would like to thank A*STAR for the award of the A*STAR Graduate Scholarship, which has supported me to the work described in this thesis. In addition, I would also like to say a big thank you to all the staff of the NGS department in NUS, especially to Gloria, Ivy and Madeline, for their administrative support, friendship and encouragement the past few years. I am blessed to have the friendship of my classmates from NGS, good friends from Fairfield Methodist Primary and Secondary School and Faith Methodist Church, who have always been there for me and kept me sane. In addition, I thank God for two of my buddies from ORI, Ti Ling and Michelle, who shared laughter, tears and joy with me in ORI. Lastly, thank you to all my Sunday school kids from Faith Methodist Church who have, in their special way, been my source of joy. Finally I would like to express my greatest and deepest gratitude to my family my fiancé, Wei Jie, who has always been there for me through all my ups and downs; my beloved and charming brother, Wei Kit, who is the best brother anyone could ever ask ii for; my beloved mum and dad, for their immense encouragement and unwavering support. They raised me, taught me, supported me, inspired me, loved me, provided me with the best education and moulded me into the person I am today. To them, I dedicate this thesis. iii Table of Contents Acknowledgements Table of Contents Summary List of Tables List of Figures List of Abbreviations i iv vii ix x xii Chapter 1: Introduction 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10 1.11 1.12 Prologue History and Discovery of RUNX Evolutionary conservation of RUNX genes The Runt domain transcription factor, PEBP2/CBF RUNX1, 2, Gain or loss of RUNX genes in cancer RUNX3 as a tumour suppressor 1.7.1 Role of RUNX3 in the TGF-β pathway 1.7.2 RUNX3 and the chromosomal locus 1p36 1.7.3 Other evidences Knockout phenotype of RUNX3 Biology of the intestinal epithelium Intestinal neoplasia – colorectal cancer Signalling pathways involved in intestinal development and cancer 1.11.1 Wnt signalling 1.11.2 TGF-β signalling 1.11.3 BMP signalling Interaction between BMP and Wnt signalling pathways in colorectal cancer Chapter 2: Results 2.1 Characterization of colorectal cancer cell lines 2.1.1 Gene expression profile of BMP receptor and components of BMP signalling pathway 2.1.2 Gene expression profile of members of RUNX family 2.1.3 Protein expression profile of members of RUNX family 2.1.4 Immunofluorescence analysis of endogenous RUNX3 in colorectal cancer cell lines 11 13 13 21 22 25 27 30 31 32 38 41 50 56 57 57 60 62 63 iv 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14 2.15 2.16 BMP-SMAD signalling is intact in HT-29 colon cancer cells Growth suppressive effect of BMP Growth suppression by BMP is not mediated by apoptosis Growth suppression by BMP is not mediated by p21CIP1/WAF1 BMP treatment enhances RUNX3 expression in HT-29 cells RUNX3 negatively regulates Wnt signalling and forms a ternary complex with β-catenin and TCF4 RUNX3 attenuates the transactivational potential of β-catenin/TCF4 in Wnt signalling RUNX3 attenuates the DNA binding activity of β-catenin/TCF4 BMP treatment represses c-Myc expression BMP targets c-Myc expression by transcriptional mechanisms BMP attenuates the transcriptional potential of β-catenin/TCF4 in Wnt signalling Mutations in RUNX- and TCF- binding elements