Role of protein misfolding pathway in HBX induced hepatocellular carcinoma

ROLE OF PROTEIN MISFOLDING PATHWAY IN HBX-INDUCED HEPATOCELLULAR CARCINOMA (HCC) TAN SU YIN NATIONAL UNIVERSITY OF SINGAPORE 2011 ROLE OF PROTEIN MISFOLDING PATHWAY IN HBX-INDUCED HEPATOCELLULAR CARCINOMA (HCC) TAN SU YIN (B.Sc.(Hons.), NTU) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF MEDICINE YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2011 I ACKNOWLEDGEMENTS It would not have been possible to complete my journey in this masters thesis without the help and support from many kind people around me. I would like to express my deepest appreciation to my supervisor, Dr Matiullah Khan, for his invaluable guidance, advice and insightful discussions throughout my university years and in this course of research. My thanks also extend to Cancer Science Institute as well as National University of Singapore, where the project was carried out, for the opportunity to pursue this degree through their sponsorship and support. My heartfelt thanks to Dr Azhar Ali and Dr Angela Ng for their valuable discussion and technical advices whenever needed. I have benefited greatly from their wisdom and sharing of expertise in scientific knowledge as well as in life experiences. Special thanks to Dr Azhar Ali, Dawn and Sarawut for taking their precious time to proof-read this thesis. I would like to thank all the past and present members of this lab for the wonderful working experience, especially Angie, Jess, Lizan, Dawn, Haris, Lifeng and Wanqiu whom all have contributed to the success of this thesis in one way or another. It has been a real pleasure working with all of you throughout these years. My deepest gratitude to my dearest friends, Meg, Li Ren, Pei Li, John, Ben, Mei Ling and Sarawut for their care and concern towards my well-being. Thanks for being such wonderful colleagues and friends and I really treasure all the joys and laughter that we have shared. Lastly, I would like to express my most sincere thanks to my family. I could not have completed without the immense love and unequivocal support from them. Heartfelt thanks to my dad and my little sister for cheering me up and ensuring that I achieve the goals in my life. Thank You. Tan Su Yin August 2011 II TABLE OF CONTENTS ACKNOWLEDGEMENTS TABLE OF CONTENTS I II VII SUMMARY LIST OF TABLES VIII LIST OF FIGURES IX ABBREVIATIONS XI Chapter 1. Introduction 1 1.1 Liver Cancer 1 1.2 Hepatocellular Carcinoma (HCC) 1 1.2.1 Epidemiology 1 1.2.2 Aetiological factors of HCC 2 1.2.2.1 Hepatitis B virus (HBV) 2 1.2.2.2 Hepatitis C virus (HCV) 3 1.2.2.3 Aflatoxin B1 (AFB1) 4 1.2.2.4 Alcohol 5 Diagnosis and treatment 6 1.2.3.1 Screening tests 6 1.2.3.2 Treatment 7 Challenges in understanding HCC 8 1.2.3 1.2.4 1.3 Acute promyelocytic leukemia (APL) 9 1.3.1 PML-RARα 9 1.3.2 Nuclear hormone co repressor (N-CoR) 10 III 1.3.3 1.4 Current knowledge of PML-RARα,  N-CoR and its perceived mechanism in APL 10 ER stress and unfolded protein response (UPR) 12 1.4.1 Protein folding 12 1.4.2 Unfolded protein response 13 1.4.3 UPR-induced apoptosis 15 1.5 Ubiquitin Proteasome Pathway (UPP) 19 1.6 Autophagy 19 1.6.1 Macroautophagy 20 1.6.2 Chaperone-mediated autophagy (CMA) 22 1.7 Current perspective in APL and study hypothesis and objectives 23 1.7.1 Current perspective in APL 23 1.7.2 Study hypotheses and objectives 24 Chapter 2. Materials and Methods 27 2.1 Materials 27 2.1.1 General Reagents 27 2.1.2 Antisera 28 2.1.2.1 Western Blotting 28 2.1.2.2 Immunofluorescence Staining 29 Primer Sequences 29 2.1.3.1 Semi-Quantitative RT-PCR primers 29 2.1.3.2 siRNA sequences 30 2.1.4 Drugs and Inhibitors 30 2.1.5 Hepatocellular Carcinoma Tissues 31 2.1.5.1 Human Samples 31 2.1.3 IV 2.1.6 2.1.7 2.2 2.1.5.2 Tissue Microarray 32 Cell lines 32 2.1.6.1 Liver cancer cell lines 32 2.1.6.2 HEK 293T and HeLa cell lines 32 Plasmids 32 2.1.7.1 pACT vector 32 Methods 33 2.2.1 Cell Culture Treatment 33 2.2.1.1 Cell culture 33 2.2.1.2 Treatment with drugs and inhibitors 33 2.2.1.3 Transfection 33 2.2.2 2.2.1.3.1 DNA transfection of 293T cells and HeLa cells 33 2.2.1.3.2 N-CoR siRNA knockdown in HCC cells 34 Protein extraction 34 2.2.2.1 Mammalian cells 34 2.2.3 Immunoblotting/Western Blotting 35 2.2.4 Immunohistochemistry 36 2.2.5 Immunofluorescence and confocal microscopy 36 2.2.6 RNA extraction 37 2.2.6.1 Mammalian cells 37 Reverse transcription polymerase chain reaction (RT-PCR) amplification 38 2.2.7.1 DNA agarose gel electrophoresis 39 Biological Assays 39 2.2.8.1 Luciferase assay 40 2.2.7 2.2.8 2.2.8.2 ATP assay 40 V 2.2.8.3 Ubiquitination assay/ Immunoprecipitation 40 2.2.8.4 Solubility assay 41 Chapter 3. Results 42 3.1 Analysis of N-CoR status in human liver cancer cells and tumours 42 3.1.1 Loss of N-CoR protein in multiple human liver cancer cell lines 42 3.1.2 Loss of N-CoR protein in human liver cancers 42 3.2 3.3 Elucidating the mechanism of N-CoR loss 44 3.2.1 N-CoR loss in HCC cells is linked to misfolding 44 3.2.2 Inverse relationship between N-CoR and HBX expression levels 45 3.2.3 Co-expression with HBX preferentially localizes N-CoR to the cytosol 46 3.2.4 N-CoR in HBX positive HCC cells is found to be co-localized with HBX in the cytosol 48 3.2.5 N-CoR protein in liver tumour sections is also found to be localized in the cytosol 52 3.2.6 HBX induces N-CoR insolubility 52 3.2.7 HBX promotes degradation of ectopic N-CoR in transfected 293T cells 53 3.2.8 HBX promotes ubiquitin-proteasome mediated degradation of N-CoR 54 3.2.9 N-CoR loss in HBX positive HCC cells is mediated by the ubiquitin-proteasome pathway 55 The role of misfolded N-CoR in HBX-induced UPR 58 3.3.1 HBX positive HCC cells exhibit amplification of ER stress 58 3.3.2 HBX promotes ATF6 activation 62 VI 3.3.3 3.4 HBX abrogates N-CoR mediated repression of the ATF6 promoter 63 Investigating the molecular mechanism underlying the misfolded NCoR induced transformation of HCC cells 66 A. N-CoR misfolding and Chronic inflammation 66 3.4.1 Upregulation of APR gene transcript in HCC cells 66 3.4.2 Pro-inflammatory cytokine abrogates N-CoR function 66 3.4.3 APR genes are not repressed by N-CoR 68 B. N-CoR misfolding and Epithelial Mesechymal Transition (EMT) 69 3.4.4 The levels of EMT gene transcripts in HCC cells are high 69 3.4.5 EMT genes are not repressed by N-CoR 70 C. N-CoR misfolding and Autophagy 71 3.4.6 Autophagy is activated in HCC cells 71 3.4.7 Formation of autophagosomes in HBX positive cells 72 3.4.8 Loss of N-CoR may be linked to autophagy 76 3.4.9 HBX activates autophagy 77 3.4.10 Autophagy supports the growth of HCC cells 77 Chapter 4. Discussion 81 4.1 Role of misfolded N-CoR in HCC pathogenesis 81 4.2 HBX induces a conformational change in N-CoR 81 4.3 HBX-induced conformational change leads to instability and N-CoR protein degradation 82 4.4 Role of autophagy in the survival and growth of HCC cells 83 4.5 Role of HBX-induced misfolded N-CoR in the activation of UPR and autophagy 84 VII 4.6 Future areas of research 86 4.6.1 Mechanism of autophagy mediated N-CoR loss in liver cancer pathogenesis 86 4.6.2 Identification of misfolded N-CoR as a molecular target in HCC 86 4.6.3 Appropriate experimental controls 88 REFERENCES 90 VII SUMMARY Transcriptional control imparted by nuclear receptor co-repressor (N-CoR) plays an important role in the growth suppressive function of several tumour suppressor proteins. Abrogation of this transcriptional control due to a misfolded conformation dependent loss (MCDL) of N-CoR has been implicated in acute promyelocytic leukemia (APL). It was hypothesized that an APL like MCDL of NCoR might be involved in other malignancy. Indeed, the initial screening of N-CoR status in various liver cancer (HCC) cell lines revealed an APL like MCDL of N-CoR protein. The N-CoR protein presented in these HCC cells was misfolded and was linked to the amplification of endoplasmic reticulum (ER) stress and cytoprotective unfolded protein response (UPR). siRNA-induced N-CoR ablation led to selective reduction of intracellular ATP level and significantly compromised the growth of HCC cells, suggesting an important role of energy, possibly derived from N-CoR catabolism, in HCC cell growth. These findings identify an important role of autophagy-induced recycling of misfolded N-CoR protein in the selective activation of autonomous survival and growth in HCC cells. VIII LIST OF TABLES Table 2.1. List of Chemicals, Reagents and Kits 27 Table 2.2. List of Primary Antibodies 28 Table 2.3. List of Secondary Antibodies 28 Table 2.4. List of Primary Antibodies 29 Table 2.5. List of Secondary Antibodies 29 Table 2.6. List of semi-quantitative RT-PCR primers 29 Table 2.7. List of siRNA sequences used in siRNA mediated gene knockdown 29 Table 2.8. List of drugs and inhibitors 30 Table 2.9. Clinical characteristics of patient samples used in this study 31 IX LIST OF FIGURES Figure 1.1. Model of bifunctional role of nuclear receptor corepressor in acute promyelocytic leukemia pathogenesis 11 Figure 1.2. UPR signalling pathways in mammalian cells 16 Figure 1.3. ER stress pathways implicated in mediating cell apoptosis 18 Figure 1.4. Kinetics of N-CoR misfolding in APL 25 Figure 3.1.A-B Selective loss of N-CoR protein in HCC cells 43 Figure 3.2. Evidence of N-CoR loss in primary human liver cancer samples 44 Figure 3.3. Genistein promotes stabilization of N-CoR protein in SK Hep1 cells 45 Figure 3.4. N-CoR protein level in HCC cells is inversely related to HBX transcript level 46 Figure 3.5. HBX promotes cytosolic retention of N-CoR protein 47 Figure 3.6. N-CoR of HBX positive HCC cells is found in the cytosol 51 Figure 3.7. N-CoR protein is mainly localized in the cytosol in tumour sections 51 Figure 3.8. HBX can induce N-CoR insolubility 53 Figure 3.9. HBX promotes N-CoR degradation 54 Figure 3.10. HBX promotes ubiquitination of N-CoR protein 56 X Figure 3.11.A-B Ubiquitin-proteasome mediated degradation of N-CoR protein in HBX positive HCC cells 57 Figure 3.12.A-D HBX positive cells exhibit high level of ER stress 59 Figure 3.13.A-C Regulation of UPR by HBX and N-CoR 64 Figure 3.14. Ectopic expression of HBX in HeLa cells repressed ATF6 promoter activity 65 Figure 3.15. N-CoR loss in HCC cells might be associated with an upregulation of APR genes 67 Figure 3.16. Pro-inflammatory cytokine promotes degradation of N-CoR 68 Figure 3.17. APR genes were up-regulated in a dose dependent manner after genistein treatment 69 Figure 3.18. Expression level of EMT genes 70 Figure 3.19. N-CoR did not repress EMT gene expression after genistein treatment 71 Figure 3.20. Level of LC3-II in HBX positive cells is elevated 73 Figure 3.21. Autophagosomes are seen in HBX positive cells 73 Figure 3.22. Bafilomycin promotes stabilization of N-CoR protein in SK Hep1 cells 76 Figure 3.23.A-B HBX induced activation of autophagy is mediated by misfolded N-CoR 78 Figure 3.24. N-CoR loss is linked to ATP mediated growth of SK Hep1 cells 80 Figure 4.1. Schematic representation of the regulation of UPR and autophagy in normal and HCC cells. 86 XI ABBREVIATIONS AFB1 aflatoxin B1 AFP alpha-fetoprotein AML acute myeloid leukemia APL acute promyelocytic leukemia ASK1 apoptosis-signal-regulating kinase 1 Atg Autophagy-related genes ATF activating transcription factor BA-1 Bafilomycin A1 BiP binding Ig protein BSA bovine serum albumin CHOP C/EBP homologous protein CMA chaperone-mediated autophagy CT computed tomography DCP des-gamma-carboxy prothrombin DAPI 4,6-diamidino-2-phenylindole DMSO dimethyl sulfoxide DNA deoxyribonucleic acid EDEM ER-degradation-enhancing  α-mannosidase-like protein EDTA ethylenediaminetetraacetic acid ERAD ER associated degradation eIF2α elongation factor 2α EM electron microscopy EMT epithelial-mesenchymal transition ER endoplasmic reticulum ERSE endoplasmic reticulum stress element ERAD ER-associated degradation FAB French American and British FBS fetal bovine serum XII GFP green fluorescent protein GRP glucose-regulated protein HBV hepatitis B virus HBX hepatitis B virus X protein HCC hepatocellular carcinoma HCV hepatitis C virus HDACs histone deacetylase complexes HMW high molecular weight HRAS Harvey-ras proto-oncogene Hsp heat shock protein IF immunofluorescence IHC immunohistochemistry IRE1 inositol requiring kinase 1 JNK c-Jun N-terminal kinase kb kilo base kDa kilo Dalton LAMP2A lysosomal-associated membrane protein 2A MCDL misfolded conformation dependent loss mins minutes mRNA messenger RNA MRI magnetic resonance imaging mTOR rapamycin N-CoR nuclear receptor co-repressor OSGEP O-Sialoglycoprotein endopeptidase PBS phosphate buffered saline PDI protein disulpide isomerase PERK RNA-activated protein kinase like endoplasmic reticulum kinase PI3K phosphatidyl-inositol 3 kinase PI3KC3 class III phosphatidyl inositol 3-kinase PLB passive lysis buffer XIII PML promyelocytic leukemia POD PML oncogenic domains PtdIns phosphatidyl inositol PtdIns(3,4,5) P3 phosphatidylinositol-3,4,5- triphosphate PTM post translational modification PVDF polyvinylidene difluoride RA retinoic acid RARα   retinoic  acid  receptor  α RFA radiofrequency ablation ROS reactive oxygen species RT-PCR reverse transcription-polymerase chain reaction S1P site-1 protease SDS sodium dodecyl sulphate SDS-PAGE SDS-Polyacrylamide gel electrophoresis siRNA small interfering RNA SMRT silence mediator of retinoic and thyroid receptors TRAF2 TNF receptor-associated factor 2 Ub Ubiquitin UGGT UDP-glucose:glycoprotein glucosyltransferase UPP ubiquitin proteasome pathway UPR unfolded protein response UPRE unfolded protein response element US ultrasound Vps34 vesicular protein sorting 34 XBP1 X-box DNA-binding protein 1 1 Chapter 1. Introduction 1.1 Liver Cancer Liver cancer is one of the most lethal malignancies worldwide. Globally it ranks the second most frequent cause of cancer death in men and the sixth in women (Ahmedin et al., 2011). Liver cancer consists of several histologically different primary hepatic malignancies, such as cholangiocarcinoma (bile duct cancer), hepatoblastoma (liver cancer affecting young children) and haemangiosarcoma (cancer arising from the blood vessels of the liver). Hepatocellular carcinoma (HCC), also called malignant hepatoma, is by far the most common type. 1.2 Hepatocellular Carcinoma (HCC) 1.2.1 Epidemiology Hepatocellular carcinoma (HCC) is a major cause of morbidity and mortality in human. It is the fifth most common cancer worldwide, and the third leading cause of cancer-related deaths (Ferlay et al., 2008) with an estimate of one million deaths annually (Bosch et al., 2004; Cha and Dematteo, 2005). The incidence of liver cancer varies around the world and is highest in Mongolia with 116.6 cases per 100,000 person-years for men; 74.8 cases per 100,000 person-years for women (Ferlay et al., 2008). Over 80% of HCC cases occur in developing countries such as sub-Saharan Africa, Southeast Asia, and East Asia (including Mongolia). In contrast, the incidence of HCC is much lower in developed countries such as North America with 6.8 cases per 100,000 person-years for men; 2.2 cases per 100,000 person-years for women. 2 In Singapore, HCC is the fourth most frequently occurring cancer in men with an overall incidence of 18.9 per 100 000 person-year (Singapore Cancer Society). The incidence is highest in the Chinese population as compared to the other ethnic groups (Yuen et al., 2009). 1.2.2 Aetiological factors of HCC Significant differences in HCC incidence across different countries reflect regional differences in the prevalence of specific aetiological factors. The most prominent factors associated with an increased risk of HCC include chronic hepatitis B and C viral infection, aflatoxin B1 contamination and severe alcohol abuse. Other aetiological factors which occurred at a lower frequency include demographic characteristics (gender and age), lifestyle (smoking) and clinical factors (metabolic abnormalities, diabetes, and obesity). Overall, viral infection has been indexed as the major risk factor because more than 80% of HCC in humans are attributable to infection with either hepatitis B or C virus or both (Parkin et al., 2006). Nevertheless, it should be highlighted that the risk of HCC increases in the event of multiple risk factors. 1.2.2.1 Hepatitis B virus (HBV) Hepatitis B virus (HBV) is a 3.2kb partially double stranded DNA virus that can elicit an acute and chronic inflammatory response in the liver. It infects approximately 2 billion individuals globally and causes an estimated 320 000 deaths annually (Lavanchy, 2004). As such, HBV-associated HCC is a disease of global importance and is ranked the top 5 most frequent cancers worldwide (Marrero, 2006). In Singapore, one third of HCC patients are HBV positive (Yuen et al., 1999). 3 An array of processes is involved in HBV-induced hepatocarcinogenesis. These include host-viral interactions (Block et al., 2003; Nowak et al., 1996; Wieland et al., 2004; Rehermann et al., 2004), sustained cycles of necrosis-inflammationregeneration (Block et al., 2003; Lok et al., 2001), viral-endoplasmic reticulum interactions (Kojima, 1982), viral integration into the host genome (Brechot, 2004) and targeted activation of oncogenic pathways by viral proteins (Matsubara et al., 1990). The 16.5 kDa HBV X protein (HBX) is suspected to be an important oncogenic factor in hepatocarcinogenesis (David et al., 2006). In vivo studies have shown that it can trans-activate a large number of promoters related to inflammation and cell proliferation through protein-protein binding. This mechanism allows HBX to undergo favourable alteration in the cellular environment for further viral replication (Tang et al., 2006). In the infected liver host cells, HBX appears to induce variety of responses such as genotoxic stress response, transcription modulation, protein degradation, cellular signalling pathways, cell cycle checkpoints and apoptosis (Murakami, 2001). HBX has since been proposed to be strongly related to the progression of HCC however its exact role in malignant transformation has yet to be fully elucidated. 1.2.2.2 Hepatitis C virus (HCV) Hepatitis C virus (HCV) is a 9.6kb non-cytopathic virus of the flaviviridae family. It possesses a positive single-stranded RNA genome encoding non structural proteins (NS2, NS3, NS4A, NS5A and NS5B) and structural proteins (core, E1, E2). HCV has been implicated to be responsible for an estimate of 170 million chronic infections worldwide. Further, it is a major risk factor for the development of HCC 4 (McGlynn and London, 2005), predominantly in developed nations such as Europe, United States and Japan (Bosch et al., 1999) Analogous to HBV infection, HCV induces liver inflammation followed by a continuous cycle of hepatocyte cell death and regeneration. However, unlike HBV, its genome is RNA-based, thus it is unable to integrate to host genome to induce hepatocarcinogenesis (Rehermann et al., 2005). Instead, HCV has been shown to possess the   propensity   to   evade   the   host’s   immune response to promote tumourigenesis (Gale and Foy, 2005). The HCV’s  RNA and core proteins are found to impair dendritic functions which are important for T-cell activation (Pachiadakis et al., 2005) and evade immune-mediated cell death by interacting with interferon-α  and   tumour necrosis factor-α  (Melén et al., 2004; Gale and Foy, 2005). Additional roles of the HCV core proteins in the pathogenesis of HCC include inhibition of apoptosis (Kamegaya et al., 2005), interference with cell signalling pathways to modulate cell proliferation (Hino O. et al., 2002; Macdonalds et al., 2003) and modulate p53 transcription thereby affecting the p53-regulated pathways (Majumder et al., 2001). In mouse models, HCV core proteins have been shown to induce hepatic steatosis as well as reactive oxygen species (ROS) and oxidative stress (Moriya et al., 1998; 2001). These data collectively suggest that viral proteins play a direct role in inducing hepatocarcinogenesis. 