abrogate BMP-mediated repression of c-Myc promoter activity BMP reduces the DNA-binding ability of β-catenin/TCF4 on the c-Myc promoter RUNX3 expression plays a role in growth suppression by BMP Summary 65 67 70 71 72 77 83 85 88 89 95 96 101 103 116 Chapter 3: Discussion 118 Chapter 4: Conclusion and Perspectives 157 Chapter 5: Materials and Methods 161 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 Cell culture and cell lines Recombinant proteins Transfections Lentiviral Infection Promoter studies Site-directed mutagenesis RNA isolation and reverse transcription Real-time PCR analysis Preparation of nuclear and cytoplasmic extracts Western Blot analysis Immunoprecipitation Immunofluorescence 162 162 162 164 165 166 167 168 168 169 170 171 v 5.13 5.14 5.15 5.15 Measurement of cell proliferation Apoptosis detection Chromatin Immunoprecipitation (ChIP) assay Statistical analysis 171 171 172 174 Bibliography 175 Appendices 215 vi Summary Bone Morphogenetic Protein (BMPs), a member of the Transforming Growth Factor-β (TGF-β) superfamily, are multifunctional cytokines which regulate a broad spectrum of biological functions including development, morphogenesis, proliferation, differentiation and apoptosis. There is growing evidence that aberrations in BMP signalling play an important role in intestinal cancer pathogenesis. Recent studies show the presence of BMP receptor 1a mutations in juvenile polyposis and frequent Smad4 mutations in colon cancer. However, the exact molecular mechanisms remain poorly understood. The Runt domain transcription factor, RUNX3, is an integral component of signalling pathways mediated by both the TGF-β and BMPs in various biological systems. RUNX3 has been shown to be a gastric tumour suppressor, functioning downstream of TGF-β pathway. Recently we demonstrated the tumour suppressive effects of RUNX3 by its ability to attenuate β-catenin/TCFs transactivation in intestinal tumorigenesis. The objective of this study is to explore the molecular basis of the tumour suppressive function of the BMP pathway through RUNX3 in colorectal carcinogenesis. I observed that BMP2/4 exerted a growth suppressive effect in HT-29, a human colorectal cancer cell line, which retains an intact BMP signalling pathway. c-Myc, a well-known oncogene in colorectal cancers and a target of the Wnt signalling pathway, was found to be down-regulated by BMP2/4 and/or RUNX3 in HT-29. Evidence obtained by this study suggests that up-regulation of RUNX3 by BMP reduces c-Myc expression. A mutational analysis of the human c-Myc promoter showed that RUNX3 reduces the promoter activity through RUNX- and TCF-consensus sites. Taken together, vii these results suggest that RUNX3 down-regulates c-Myc expression directly at the transcriptional level, and through attenuation of β-catenin/TCFs, downstream of BMPs in colorectal epithelial cells. viii GAPDH glyceraldehyde-3phosphate dehydrogenase NM_002046 Hs99999905_m1 β-actin actin, beta NM_001101 Hs99999903_m1 218 H T H 29 CT 11 SW 48 D LD W iD Ls r 17 Co T lo 20 Co lo 32 Lo Vo RK Co O lo 20 SW 40 Ca Co O UM S2 SW 83 CC K 81 Co CM I Ls 51 Ls 10 34 Sw 62 H CC 56 RC M Relative expression of RUNX1 mRNA A B H T H 29 CT 11 SW 48 D LD W iD Ls r 17 Co T lo 20 Co lo 32 Lo Vo RK Co O lo 20 SW 40 Ca Co O UM S2 SW 83 CC K 81 Co CM I Ls 51 Ls 10 34 Sw 62 H CC 56 RC M Relative expression of RUNX2 mRNA Appendix Relative