1.2.2.3 Aflatoxin B1 (AFB1) Aflatoxin B1 (AFB1) is a mycotoxin that is produced by the Aspergillus fungi. Epidemiological studies have shown that AFB1 consumption increases HCC risk by approximately four-fold. Further, it has been observed that areas of high HCC incidence and high AFB1 intake correspond to areas where HBV is endemic 5 (Groopman et al., 1996; Qian et al., 1994). Previous studies have also shown that the combination of AFB1 and HBV is likely to increase the risk of HCC by 60-fold (ElSerag and Ruldolph, 2007; McGlynn and London, 2005). Once ingested, AFB1 is metabolized to form a pro-mutagenic DNA adduct which results in a specific point mutation from guanine to thymine at codon 249 (AGG to AGT) in the tumour suppressor, p53 (Moradpour and Blum, 2005; Wild and Montesano, 2009). To some extent, the loss of p53 function via mutation is believed to induce loss of cell growth control and eventually promote hepatocarcinogenesis. Further, this loss of p53 function has also been reported to assist the mutational activation of oncogenes such as human Harvey-ras proto-oncogene (HRAS) (Riley et al., 1997). Together, the mutational actions of AFB1 may contribute to the development of HCC. 1.2.2.4 Alcohol Chronic alcohol intake has long been recognized as an important HCC risk factor. Epidemiology data suggests that alcohol has been found to be a more prominent risk factor for HCC in low incidence areas than in high incidence areas. This may be due to the lower mean alcohol consumption and/or the dominant effect of HBV infection in the high-risk population (El-Serag and Ruldolph, 2007; Bosch et al., 2005; McGlynn and London, 2005). Alcohol-induced hepatocarcinogenesis has been associated with the production of inflammatory cytokines through monocytic activation (McClain et al., 2002). Consequently, Kupffer cells are activated with the eventual release of chemokines and cytokines, leading to hepatocytes necrosis. Alcohol also damages the liver through an oxidative-stress mechanism. First, it promotes the development of 6 fibrosis and cirrhosis and creates a conducive HCC microenvironment (Campbell et al., 2005). Second, oxidative stress may decrease STAT1-directed  activation  of  IFNγ   with consequent hepatocyte damage and constant tissue destruction ultimately leads to hepatocarcinogenesis (Osna et al., 2005). 1.2.3 Diagnosis and treatment HCC patients are commonly diagnosed at very late stages due to the asymptomatic features during the course of neoplastic development and the lack of reliable biomarkers. Thus, this disease has a very poor prognosis with more than 50% of the patients dying within 1 year and less than 6% with a 5 year survival rate (Hoofnagle, 2004). 1.2.3.1 Screening tests The most widely used marker today for HCC is alpha-fetoprotein (AFP). In recent years, clinical research has discovered a fucosylated AFP (AFP-L3) to be a new tumour marker with sensitivity of about 56% and specificity of >95% (Li et al., 2001). More importantly, it has been suggested to be a specific indicator for poorlydifferentiated and unfavourable diagnosis (Tateishi et al., 2006). Another established serum biomarker for HCC is an abnormal prothrombin, des-gamma-carboxy prothrombin (DCP) (Liebman, 1989; Aoyagi et al., 1996). DCP has been reported to be the most significant predisposing factor for the development of portal vein invasion which is an indicator of end-stage liver disease (Koike et al., 2001). Hence, it is more commonly used as a diagnostic marker rather than for surveillance. Together, DCP may complement AFP or AFP-L3 for HCC diagnosis purposes. Other promising biomarkers for HCC include Golgi-protein 73 (Kladney et 7 al., 2000; Marrero et al., 2005), glypican-3 (Nakatsura et al., 2003; Capurro et al., 2003), hepatocyte-growth factor (Yamagamim et al., 2002), insulin growth factor 1 (Mazziotti et al., 2002), vascular endothelial growth factor (Poon et al., 2004; Schoenleber et al., 2009) and transforming growth  factor  β-1 (Song et al., 2002; Yao et al., 2007). Other than biomarkers, imaging studies play a crucial role in the diagnosis of HCC. Imaging techniques most commonly used for the diagnosis of HCC include ultrasound (US), computed tomography (CT), magnetic resonance imaging (MRI), and angiography. While ultrasonography is widely accepted for HCC surveillance, spiral computed tomography (CT) or dynamic magnetic resonance imaging are the required imaging techniques for diagnostic confirmation and intrahepatic tumor staging. Currently, with the development of imaging modalities, invasive biopsy is infrequently required prior to treatment, and the diagnosis of HCC is strongly dependent on hemodynamic features (arterial hypervascularity and washout in the venous phase) on dynamic imaging. 1.2.3.2 Treatment To date, the only proven cures for HCC are surgical resection or liver transplantation. However, for liver transplantation, the scarcity of donor liver remains a universal problem. In addition, a major concern for resected HCC patients is the possibility of recurrence that can occur in about 75% of the resected patients (Llovet and Bruix, 1999). In cases when HCC lesions are unresectable, an alternative option is nonsurgical treatment. For example, radiofrequency ablation (RFA) uses a needle with an electrode placed percutaneously into targeted tumour in the presence of ultrasound 8 scanning. The local heat generated then melts the tumour tissues (Gough-Palmer and Gedroyc, 2008). Other treatments include conventional or molecular targeted chemotherapy and radiotherapy. 1.2.4 Challenges in understanding HCC Hepatocarcinogenesis is a multistep process involving diverse aetiologies, ranging from metabolic disorders to hepatoxins to viruses. As documented by numerous independent HCC studies, the genetic aberrations in HCCs are heterogenous whereby distinct molecular players but related genetic pathways are affected (Thorgeirsson et al., 2006). Nevertheless, the exact underlying mechanism of hepatocarcinogenesis remains elusive. To aggravate the situation, early hepatocellular carcinoma is characteristically silent and slow growing with few symptoms until late in disease. The lack of clinically validated biomarkers and clinical identification of hepatocellular carcinoma at advanced stages present difficulties for diagnosis and treatment. While advances in computed tomography and magnetic resonance imaging have markedly increased the sensitivity and specificity of testing, they are still flawed with a relatively high false-positive rate. Several surgical and nonsurgical therapies have been developed, but used with varying degrees of success. Hepatocellular carcinoma in general is a disease of multifactorial etiology and confers many management challenges. It is therefore imperative to identify and characterize new molecular mechanisms associated to HCC. This allows a better understanding of the disease for the identification of effective diagnostic and prognostic markers. 9 1.3 Acute promyelocytic leukemia (APL) Acute promyelocytic leukemia (APL), designated as AML-M3 (acute myeloid leukemia) in FAB (French American and British) classification (Bennett et al., 1976), is a disease characterized by the chromosomal translocation involving retinoic acid receptor-alpha   (RARα)   gene   on   chromosome   17   with   the   promyelocytic   leukemia   gene (PML) on chromosome 15. It represents about 10% of all AML in adults with a lower incidence reported in children (Stone et al., 1990; Chan et al., 1981). 1.3.1 PML-RARα The ramification of chromosomal translocation mentioned earlier is the generation of a fusion oncoprotein PML-RARα. Since its discovery in the 1990s, the transformation role of PML-RARα  in  APL  has  been  well studied. One direct evidence shown in two studies is the generation of an APL-like disease when this fusion protein was targeted to the myelocytic compartment in transgenic mice (He et al., 1997; Grisolano et al., 1997). Studies have elucidated the role of PML-RARα  as  a  dominant   negative transcription repressor. PML-RARα  has  been  proposed  to  recruit  the  N-CoR containing chromatin-remodeling complex to their promoters which eventually repress the target genes for differentiation of granulocytes (Lin et al., 1998; Grignani et al., 1998). It also recruits methylating enzymes and hypermethylates promoters of retionic acid (RA) target genes resulting in transcriptional repression. The overall outcome of this PML-RARα-induced transcriptional inhibition is differentiation arrest of APL cells. 10 1.3.2 Nuclear hormone co repressor (N-CoR) N-CoR is a key component of the co-repressor complex involved in transcriptional control. It was originally identified as a co-repressor of un-liganded nuclear hormone receptors such as thyroid-hormone and retinoic acid (Heinzel et al., 1997; Laherty et al., 1997). Later, it was shown to interact with different corepressors and histone deacetylase complexes (HDACs) to mediate repression by steroid hormone receptors as well as by tumour suppressor Mad to suppress proliferation (Khan et al., 2001). 1.3.3 Current knowledge of PML-RARα,  N-CoR and its perceived mechanism in APL It has now been recognized that N-CoR recruitment by PML-RARα contributes to transformation of APL cells by repressing retinoic acid (RA) target genes essential for the maturation of promyelocytic cells (Lin et al., 1998; Grignani et al., 1998). Nevertheless, this may not be the only mechanism behind APL pathogenesis. It has been proposed recently that PML-RARα could also abrogate NCoR-mediated repressor pathway. In APL cells, two distinct forms of N-CoR protein existed, the natively folded and stable N-CoR localized in the nucleus, and the misfolded and unstable N-CoR found in the cytosol (Ng et al., 2006). Similar to N-CoR, at least two distinct forms of PML-RARα protein, nuclear and cytosolic, are observed in most APL-derived cells. It was thus hypothesized that each form of PML-RARα engages the respective form of N-CoR protein to regulate two opposing transcriptional mechanisms that ultimately leads to derailment of both the activation and repression pathway in a synchronized manner (Matiullah, 2010). Most probably, the nuclear PML-RARα recruits the 11 natively folded N-CoR on the RA promoter and turn off the expression of the RAregulated genes. Simultaneously, the cytosolic form of PML-RARα/N-CoR complex leads to de-repression of self-renewal genes (Fig 1.1). The combined outcome of concurrent deregulation of these two pathways leads to ectopic re-activation of selfrenewal potentials and eventually contributes to transformation in APL. Figure 1.1. Model of bifunctional role of nuclear receptor corepressor in acute promyelocytic leukemia pathogenesis Two distinct forms of PML-RAR, one nuclear another cytosolic, could engage the NCoR protein in a bifunctional manner, which would have two opposing transcriptional outcomes. The nuclear PML-RAR could repress the RA target genes by recruiting the natively folded and fully functional N-CoR to the RA promoter, while the selfrenewal genes originally repressed by N-CoR could be de-repressed owing to PMLRARα-induced misfolding and cytosolic export of N-CoR protein. 12 1.4 ER stress and unfolded protein response (UPR) 1.4.1 Protein folding Protein folding occurs in a specialized compartment- the endoplasmic reticulum (ER). The ER is an oxidising environment that contains high amounts of ATP and Ca2+ required for its proper function (Gaut et al., 1997). In the ER, only correctly folded molecules can proceed further into the secretory pathway and that persistently misfolded proteins are targeted for degradation. Three classes of proteins in the ER assist nascent proteins to fold properly – molecular chaperones like glucose-regulated proteins (GRPs) GRP78, GRP94 and lectin-like proteins, folding enzymes like protein disulpide isomerase (PDI) and ERdegradation-enhancing  α-mannosidase-like protein (EDEM) (Schroder et al., 2005). One of the most abundant proteins in the ER is binding Ig protein (BiP) BiP/GRP78- a member of the heat shock protein (Hsp)70 family of chaperones (Mayer et. al., 1998) that transiently binds a wide variety of newly synthesized secretory proteins but associates more permanently with misfolded or unassembled proteins. The association and dissociation of peptides to BiP/GRP78 are thought to be coupled to the binding and hydrolysis of ATP (Flynn G.C. et al., 1999). The binding of peptides to BiP/GRP78, an ATP-dependent conformational change strengthens the interaction between GRP78 and the unfolded/misfolded protein. Under this condition, protein disulphide isomerase (PDI) can also work to promote disulfide reduction, rearrangement, and re-oxidation until the correct protein conformation is achieved. Once the protein conformation is corrected, refolded proteins will dissociate from BiP/GRP78 (Mayer et al., 2000). 13 Partially folded monoglucosylated N-linked glycans are bound to lectin like proteins - calnexin (transmembrane ER protein) and calreticulin (soluble luminal ER protein) (Bergeron et al., 1998). Glucosidase II then removes the glucose residue on the oligosaccharide and releases the bound glycoprotein. If the glycoprotein remains unfolded, it is re-glucosylated by UDP-glucose:glycoprotein glucosyltransferase (UGGT) for interaction with the lectin like proteins. Numerous cycles of binding to and release from calnexin and calreticulin alternates until the protein is correctly folded or is sentenced for degradation by EDEM by removing its mannose residue (Ellgaard et al., 2003). GRP94, another abundant chaperone protein has been reported to bind partially folded proteins that are unfolded by GRP78 or calnexin/calreticulin and retaining them in the ER (Argon et al., 1999). On the other hand, PDI interacts with the underperforming proteins for retrograde transport into the cytosol for ERassociated protein degradation (ERAD) via the proteasome (Gillece et al., 1999). In brief, misfolded proteins loiter in the ER for re-folding; otherwise, they are translocated in a retrograde manner back to the cytosol and targeted to proteasomal degradation by the 26S proteasome (Ellgaard et al., 1999; Brodsky et al., 1999). 1.4.2 Unfolded protein response ER stress occurs when the processing capacity of the ER is overwhelmed. The normal physiological state of the ER is thus perturbed. Unfolded protein response (UPR) is then activated in response to restore a favourable physiological state. UPR involves three main signalling pathways: the activating transcription factor 6 (ATF6) pathway, the inositol requiring kinase 1 (IRE1) pathway and the 14 double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) pathway. UPR is initiated when GRP78 dissociates from the intracellular receptors and binds preferentially to the misfolded proteins (Bertolotti et al., 2000). Upon sensing ER stress, PERK, a type I transmembrane kinase dissociates from GRP78 and activates itself through dimerization and autophosphorylation (Liu et al., 2000). Once activated, PERK phosphorylates elongation factor 2α (eIF2α),  which  transiently  slows   down the rate of protein translation and attenuates protein synthesis (Harding et al., 1999; Shi et al., 1998). Simultaneously,  phosphorylated  eIF2α  preferentially  initiates   the translation of ATF4 mRNA, which contains an open reading frame within its 5’   untranslated region. ATF4 then translocates into the nucleus and up-regulates ER stress target genes including amino acid transporters and cellular redox control genes (Zhang et al., 2004). More importantly, ER stress will be relieved as ATF4 activation induces the expression of C/EBP homologous protein (CHOP) leading to growth arrest and apoptosis (Ma et al., 2002). Similar to PERK, IRE1 dissociates from GRP78, homodimerizes and transphosphorylates (Shamu and Walter, 1996; Welihinda et al., 1996) and triggers its endo-ribonuclease activity. Its cytosolic activated RNase domain then catalyses the splicing of 26-nucleotide intron sequence from X-box DNA-binding protein 1 (XBP1) mRNA (Sidrauski and Walter, 1997). This resulting XBP1 is a potent activator of UPR related genes which will migrate to the nucleus and bind upstream DNA UPR element (UPRE) or ER stress element (ERSE) (Lee et al., 2003; Yoshida et al., 1998). The UPR genes regulating XBP1 are essential for protein folding, maturation and degradation (Keisuke et al., 2003). 15 Finally, ATF6, a type II transmembrane protein, upon dissociation from GRP78, is translocated from the ER to the Golgi (Chen et al., 2002; Shen et al., 2002). The 50 kDa cytosolic region of ATF6 is then proteolytically cleaved by site-1 protease (S1P) and site-2 protease (S2P) (Shen, 2004). This active cytosolic ATF6 domain then migrates to the nucleus to promote the transcription of ER-resident molecular chaperones and other assistant folding enzymes (Wang et al., 2000; Yoshida et al., 1998). ATF6 may also work in tandem with XBP1 to promote proteins needed to assist in the folding of unfolded proteins in the ER (Keisuke et al., 2003). Another function of the UPR that does not involve any signalling pathway is the degradation of accumulated misfolded proteins to alleviate ER stress as well as to supply nutrients to the cell. This function is achieved by two main pathways. The first is ER-associated protein degradation (ERAD) of misfolded proteins which fail to be refolded, via the ubiquitin proteasome pathway (UPP). The second is autophagy, an alternative non-proteasomal degradation system that has recently been shown to be triggered by ER stress, to remove and recycle misfolded proteins. (Kawakami et al., 2009; Yorimitsu et al., 2006). These two pathways will be further discussed in the following sections. 1.4.3 UPR-induced apoptosis Signalling may switch from pro-survival to pro-apoptotic in conditions of prolonged and severe ER stress. Given the central role of PERK, IRE1 and ATF6 in UPR signalling, it has been suggested that these UPR mediators may be fundamental in facilitating the switch. 16 Figure 1.2. UPR signalling pathways in mammalian cells The UPR is mediated by three ER resident transmembrane proteins that sense ER stress and signal downstream pathways. The PERK kinase is activated by dimerization and phosphorylation. Once activated, it phosphorylates eIF2α, resulting in translation attenuation. Phosphorylated eIF2α selectively enhances translation of the ATF4 transcription factor that induces expression of UPR target genes. Activation of IRE1 by dimerization and phosphorylation causes IRE1 mediated splicing of XBP1 mRNA. Translation of spliced XBP1 mRNA produces a transcription factor that upregulates target genes via the ERSE promoter. ATF6 activation involves regulated intramembrane proteolysis. The protein translocates from the ER to the Golgi where it is proteolytically processed to release a 50 KDa transcription factor that translocates to the nucleus and binds the ERSEs of UPR target genes. All three ER resident transmembrane proteins are thought to sense ER stress through Grp78 binding/release via their respective luminal domains, although structural studies have also suggested that IRE1 may interact with infolded proteins directly. The GADD34 protein, a protein phosphatase up-regulated by the PERK pathway, dephosphorylates eIF2α to restore global protein synthesis. 17 In response to ER stress, IRE1 has been found to recruit the adaptor protein TNF receptor-associated factor 2 (TRAF2) to the ER membrane (Urano et al., 2000).   