expression of RUNX genes in colorectal cancer cell lines 4.5 3.5 2.5 1.5 0.5 4.5 3.5 2.5 1.5 0.5 219 H T H 29 CT 11 SW 48 D LD W iD Ls r 17 Co 4T lo Co 05 lo 32 Lo Vo RK Co O lo 20 SW 40 Ca C O o2 UM S2 SW 83 CC K Co CM I Ls 51 Ls 10 34 Sw 62 H CC 56 RC M Relative expression of PEBP2B cDNA H T H 29 CT 11 SW 48 D LD W iD Ls r 17 Co 4T lo 20 Co lo 32 RK O Lo Vo SW 40 Co lo 20 Ca Co O UM S2 SW 83 CC K Co CM I Ls 51 Ls 10 34 Sw 62 RC M H CC 56 Relative expression of RUNX3 mRNA C D 9000 8000 7000 6000 5000 4000 3000 2000 1000 Expression levels of RUNX genes in colorectal cancer cell lines by real-time PCR analysis. (A) RUNX1; (B) RUNX2; (C) RUNX3; (D) PEBP2β. Values were normalised against endogenous housekeeping gene, GAPDH, and expressed as a fold change over the value for HT-29. 220 Appendix Changes of RUNX3 and c-Myc gene expression in response to BMP treatment HT-29 14 1.2 Relative expression of c-Myc mRNA Relative expression of RUNX3 mRNA 12 10 0.8 0.6 0.4 0.2 + - Control BMP2 BMP4 + + - Control BMP2 BMP4 + - + - + HCT116 1.4 2.5 Relative expression of c-Myc mRNA Relative expression of RUNX3 mRNA 1.5 0.5 0.8 0.6 0.4 0.2 + - Control BMP2 BMP4 1.2 + + - Control BMP2 BMP4 + - + + - SW480 1.2 Relative expression of c-Myc mRNA Relative expression of RUNX3 mRNA 2.5 1.5 0.5 0.8 0.6 0.4 0.2 Control BMP2 BMP4 + - + - + Control BMP2 BMP4 + - + - + 221 1.4 1.2 1.2 0.8 0.6 0.4 of c-Myc mRNA 1.4 Relative expression Relative expression of RUNX3 mRNA DLD1 0.8 0.6 0.4 0.2 0.2 + - Control BMP2 BMP4 + - + Control BMP2 BMP4 + - + - + + - + - + + - + - + WiDr 4.5 1.2 3.5 Relative expression of c-Myc mRNA Relative expression of RUNX3 mRNA 2.5 1.5 0.5 Control BMP2 BMP4 0.8 0.6 0.4 0.2 + - + - + Control BMP2 BMP4 Ls174T 1.6 1.4 1.2 1.2 0.8 0.6 0.4 of c-Myc mRNA 1.4 Relative expression Relative expression of RUNX3 mRNA 1.6 0.8 0.6 0.4 0.2 0.2 Control BMP2 BMP4 + - + - + Control BMP2 BMP4 222 Colo205 1.4 1.2 Relative expression of c-Myc mRNA Relative expression of RUNX3 mRNA 2.5 1.5 0.5 0.8 0.6 0.4 0.2 + - Control BMP2 BMP4 + + - Control BMP2 BMP4 + - + - + + - + - + Colo320 1.6 1.2 Relative expression of c-Myc mRNA RUNX3 mRNA Relative expression of 1.4 0.8 0.6 0.4 0.2 1.2 0.8 0.6 0.4 0.2 + - Control BMP2 BMP4 + + - Control BMP2 BMP4 RKO 1.6 Relative expression of c-Myc mRNA 1.4 1.2 0.8 0.6 0.4 0.2 Control BMP2 BMP4 + - + - + *RUNX3 expression undetected 223 LoVo 1.2 2.5 Relative expression of c-Myc mRNA Relative expression of RUNX3 mRNA 1.5 0.5 0.8 0.6 0.4 0.2 Control BMP2 BMP4* + - Control BMP2 BMP4 + + - + - + - + + - + - + + - + - + *RUNX3 expression undetected SW403 1.2 1.6 Relative expression of c-Myc mRNA Relative expression of RUNX3 mRNA 1.4 1.2 0.8 0.6 0.4 0.2 0.8 0.6 0.4 0.2 Control BMP2 BMP4 + - + - + Control BMP2 BMP4 Colo201 2.5 Relative expression of c-Myc mRNA RUNX3 mRNA Relative expression of 3.5 1.5 0.5 2.5 1.5 0.5 Control BMP2 BMP4 + - + - + Control BMP2 BMP4 224 CaCo2 1.2 Relative expression of c-Myc mRNA Relative expression of RUNX3 mRNA 1.2 0.8 0.6 0.4 0.2 0.8 0.6 0.4 0.2 + - Control BMP2 BMP4 + + - Control BMP2 BMP4 + - + + - OUMS23 3.5 1.2 Relative expression of c-Myc mRNA Relative expression of RUNX3 mRNA 2.5 1.5 0.5 0.8 0.6 0.4 0.2 + - Control BMP2 BMP4 + - + Control BMP2 BMP4 + - + - + + - + - + SW837 1.2 1.2 Control BMP2 BMP4 Relative expression of c-Myc mRNA Relative expression of RUNX3 mRNA 1 0.8 0.6 0.4 0.8 0.6 0.4 0.2 0.2 + - + - + Control