The IRE1-TRAF2 complex then recruits the apoptosis-signal-regulating kinase 1 (ASK1), which in turn phosphorylates and activates c-Jun N-terminal kinase (JNK) (Nishitoh et al., 1998). Activated JNK inactivates anti-apoptotic BCL2 through phosphorylation, thus causing it to malfunction in sequestering pro-apoptotic molecules and control Ca2+ influx (Bassik et al., 2004). JNK also phosphorylates BH3, releasing them from inhibition thus allowing them to exert their pro-apoptotic effects (Lei et al., 2003). Concurrently, there is CHOP induction by PERK through ATF4 (Szegezdi E. et al., 2006). Expression of the anti-apoptotic BCL2 protein is directly suppressed by CHOP (McCullough et al., 2001). CHOP also enhances the induction of pro-apoptotic proteins such as GADD34 and ERO1 (Marciniak et al., 2004). Additionally, UPR-induced apoptosis is also mediated through caspases. Procaspase 12 (pCP12) is activated when its association with TRAF2 is disrupted (Yoneda et al., 2001). During ER stress, conformational changes in Bax and Bak facilitate the entry of Ca2+ into cytosol, thereby activating m-Calpain, which subsequently cleaves and activates pCP12 (Nakagawa et al., 2000; Zong et al., 2003). Activated caspase-12 (CP12) then cleaves and activates procaspase-9, which in turn activates the downstream caspase cascade including caspase-3 (Morishima et al., 2002; Tan, 2006). Further, the mitochondria can take up Ca2+ released from the ER and trigger release of cytochrome c into the cytoplasm (Crompton, 1999). This permits the formation of an apoptosome which activates caspase-9 and the 18 executioner caspase 3 (Rutkowski and Kaufman, 2004). Together, JNK, CHOP and caspases act synergistically to regulate the apoptotic effect during ER stress. Figure 1.3. ER stress pathways implicated in mediating cell apoptosis I: Activation of the PERK and ATF6 (not shown) pathways leads to the induction of CHOP, which downregulates the expression of the anti apoptotic protein Bcl2 and induces GADD34 and ERO1α. The latter promotes ER stress by increasing ER protein load (via translational recovery by eIF2α dephosphorylation) and altering ER redox conditions. II: Activated IRE1 binds JIK and recruits TRAF2, which leads to the activation of ASK1 and JNK. JNK phosphorylates Bcl2 and BH3 only protein (Bim), initiating mitochondria mediated apoptosis (not shown). III: Recruitment of TRAF2 to IRE1 also permits TRAF2 to dissociate from procaspase 12 (pCP12) residing on the cytoplasmic side of ER membrane, allowing pCP12 activation. During ER stress, Bax and Bak in the ER membrane oligomerize and allow the release of Ca2+ from the ER to the cytosol, which activates m Calpain, which subsequently cleaves and activates pCP12. Active caspase 12 (CP12) cleaves and activated procaspase 9, which in turn activates downstream caspases, including caspase 3. In addition, Ca2+ released from the ER is taken up by the mitochondria, causing mitochondrial inner membrane depolarization and cytochrome c release into the cytoplasm. This allows the formation of the apoptosome (consisting of Apaf1, cytochrome c, ATP and procaspase 9), activation of procaspase 9 and subsequent downstream caspases leading to cell apoptosis. 19 1.5 Ubiquitin Proteasome Pathway (UPP) The Ubiquitin Proteasome Pathway (UPP) consists of concerted actions of enzymes that attach multiple ubiquitin (Ub) molecules to the target protein to mark them for degradation (Glickman et al., 2002; Pickart, 2004). This tagging process leads to their recognition by the 26S proteasome, a very large multi-catalytic protease complex that degrades ubiquitinated proteins to small peptides (Baumeister et al., 1998). Covalent conjugation of ubiquitin onto proteins that are destined for degradation proceeds via a three step mechanism. Initially, E1 (Ub-activating enzyme) modifies and activates ubiquitin so that the C-terminal glycine on Ub will react with the lysine side-chains on the substrate protein. Following activation, E2 (Ub-conjugating enzyme) catalyzes the attachment of Ub to the substrate protein. Finally, E3 (Ub-protein ligase), also the key enzyme in this process, works in concert with E2 enzymes in attachment of Ub to the substrate. 1.6 Autophagy Autophagy is a self-catabolic process where bulk cytoplasmic components and intra-cellular organelles are sequestered in double membrane vesicles called autophagosomes. The mature autophagosome eventually fuses with the lysosome, forming an autolysosome, in which its contents are degraded by lysosomal enzymes. In general, autophagy is seen to play a housekeeping role in removing misfolded or aggregated proteins, clearing damaged organelles, such as mitochondria, endoplasmic reticulum and peroxisomes, as well as eliminating intracellular pathogens. Currently, three types of autophagy have been identified: microautophagy, macroautophagy, and chaperone-mediated autophagy. 20 In microautophagy, cytosolic components are directly taken up by the lysosome itself through invagination of the lysosomal membrane (Kunz et al., 2004). Nevertheless, this function in higher eukaryotes is unclear. Macroautophagy is the major regulated catabolic mechanism that eukaryotic cells use to degrade long-lived proteins and organelles and will be further discussed in the following section. 1.6.1 Macroautophagy This form of autophagy begins with an isolation membrane or phagophore, which expands to engulf the cytoplasmic material to be degraded, and sequesters the cargo in a double membrane autophagosome. The loaded autophagosome then fuses with lysosomes, forming autolysosomes. This allows the lysosomal enzymes to degrade the autophagosomal contents. Lysosomal permeases and transporters then export amino acids and other by-products of degradation back out to the cytoplasm, where they can be re-used for building macromolecules and for metabolism (Mizushima, 2007). Thus, this catabolic activity is often thought of as a cellular “recycling  factory”  that  enables  cells  to  restore  sufficient  energy  levels  in  the  absence   of nutrients and consequently promotes viability (Huang et al., 2002). This process is also orchestrated at the molecular level. The class III phosphatidyl inositol 3-kinase (PI3KC3), an ortholog of vesicular protein sorting 34 (Vps34) mediates the initial formation of autophagic vacuoles (Kihara et al., 2001). Together with Beclin1 and other Autophagy-related genes (Atg) proteins, this complex controls the formation and elongation of phagophores (Suzuki et al., 2007; Obara et al., 2008). Conversely, the class I PI3K complex activates the AKT1 pathway thereby activating the mammalian target of rapamycin (mTOR) and shuts off autophagy (Yorimitsu et al., 2005). mTOR functions by hyperphosphorylating Atg13 21 and decreases its affinity for Atg1, a serine/threonine kinase. In yeast, under conditions of nutrient deprivation, TOR is found to be deactivated and dephosphorylated by Atg13 which then binds Atg1 to activate autophagy (Kamada et al., 2000). However, the exact mechanism by which mTOR inhibits autophagy in mammalian cells remains unclear. Another negative regulator of autophagy is Bcl-2. Under normal conditions, Beclin 1 binds to the groove of Bcl-2 and Bcl-XL at the BH3 domain (Maiuri et al., 2007). Hence under stressful conditions, the dissociation of Beclin1 from Bcl-2/Bcl-XL allows Beclin1 to bind to Vps34 and induces autophagy (Pattingre et al., 2005). Elongation of the autophagic vacuole membrane is mediated by the Atg5– Atg12 conjugation step and the LC3 processing step (Kirkin et al., 2009). The Atg12– Atg5 dimer is recruited to the outer autophagosomal membrane, presumably via interaction with Atg16. Atg 4, 7 and 3 cleave LC3 into LC3-II in a sequential manner whereby phosphatidylethanolamine is conjugated to. Finally, the Atg5–Atg12–Atg16 complex induces hemi-fusion of membranes through a symmetric recruitment of processed LC3-II. The synthesis and processing of LC3 into LC3-II is increased during autophagy, making it a key readout of levels of autophagy in cells (Barth et al., 2010). Autophagy is also identified by a punctuated immunofluorescence staining pattern. Recently, autophagy has been shown to be a backup degradation system induced by ER stress and UPR. It is widely believed to be a compensatory protective mechanism to remove and recycle misfolded proteins and provide nutrients for the cell (Kawakami et al., 2009; Yorimitsu et al., 2006). On the other hand, during prolonged ER stress, autophagy can instead lead to cell death (Yorimitsu and 22 Klionsky, 2007). Till now, it remains controversial as to how ER stress activates autophagy. Nevertheless, strong evidence has shown to suggest that PERK which is activated by ER stress and is part of the UPR might be essential for ER stressedinduced   autophagy   through   phosphorylation   of   eIF2α   and   activation   of   Atg12   in   mouse embryonic fibroblast (MEF) and differentiated p19 embryonal carcinoma C2C5 cells (Kouroku et al., 2007). Overall, more research is still required to define the molecular mechanisms underlying ER stress-induced autophagy. 1.6.2 Chaperone-mediated autophagy (CMA) Unlike microautophagy and macroautophagy, vesicular traffic is not involved in chaperone-mediated autophagy (CMA) (Cuervo et al., 1998; Dice, 2000; Franch et al., 2001). Substrate proteins with an exposed pentapeptide sequence related to KFERQ are preferentially recognised by a molecular chaperone complex consisting of the constitutive form of the heat shock 70 kDa protein (hsc70), the heat shock protein of 40 kDa (hsp40), the heat shock protein of 90 kDa (hsp90), the hsc70-interacting protein (hip), the hsc70-hsp90 organizing protein (hop), and the Bcl2-associated athanogene 1 protein (bag-1) (Agarraberes and Dice, 2001). The chaperone/proteins complex then interacts with the lysosomal membrane receptor lysosomal-associated membrane protein 2A (LAMP-2A) (Cuervo et al., 1996). Once bound to the lysosomal membrane, the substrate is unfolded (Salvador et al., 2000), presumably assisted by the molecular chaperone complex and with the help of lysosomal hsc70 (ly-hsc70), the protein is pulled into the lysosomal lumen (Cuervo et al., 1997; Agarraberes et al., 1997). The ultimate fate of this protein is degradation by proteases present in the lysosomes (Majeski and Dice, 2004). 23 1.7 Current perspective in APL and study hypothesis and objectives 1.7.1 Current perspective in APL It has previously been shown that misfolded conformation dependent loss (MCDL) of N-CoR played a role in the pathogenesis of APL. This loss of N-CoR is attributed to the expression of PML-RARα. Khan et al. have reported that PMLRARα   could bind aberrantly to the N-CoR molecule. Such interaction could induce the N-CoR protein to adopt an abnormal conformation and subsequent degradation, thus disrupting its important role in the transcriptional control of several tumour suppressor proteins (Khan et al., 2001; 2004). In addition to the loss of function effect due to misfolding, misfolded N-CoR could also contribute to APL pathogenesis through an aberrant gain of function properties. In an earlier study, a glycoprotease selectively expressed in APL cells has been shown to regulate the response of APL cells to UPR induced apoptosis through the processing of misfolded N-CoR protein (Ng et al., 2006). Ng et al. found that NCoR cleavage in APL cells by O-Sialoglycoprotein endopeptidase (OSGEP) protease reduced the ER stress to a level that was below the threshold needed to evoke the stress mediated apoptosis pathway thus allowing the cells to escape cell death. This unique cellular defense mechanism was termed   as   ‘late’   cytoprotective UPR which was activated when the early cytoprotective UPR failed to protect the cells from the harmful effect of misfolded proteins. The net outcome was resistance to UPR-induced apoptosis and uncontrolled proliferation of promyelocytic cells. Further, there is preliminary evidence in our laboratory that indicated processing of misfolded N-CoR through a cytoprotective autophagic response not only protects APL cells from UPR-induced apoptosis, but also provides support for 24 uncontrolled growth and proliferation of APL cells. It has been hypothesized that energy derived from the degradation of misfolded N-CoR protein through autophagy could sustain the growth of APL cells. A schematic representation of the regulation of UPR in APL cells is shown in Figure 1.4. Apart from the elucidation of underlying molecular mechanism in APL pathogenesis, therapeutic modulation was also investigated. Ng et al. previously reported that PML-RARα mediated misfolding of N-CoR protein could be inhibited by genistein, a potent modifier of N-CoR protein conformation. The mode of action exerted by genistein was thought to be through the inhibition of selective phosphorylation-dependent binding of N-CoR to PML-RARα. This could have resulted in the dissociation of PML-RARα from N-CoR and restored N-CoR’s  growth   suppressive function (Ng et al., 2007). More recently, Ng and colleagues demonstrated that curcumin promoted net accumulation of aberrantly phosphorylated misfolded N-CoR protein by blocking its ERAD and protease-mediated degradation. This resulted in the activation of UPR-induced apoptosis in APL cells (Ng et al., 2011). 1.7.2 Study hypotheses and objectives The role of N-CoR in the cancer pathogenesis has been mostly studied in APL however its role in the pathogenesis in other solid tumours has yet to be established. Currently, there is limited information on the role of N-CoR in the pathogenesis of HCC. HBX is known to be required to play multifunctional roles such as transcription modulation, protein degradation, cellular signalling pathways, cell cycle checkpoints and apoptosis (Murakami, 2001). A study has also shown that the   25 integrity of PML oncogenic domains (POD), of which N-CoR is an important component, was altered in several cases of viral infections (Day et al., 2004).   The findings provided clues that HBX, similar to PML-RARα might function as a transforming protein which may interfere with N-CoR protein. Further, the role of UPR in hepatocarcinogenesis has been demonstrated by Mashiro and colleagues where they found that UPR caused the activation of ATF6, XBP1 and GRP78 genes in human HCC (Masahiro et al., 2003). Figure 1.4. Kinetics of N-CoR misfolding in APL ER accumulated misfolded N-CoR cooperates with PML-RARα to trigger cell death initially, whereas this toxic effect is neutralised due to structural and functional modification of misfolded N-CoR which will then induce cell survival and growth. 26 Given the apparent similarities and correlations between APL and HCC, we plan to investigate the role of N-CoR in HCC. The objectives of this study are as follows: 1. to investigate if viral proteins can promote misfolding/loss of N-CoR; 2. to elucidate the molecular mechanisms underlying HCC   cells’   resistance   to   UPR induced apoptosis; 3. to investigate the molecular mechanism underlying the induction of autophagy and their effects on growth of HCC cells and; 4. to evaluate the feasibility of therapeutic targeting of ER stress, UPR and autophagy in HCC cells. 27 Chapter2. Materials and Methods 2.1 Materials 2.1.1 General Reagents Table 2.1. List of Chemicals, Reagents and Kits Chemicals/Reagents/Kits Company Country 2-Mercaptoethanol Bio-Rad CA, USA 4,6-diamidino-2 phenylindole (DAPI) Sigma Aldrich MO, USA 1 kb DNA ladder Promega WI, USA 30% Acrylamide-Bis Solution Bio-Rad CA, USA Agarose Bio-Rad CA, USA Ammonium Persulfate Bio-Rad CA, USA Bovine Serum Albumin (BSA) Sigma Aldrich MO, USA Diethylpyrocarbonate (DEPC) Sigma Aldrich MO, USA Dimethyl sulfoxide (DMSO) Sigma Aldrich MO, USA Dulbucco’s  Modified  Eagle’s  Medium  (DMEM) Sigma Aldrich MO, USA Ethanol Merck Darmstadt Fetal Bovine Serum (FBS) Hyclone Lab Logan, UT Fugene 6 Roche Germany Glacial Acetic Acid Merck Darmstadt Glycine Bio-Rad CA, USA Isopropanol Merck Darmstadt Lipofectamine 2000 Invitrogen CA, USA Luciferase Assay System (Dual) Promega WI, USA Methanol Sigma Aldrich MO, USA Murine Reverse Transcriptase (MMLV) Promega WI, USA Normal Goat IgG Santa Cruz CA, USA Normal Mouse IgG Santa Cruz CA, USA Normal Rabbit IgG Santa Cruz CA, USA Paraformaldehyde Sigma Aldrich MO, USA Phosphatase Inhibitor Cocktails 1 and 2 Sigma Aldrich MO, USA Precision Dual Colour Standard Protein Marker Bio-Rad CA, USA Prolong Gold Antifade Reagent Invitrogen CA, USA Protein G Sepharose Beads Roche Germany PVDF Membrane Bio-Rad CA, USA Qiagen plasmid DNA purification Kit Qiagen GmBH Hilden (maxi and miniprep kit) 28 Chemicals/Reagents/Kits Company Country Qiagen RNasey RNA extraction Kit Qiagen GmBH Hilden Skim Milk powder Sigma Aldrich MO, USA Sodium Dodecyl Sulfate (SDS) Bio-Rad CA, USA Sodium Fluoride Sigma Aldrich MO, USA Tetramethylethylenediamine (TEMED) Bio-Rad CA, USA Tris-Base Bio-Rad CA, USA Trizol Sigma Aldrich CA, USA Tween 20 Sigma Aldrich MO, USA 2.1.2 Antisera 2.1.2.1 Western Blotting Table 2.2. List of Primary Antibodies Antibodies Description Ratio Source β-actin Mouse Monoclonal 1:10 000 Sigma Aldrich ATF6α Rabbit Polyclonal 1:5000 AbCam eIF2α Rabbit Polyclonal 1:1000 Cell Signalling FLAG Mouse Monoclonal 1:20 000 Sigma Aldrich HA Rabbit Polyclonal 1:1000 Santa Cruz GRP78 Goat Polyclonal 1:2000 Santa Cruz LC3 Rabbit Polyclonal 1:5000 Novus Biologicals Myc Rabbit Polyclonal 1:4000 Santa Cruz N-CoR Goat Polyclonal 1:500 Santa Cruz PDI Rabbit Polyclonal 1:2000 Santa Cruz Phospho- eIF2α  (Ser51) Rabbit Polyclonal 1:1000 Cell Signalling Table 2.3. List of Secondary Antibodies Description Ratio Source HRP-Goat anti-Rabbit 1:10 000 Zymed Laboratories HRP-Goat anti-Mouse 1:10 000 Zymed Laboratories HRP-Rabbit anti-Goat 1:10 000 Zymed Laboratories 29 2.1.2.2 Immunofluorescence Staining Table 2.4. List of Primary Antibodies Antibodies Description Ratio Source FLAG Mouse Monoclonal 1:1000 Sigma Aldrich HA Rabbit Polyclonal 1:1000 Santa Cruz HBX Mouse Monoclonal 1:100 Santa Cruz LC3 Rabbit Polyclonal 1:300 Novus Biologicals N-CoR Goat Polyclonal 1:100 Santa Cruz PDI Rabbit Polyclonal 1:100 Santa Cruz Table 2.5. List of Secondary Antibodies Description Ratio Source Alexa Flour Chicken Anti-Goat 488 1:200 Invitrogen Alexa Flour Chicken Anti-Goat 594 1:200 Invitrogen Alexa Flour Chicken Anti-Mouse 488 1:200 Invitrogen Alexa Flour Chicken Anti-Mouse 594 1:200 Invitrogen Alexa Flour Chicken Anti-Rabbit 488 1:200 Invitrogen Alexa Flour Chicken Anti-Rabbit 594 1:200 Invitrogen 2.1.3 Primer Sequences 2.1.3.1 Semi-Quantitative RT-PCR primers Table 2.6. List of semi-quantitative RT-PCR primers Gene B2M Sequence F:  5’-ATCCAGCGTACTCCAAAGAT-3’ Annealing Temperature (oC) Cycles 58 30 56 38 60 30 60 35 60 35 60 35 R:  5’-TTACATGTCTCGATCCCACT-3’ HBX F:  5’-GTACTGCCAACTGGATCCTTC-3’ R:  5’-CCTCCCAGTCCTTAAACACAC-3’ N-CoR F:  5’-TACCGCAGGAGCCATACAAGA-3’ R:  5’-GCTCAGTTGTGCTTTGGGAGC-3’ Haptoglobin F:  5’-GCCTGGGCAACAGGAGTGAAA-3’ R:  5’-CTTGGTTGGTCTTGCCTCTGG-3’ PAA-1 F:  5’-ACACATGCCTCAGCAAGTCC-3’ R:  5’-TCTTCTTGACAGCGCTCTTG-3’ SAA F:  5’-GCACAACTGGGATAA-3’ R:  5’-ATCTGTGCTGTAGCT-3’ 30 Gene Sequence Annealing Temperature (oC) Cycles Snail F:  5’-GCGAATTCTAGCGAGTGG 60 35 60 35 60 35 60 35 TTCTTCTGCGCTACTGCT-3’ R:  5’-ATAGCGGCCGCCAGGTAT GGAGAGGAAGAGGGAGC-3’ Slug F:  5’-TGTGTCCAGTTCGCT-3’ R:  5’-ATGCCGCGCTCCTTCCT-3’ Vimentin F:  5’-GAGAACTTTGCCGTTGAAGC-3’ R:  5’-TCCAGCAGCTTCCTGTAGGT-3’ Twist-1 F:  5’-AGCTACGCCTTCTCGGTCT-3’ R:  5’-CCTTCTCTGGAAACAATGACATC-3’ 2.1.3.2 siRNA sequences Table 2.7. List of siRNA sequences used in siRNA mediated gene knockdown 2.1.4 Target Sequence N-CoR 5’-AATGCTACTTCTCGAGGAAACA-3’ Luciferase 5’-CGTACGCGGAATACTTCGA-3’ Drugs and Inhibitors All drugs and inhibitors used were prepared as stock solutions and stored as aliquots at -20°C. Table 2.8. List of drugs and inhibitors