BMP2 BMP4 225 CCK81 2.5 1.2 Relative expression of c-Myc mRNA RUNX3 mRNA Relative expression of 0.8 0.6 0.4 0.2 1.5 0.5 + - Control BMP2 BMP4 + - + Control BMP2 BMP4 + - + - + Ls513 1.4 1.4 1.2 Relative expression of c-Myc mRNA RUNX3 mRNA Relative expression of 1.2 0.8 0.6 0.4 0.8 0.6 0.4 0.2 0.2 + - Control BMP2 BMP4 + - + Control BMP2 BMP4 + - + + - Ls1034 1.6 1.4 1.4 Relative expression of c-Myc mRNA Relative expression of RUNX3 mRNA 1.2 0.8 0.6 0.4 1.2 0.8 0.6 0.4 0.2 0.2 Control BMP2 BMP4 + - + - + Control BMP2 BMP4 + - + - + 226 Relative expression of c-Myc mRNA CoCM1 1.4 Relative expression of RUNX3 mRNA 1.2 0.8 0.6 0.4 0.2 + - Control BMP2 BMP4 + - + 2.5 1.5 0.5 Control BMP2 BMP4 + - + - + 1.4 1.2 1.2 Relative expression of c-Myc mRNA Relative expression of RUNX3 mRNA SW620 0.8 0.6 0.4 0.2 0.8 0.6 0.4 0.2 + - Control BMP2 BMP4 + - + Control BMP2 BMP4 + - + - + + - + - + RCM1 1.4 1.6 1.2 Relative expression of c-Myc mRNA Relative expression of RUNX3 mRNA 1.4 1.2 0.8 0.6 0.4 0.6 0.4 0.2 0.2 Control BMP2 BMP4 0.8 + - + - + Control BMP2 BMP4 227 HCC56 1.2 Relative expression of c-Myc mRNA Relative expression of RUNX3 mRNA 2.5 1.5 0.5 Control BMP2 BMP4 0.8 0.6 0.4 0.2 + - + - + Control BMP2 BMP4 + - + - + Effect of BMP2/BMP4 on RUNX3 and c-Myc expression levels in 22 colorectal cancer cell lines. Colorectal cancer cells were treated with 100ng/ml BMP2 or BMP4 for 48 hours. RUNX3 mRNA expression levels were measured by real-time quantitative PCR using Taqman probes for human RUNX3 and GAPDH, which was used for normalization. 228 Appendix A Relative Luciferase Activity 25 pGL3 c-Myc Pro 20 15 10 Control BMP2 BMP4 B + - + - + Mutant RUNX promoter 1.4 Relative Luciferase Activity Mutant TCF promoter 1.2 0.8 0.6 0.4 0.2 Control BMP2 BMP4 + - + - + 229 C Relative Luciferase Activity 4.5 3.5 2.5 1.5 0.5 pGL3 Control BMP2 BMP4 + - + - c-Myc Pro + + - + - + mR12 + - + - + mT123 + - + - + Luciferase reporter assays using the human wild-type c-Myc promoter. (A) Repression of the c-Myc promoter activity by BMP2/BMP4 in HCT116 cells. HCT116 cells were transfected with either the control parental pGL3 basic vector or with a pGL3 vector containing the 2.5kB human c-Myc promoter. After transfection, HCT116 cells were either unstimulated or stimulated with 100ng/ml BMP2 or 100ng/ml BMP4. (B) Mutational analysis of the human c-Myc promoter. Mutations of either the RUNX- or TCF-binding elements abolished the repression of the human c-Myc promoter activity by BMP2 and BMP4. HCT116 cells were transfected with the mutant c-Myc promoter constructs. 48 hours post-transfection, cells were treated with 100ng/ml of BMP2 or BMP4. (C) HCT116 cells were transfected with the indicated promoter constructs. Only wild-type c-Myc promoter activity was inhibited by BMP2 and BMP4. BMP treatment did not repress the activity of the c-Myc promoters with mutant RUNX-binding sites (mR12) and mutant TCF-binding sites mT123). Relative luciferase activity was measured after 24 hours. All firefly luciferase activities were normalised to the Renilla luciferase activity of pRL-basic, which was used as an internal transfection control. 