Drugs/Inhibitors Stock Conc./ Solvent Preparation Source Bafilomycin (BA-1) 10µM with DMSO Sigma Aldrich DTT 1 M with deionised water Sigma Aldrich Genistein 20mM with DMSO Sigma Aldrich MG132 20mM with DMSO Sigma Aldrich 31 2.1.5 Hepatocellular Carcinoma Tissues 2.1.5.1 Human Samples Human liver tissues used in this study were obtained from 10 patients diagnosed with either moderately differentiated HCC or poorly differentiated HCC. All patients suffered from cirrhosis with chronic HBV infection. Tissues were collected from the tissue repository of National University Hospital (Singapore) and the use of these samples has been approved by NUS Institutional Review Board (IRB). Basic clinical data of the patients are presented in Table 2.9. Paired tissues were obtained from each patient, one from the tumour region of the resected liver and the other matching control from adjacent non-tumour (normal) region. Table 2.9. Clinical characteristics of patient samples used in this study Specimen Diagnosis ID Age as at collection date Gender Ethnic group % Tumour cells in sample NT00/0036 73 MALE CHINESE 90% MOD DIFF NT05/0155 59 FEMALE CHINESE 100% MOD TO POORLY DIFF NT05/0158 63 MALE CHINESE 100% POORLY DIFF NT05/0170 56 MALE CHINESE 80% GRADE3/4 NT06/0049 66 MALE INDIAN 100% MOD DIFF NT06/0278 67 MALE CHINESE 100% MOD DIFF NT06/0410 46 MALE CHINESE 70% MOD DIFF NT06/0456 76 MALE CHINESE 100% MOD DIFF NT06/0467 55 MALE CHINESE 80% MOD DIFF NT07/0032 34 MALE CHINESE 100% MOD DIFF 32 2.1.5.2 Tissue Microarray The custom tissue microarray from US Biomax was selected for our immunohistochemistry (IHC) studies. This tissue array includes the hepatic carcinoma as well as the normal tissues, containing 15 cases in early stage, 55 cases in advanced stage and 10 normal tissues. 2.1.6 Cell lines 2.1.6.1 Liver cancer cell lines All HCC cell-lines: HepG2, SK Hep1, Huh7, PLC/Prf/5, Snu449, Snu387, Snu398, Snu423 and Snu475 were purchased from the American Type Culture Collection (Rockville, MD). For short-term storage, cells are suspended in freezing media [90% filtered FBS, 10% DMSO] and kept at -80°C. For long-term storage, cells were suspended in the same storage media and maintained in liquid nitrogen. 2.1.6.2 HEK 293T and HeLa cell lines Both 293T and HeLa cells were obtained from the American Type Culture Collection (Rockville, MD). Maintenance and storage of these cells were similar to liver cancer cells. 2.1.7 Plasmids 2.1.7.1 pACT vector The pACT vector (Promega, USA) is a non-viral mammalian expression vector containing CMV promoter and carries the ampicillin bacterial resistance gene. pACT-NCoR-FLAG consists of 2 tandem repeats of Flag sequence, linked in frame to the C terminus of mouse N-CoR sequence and cloned into the vector at Nco1 and Xba1 sites. 33 2.2 Methods 2.2.1 Cell Culture Treatment 2.2.1.1 Cell culture All cells were cultured in Dulbecco’s  modified  eagle  medium  (Sigma Aldrich, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Waltham, USA), at 37°C in a 5% CO2 humidified atmosphere. Media was changed every 3-4 days and cells were passaged by trypsinization with 0.5% (w/v) trypsin and 0.2% (w/v) ethylenediaminetetraacetic acid (EDTA) disodium salt when they reached 80-90% confluency. 2.2.1.2 Treatment with drugs and inhibitors Cells were seeded according to their treatment and doubling time. At the appropriate incubation or treatment time-point and confluence, cells were then supplemented with either single agent or in combination at respective concentrations and time-points. 2.2.1.3 Transfection 2.2.1.3.1 DNA transfection of 293T cells and HeLa cells 293T or HeLa cells were seeded at 200X104 per 10-cm plate or 80X104 cells per 6-cm plate or 20X104 cells per well of a 6-well plate one day prior to transfection. Approximately 18-22 hours after seeding, transfection was carried out. Briefly, Fugene 6 (Roche, Germany) was first diluted in serum free medium for 5 mins at room temperature and indicated plasmids were then added to diluted Fugene (DNA: Fugene = 1 μg:3   μl). The mixture was incubated for further 15 mins at room temperature. The final DNA-Fugene 6-serum free medium mixture was then added 34 slowly in a drop wise manner to the cells. Cells were then incubated for at least 48 hours before harvesting. 2.2.1.3.2 N-CoR siRNA knockdown in HCC cells The N-CoR siRNA was used to specifically knockdown the N-CoR gene in HCC cells. A mock siRNA targeting the luciferase sequence which could not be found in the mammalian genome was used as non-targeting siRNA control. Cells were seeded in a similar way as mentioned earlier. Roughly 18-22 hours after seeding, cells reaching about 60% confluency were transfected with either non-targeting control siRNA or N-CoR siRNA. The transfection mixture was prepared by first mixing DMEM containing the appropriate concentration of siRNA with DMEM containing LipofectAMINETM 2000 (Invitrogen, CA, USA). This mixture was then incubated at room temperature for 20 mins before adding to the respective seeding media. The transfected cells were subsequently returned to the CO2 incubator and incubated for 24-72 hours at 37°C. The effect of gene silencing was analysed by reverse transcription-polymerase chain reaction (RT-PCR). 2.2.2 Protein extraction 2.2.2.1 Mammalian cells Cells were collected after trypsinization and pelleted by centrifuging at 200g for 5 mins at 4°C. The resulting pellet was rinsed twice with ice-cold 1X PBS (pH 7.4) before centrifugation and the supernatant was then removed. Four pellet volume of 2X SDS sample buffer [250mM Tris-HCl (pH6.8), 40% glycerol, 9.2% SDS, 0.01% bromophenol blue, 20% β-mercaptoethanol] was used to re-suspend cell pellet. Cells were then sonicated twice or thrice, 10 secs each, on ice using the Branson Sonifier 150 with an output power of 5W. The protein samples were then denatured 35 by heating at 50°C for 10 mins or 95°C for 5 mins. Protein samples were then loaded and resolved on SDS-PAGE. Protein quantification was determined after staining the gel with Coomassie Blue staining solution [0.25% Coomassie Blue, 10% (v/v) acetic acid, 50% (v/v) methanol] and Coomassie destaining solution [10% (v/v) acetic acid, 50% (v/v) methanol]. Crude lysates were stored at -80°C. 2.2.3 Immunoblotting/Western Blotting Protein samples were resolved on SDS-PAGE using the Bio-Rad Mini-Protean II system. Electrophoresis was carried out at constant current of 10mA using cold 1X Laemli running buffer [25mM Tris, 192mM glycine, 0.1% (w/v) SDS] at 4°C. The proteins were then transferred onto Hybond-P Polyvinylidene Fluoride (PVDF) membrane (GE Healthcare, Uppsala, Sweden) using a wet transelectroblotting system (Bio-Rad Inc., England). The polyacrylamide gel was run at a constant current of 60mA for 150 mins at 4°C in 1X transfer buffer [48mM Tris, 39mM glycine, 0.037% (w/v) SDS and 10% (w/v) methanol (added just prior to use)]. After transfer, membranes were blocked in PBS-T (1X PBS containing 0.1% Tween 20) containing 5% milk [5% non-fat milk powder (Merck) in 1X PBS-T] for 1 hour at room temperature or overnight at 4ºC. After incubation in the appropriate primary antibody overnight at 4°C in the respective blocking buffers, the membrane was rinsed thrice with PBS-T for 10 mins each to remove any unbound antibodies. HRP-conjugated secondary antibodies with the blocking buffer were then applied for 1h at room temperature. The unbound secondary antibodies were removed by washing the membrane 5 times for 10 mins each with PBS-T before the detection of immunoreactive bands by Western Lightning Chemiluminescence Reagent Plus 36 (Perkin Elmer, CA, USA). The X-ray film was finally developed using a Konica Minolta SRX-101A film processor. After immunodetection, the bound primary and secondary antibodies were removed from the membrane, which then can be re-probed with a different antibody. The developed membrane was briefly washed with PBS-T to remove residual chemiluminescence reagent and immersed in stripping buffer [glycine-HCl (pH2), 1% (w/v) SDS] for 30 mins with agitation. The membrane was rinsed with large volumes of PBS-T thrice for 10 mins each at room temperature and subsequently blocked with respective blocking buffers. The membrane was then re-probed by a different antibody using the same protocol described above. 2.2.4 Immunohistochemistry The paraffin-embedded human liver cancer tissue array was baked at 60°C to melt the wax and improve adhesion before staining. Immunohistochemical staining of tissue microarray was done using the goat ImmunoCruzTM Staining System (Santa Cruz Biotechnology, CA, USA). Briefly, sections were deparaffinised with histoclear, rehydrated in decreasing ethanol concentrations, and the antigens unmasked using 1mM EDTA (pH8) at 95°C for 10 mins. Endogenous peroxidise activity was quenched using hydrogen peroxide, followed by incubating with 5µg goat anti-N-CoR (1:40) antibody for 2 hours at room temperature. The sections were then treated with labelled dextran polymer conjugated with HRP for 30 mins and further incubated with DAB+substrate chromogen solution for 5-10 mins. Sections were counterstained with Mayer’s  hematoxylin  and  mounted. 37 2.2.5 Immunofluorescence and confocal microscopy Cells were grown on coverslips in culture medium till 50-60% confluency before transfection or when 80-90% confluency reached for non treated cells. They were then fixed with 4% paraformaldehyde in PBS (freshly prepared and pre-warmed to 37°C) for 30mins at 37°C, washed thrice with 1XPBS before permeabilising with 0.2% Triton X-100 in PBS on ice for 5 mins. Alternatively, cells were fixed and permeabilised with methanol at -20°C for 7 mins. After permeabilisation, cells were washed thrice with 1XPBS and blocked with 5% bovine serum albumin (BSA) in PBS for 30 mins. Cells were then incubated in primary antibodies at their appropriate dilutions for 2 hours at room temperature and subsequently washed with PBS thrice, 5 mins each followed by incubation with the appropriate Alexa Fluor secondary antibodies (Invitrogen, Carlsbad, CA, USA) with 1:200 dilution, for 1 hour at room temperature in the dark. The cells were washed 3 times, 5 mins each after the incubation. The nuclei of the cells were counterstained with 150nM of 4,6-diamidino2-phenylindole (DAPI) for 5 mins. After washing the cells thrice with PBS, the coverslips with cells were mounted with SlowFade® Gold antifade reagent (Molecular Probes, CA, USA) before sealing with transparent nail polish. Images were subsequently visualized and captured using Axioplan 2 imaging fluorescence microscope (Carl Zeiss). 