230 Appendix A Relative expression of RUNX3 mRNA 1.6 1.4 1.2 0.8 0.6 0.4 0.2 HCT116 siControl siMyc siRUNX3 B RUNX3 (5G4) β-actin + - HCT116 siControl siMyc siRUNX3 SNU16 + - + - + - + + Ratio of RUNX3/B-actin 1.2 1.00 0.79 0.8 0.85 0.6 0.4 0.2 0.04 HCT116 siControl siMyc siRUNX3 231 C 1.20 Relative expression of c-Myc mRNA 1.4 1.2 1.00 0.85 0.91 + + - + + 1.00 1.01 + + - + + - 0.8 0.6 0.4 0.2 siControl siRUNX3 Control BMP2 BMP4 + + - + + Suppression of RUNX3 expression level by RUNX3-siRNA rescues BMP-induced cMyc suppression in HCT116 colorectal cancer cell line. siRNA against RUNX3, c-Myc or control siRNA were transfected into HCT116. After 48 hours, protein and total RNA were prepared. Real-time PCR was carried out to determine changes in gene expression levels of RUNX3 (A). All expression values are relative to the level of RUNX3 in the parental HCT116 cells. Western blot analysis was performed to determine changes of RUNX3 protein expression (B). The ratios of RUNX3/β-actin densitometry levels are shown below the blot. SNU16 cell line was used as a positive control for RUNX3 expression. siRNA against RUNX3, c-Myc or control siRNA were co-transfected with BLOCK-iT Fluorescent Oligo into HCT116. RNA was prepared from FITC-positive cells. Real-time PCR was performed to determine changes in c-Myc expression and expression levels of all samples are expressed relative to the c-Myc levels in the sample transfected with siControl, and treated with vehicle control (C). c-Myc expression levels are expressed relative to the untreated samples of siControl and siRUNX3, to determine the effect of BMP treatment on changes in c-Myc expression. 232 Appendix A Relative Luciferase Activity 1.4 1.2 c-Myc Pro 1.10 1.06 1.00 0.90 * 0.8 0.64 0.6 0.4 0.2 pcDNA CA-ALK3 DN-ALK3 Smads1,5 + - + + + - + + + - Relative Luciferase Activity B 1.2 1.00 c-Myc Pro 0.8 0.54 0.6 0.42 0.4 0.38 0.2 0μg 0.01μg 0.02μg 0.03μg RUNX3 Luciferase reporter assays using the human wild-type c-Myc promoter in 293T cells. (A) Repression of the c-Myc promoter activity by transient co-transfections of CA-ALK3 and Smads and 5. (B) Dose-dependent repression of the human c-Myc promoter by transient transfections of RUNX3 in 293T cells. All firefly luciferase activities were rmalized to the Renilla luciferase activity of pRL-TK, which was used as an internal transfection control. 233 [...]... (ΔΨm) GADD45β interacts and activates mitogen-activated protein kinase kinase 4 (MKK4), which in turn activates Mitogen-activated protein kinase (MAPK) p38, leading to caspase-8 and Bad activation This mobilizes release of cytochrome C from the mitochondria, and initiates the apoptotic pathway (Takekawa et al., 2002; Yoo et al., 2003) Bim is an important pro-apoptotic factor and is required for normal... components of the TGF-β pathway have been demonstrated (Hanai et al., 1999) RUNX3 forms complexes with R-Smads, that are part of the TGF-β/activin pathways, as well as those part of the BMP pathways Three of the five R-Smads, Smad1, -5 and -8 mediate BMP signal transduction, while Smad2 and -3 mediate TGF-β and activin signalling (Massague and Wotton, 2000; Miyazono et al., 2001) A tight linkage between RUNX3. .. induction of apoptosis, by activating Bim (Yamamura et al., 2006) and inactivation of RUNX3 attenuated apoptosis mediated by Bim DAPK is a calcium/calmodulin-dependent serine/threonine kinase, and participates in various apoptotic systems Similarly, DAPK also acts on the mitochondria to mobilize cytochrome C release and caspase activation (Jang et al., 2002) Hence, the apoptotic response of cells to... transcription factors at the gene promoter Interaction in the nucleus includes Smad binding to an adjacent DNA sequence, association of the Smad complex with a DNAbinding transcription factor and association of R-Smads with transcriptional coactivators, such as CBP or p300 More than 30 transcription factors have been reported to interact with Smads, including RUNX, GATA-3 and c-Jun (Miyazawa et al.,... domain-only factor Bim and the death-associated protein kinase (DAPK) (Tachibana et al., 1997) TIEG1, a zinc-finger transcription factor, regulates expression of other apoptotic genes TIEG1-induced apoptosis was shown to involve Bax and Bim up-regulation, Bcl-2 19 and Bcl-XL down-regulation, release of cytochrome c from mitochondria, activation of caspase 3 and destroying the mitochondrial membrane potential... degradation through the recruitment of ubiquitin-ligases that induce proteasomal degradation (Ebisawa et al., 2001; Hayashi et al., 1997; Kavsak et al., 2000; Zhang et al., 16 2001) In addition, I-Smads can also antagonize the signalling pathway by interacting with the activated type-I receptor, thereby preventing access of the R-Smads to the type-I receptors Physical interaction between RUNX3 and... development of recombinant DNA technology in the 1970s and 1980s, has made a major impact in the field of cancer research Since then we have begun to study and understand the causes of cancer at a molecular level and to develop cancer therapies based on this knowledge We have seen an explosion in our understanding of cancer in the past fifty years and new scientific discoveries are made everyday in the laboratories... The probable causes, mechanisms, management and treatments for cancer have provoked much debate and controversy in the scientific and medical community Today, cancer is generally defined as a pathological expansion of a tissue resulting in morbidity, a disease akin to a modern day plague that kills our dearest and nearest with the capriciousness of the ancient plagues Hopefully, one day in the near future,... gene inactivation 12 1.7 RUNX3 as a tumour suppressor 1.7.1 Role of RUNX3 in the TGF-β pathway In recent years, the RUNX family of transcription factors has attracted broad interest due to its involvement in a large range of cancers As mentioned earlier, most published evidence points to the role of RUNX3 as a tumour suppressor, especially in the gastric context RUNX3 is a downstream target of the... the site of regulatory phosporylation by other signalling kinases The MH2 domain is the major protein- protein interaction domain and plays important roles in interaction with type I receptors, oligomer formation with other Smads and transcriptional activation MH1 domains are conserved only in R-Smads and Co-Smad whereas MH2 domain are highly conserved in all three classes of Smads (Miyazono et al., 2005) . understanding of cancer in the past fifty years and new scientific discoveries are made everyday in the laboratories. The probable causes, mechanisms, management and treatments for cancer have. four years. I am grateful to have been able to learn from him. His depth of knowledge and vast wisdom is a constant inspiration and motivation. In addition, I am also appreciative of his continued. say a big thank you to Dr Yasuko Yamamura, who taught me the basic research skills and techniques, which laid the foundation for my thesis project. A special word of thanks to Dr Hiroshi Ida,