2.2.6 RNA extraction 2.2.6.1 Mammalian cells Cells were collected after trypsinization and pelleted by centrifuging at 200g for 5 mins at 4°C. The resulting pellet was then washed twice with ice-cold 1X PBS (pH 7.4) and the supernatant aspirated completely after another round of 38 centrifugation. Purification of total RNA was then carried out using RNeasy Mini kit (Qiagen, Hilden, Germany)   according  to   the  manufacturer’s  protocol.   Up to 1 x 107 cells, depending on the cell line, were disrupted in Buffer RLT (10   µl   βmercaptoethanol per 1ml Buffer RLT was added before use) and homogenized using needle and syringe. 1 volume of 70% ethanol was added to the homogenized lysate, creating conditions that promote selective binding of RNA to the RNeasy membrane. The sample was then loaded to the RNeasy Mini spin column. Total RNA was bound to the membrane and contaminants were efficiently washed away with buffers RW1 then RPE. Lastly, high-quality RNA was eluted in 30-50µl of RNase-free water. All binding, washing, and elution steps were performed by centrifugation in a microcentrifuge. Purified RNA was eluted and its concentration was determined using Nanodrop Spectrophotometer ND-1000 (Thermo Fisher Scientific, Lafayette, CO, USA). 2.2.7 Reverse transcription polymerase chain reaction (RT-PCR) amplification Briefly, 3-5µg of purified RNA template was used for cDNA synthesis using RT-PCR System kit (Promega, WI, USA). Reverse transcription was performed at 42°C for 1 hour, followed by 65°C for 15 min. cDNA synthesis was completed after inactivation of transcriptase by incubation at 95°C for 5 mins. cDNA was either stored at 4°C or on ice for immediate analysis or stored at –20°C until use. PCR amplification was carried out as shown in Table 2.10 using Thermal Cycler GeneAmp®PCR System 9600 (Applied Biosystems, CA, USA). Depending on the different primers used, the annealing temperature and number of cycles were optimized accordingly. 39 Table 2.10. PCR amplification steps. *30-35cycles (Steps2-4) Steps Temperature (°C) Time 1. Initializing 94 5mins 2. Denaturation 94* 30s 3. Annealing 60* 30s 4. Extension/Elongation 72* 30s 5. Final Elongation 72 7mins 6. Final Hold 4 2.2.7.1 DNA agarose gel electrophoresis Gene expression analysis was carried out by agarose gel electrophoresis where PCR products were resolved in 1% w/v agarose gel (1g of electrophoretic grade agarose powder in 100ml 1XTAE buffer [40mM Tris-acetate (pH7.8), 1mM EDTA] stained with Gel Red at a 1:10000 dilution and expression levels visualized using Trans UV on the Gel Doc System (Bio-Rad, CA, USA). 2.2.8 Biological Assays 2.2.8.1 Luciferase assay The effect of N-CoR and viral proteins on promoter activity of ATF6luciferase activity was carried out using the Dual-Luciferase® Reporter (DLRTM) Assay System Kit (Promega, WI, USA) as described by the manufacturer. Hela cells were co-transfected with ATF6 reporter plasmid containing the firefly luciferase reporter gene under the control of the CMV promoter and tandem repeats of ATF6 transcriptional response element, together with different combination of other plasmids. After transfection, cells were lysed in 1X Passive Lysis Buffer (PLB). The firefly luciferase reporter was measured first by adding Luciferase Assay Reagent II 40 (LAR II) to generate a luminescent signal lasting for at least one minute. After quantifying the firefly luminescence, this reaction was quenched, and the Renilla luciferase reaction was initiated simultaneously by adding Stop & Glo® Reagent to the same sample. The apparent luminescence activity was measured using the Sirus Single Tube Luminometer (Berthold Detection System, Germany). The light signal was integrated for 10 secs after a delay of 1 sec. Each sample was assayed in triplicate. 2.2.8.2 ATP assay Measurement of internal ATP of the mammalian cells was performed using the ATP Bioluminescence Assay Kit HS II (Roche, Germany). After the knockdown, 1 X 105 live cells were pelleted, washed with ice-cold 1XPBS and re-suspended in dilution buffer to a cell concentration of 1X105 cells/ml. Cell lysis buffer was then added and the mixture was incubated at room temperature for 5 mins. Finally, luciferase regent was added to the crude lysate and luminescence activity was measured using the Sirus Single Tube Luminometer (Berthold Detection System, Germany). The light signal was integrated for 10 secs after a delay of 1 sec. Each sample was assayed in triplicates. 2.2.8.3 Ubiquitination assay/ Immunoprecipitation Cell were harvested in 4X pellet volume of lysis buffer [10mM Tris-HCl (pH8), 500mM NaCl, 2% SDS], heated at 95°C for 10mins and diluted with dilution buffer (lysis buffer:dilution buffer = 1:9) [10mM Tris-HCl (pH8), 500mM NaCl, 1% Triton-X-100), sonicated mildly twice for 10s on ice. The cell lysate was then incubated for 2.5 hours at 4°C with rotation with 5µg of either anti-Flag or anti-NCoR antibodies (Santa Cruz Biotechnology, CA, USA) and anti-mouse or anti-goat 41 IgG antibodies (Santa Cruz Biotechnology, CA, USA) were used as controls. After pre-adsorption with protein G- Sepharose beads (GE Healthcare Bio-Sciences AB, Sweden) for 2 hours at 4°C with rotation, the immunocomplexes were pulled down, added   to   2X   SDS   sample   buffer   containing   20%   β-mercaptoethanol and heated at 50°C for 10 mins. The eluted proteins were then subjected to SDS-PAGE. 2.2.8.4 Solubility assay Cells were harvested in cell lysis buffer [50mM Tris-HCl (pH8.8), 100mM NaCl, 5mM MgCl2, 0.5% NP-40, 5mM PMSF, 1µg/ml leupeptin, 1µg/ml pepstatin, 1µg/ml aprotinin] and lysed at 4°C with rotation for 30mins. The lysates were then centrifuged at 15 000rpm at 4°C for 10mins. The supernatant was aspirated as soluble fraction while the pellet was collected as insoluble fraction. The pellet was rinsed again with lysis buffer and supernatant was completely aspirated to avoid cross contamination. Pellet was then redissolved in pellet buffer [20mm Tris-HCl (pH8), 15mM MgCl2], treated with DNase I (5µl per 250µ of pellet buffer) for 30mins at 37°C. Prior to running on SDS-PAGE, SDS sample buffer was added to both soluble and insoluble fractions separately and heat inactivated at 50°C for 10 mins. 42 Chapter 3. Results 3.1 Analysis of N-CoR status in human liver cancer cells and tumours 3.1.1 Loss of N-CoR protein in multiple human liver cancer cell lines The role of misfolded conformation dependent loss (MCDL) of N-CoR protein in the pathogenesis of APL has been previously reported (Khan et al., 2004; Ng et al., 2006, 2007). However, loss of N-CoR by MCDL has not been explored in liver cancer (HCC). In this study, a small scale screening of multiple liver cancer cell lines was performed to examine the expression of N-CoR at the protein and transcript level through western blotting assay and semi-quantitative PCR respectively. As shown in Figure 3.1 A-B, majority of liver cancer cells displayed N-CoR loss at protein but not mRNA level, which suggested that N-CoR loss occurred at post transcriptional level. 3.1.2 Loss of N-CoR protein in human liver cancers To examine how this loss of N-CoR protein was associated with the malignant phenotype of liver cancer cells, we performed a comprehensive analysis of N-CoR expression status in human primary cancer cells using western blotting assay. A comparative analysis of endogenous N-CoR protein level in 10 primary liver cancer patient samples with a history of HBX infection was performed. Matching nonmalignant tissues were used as controls. As expected, we observed a significant level of N-CoR loss in liver tumour specimens (Figure 3.2). However, a surprising observation was that there was a complete loss of N-CoR protein in non-tumour tissue surrounding the tumor. As the normal tissue was a matching control from the same patient, it was thought that these tissues may have acquired early malignant changes and N-CoR loss could be an early oncogenic event. 43 A B Figure 3.1. Selective loss of N-CoR protein in HCC cells N-CoR expression at protein (A) and transcript (B) level in various liver cancer cells was determined through western blotting assay and RT-PCR analysis respectively. (A) Whole cell extracts of the various cell lines was resolved in SDS-PAGE and stained with N-CoR antibody in western blotting assay. Coomassie Blue staining and β2M  were  used  as  loading  controls.  Majority of HCC cells displayed loss of N-CoR at protein but not at mRNA level. 44 Figure 3.2. Evidence of N-CoR loss in primary human liver cancer samples Frozen pellets of patient samples were lysed using SDS lysis buffer. SDS-PAGE was used to resolve the whole cell lysates and N-CoR protein was visualized via western blotting with N-CoR antibody. HepG2 was included as a positive control. N denotes normal (adjacent non-tumour) while T denotes tumour. Similar to the cell line system, full length N-CoR protein was not present in these patient samples. 3.2 Elucidating the mechanism of N-CoR loss 3.2.1 N-CoR loss in HCC cells is linked to misfolding It has been previously reported in APL that genistein, a tyrosine kinase inhibitor, from soybean can effectively stabilize N-CoR protein by restoring its native conformation (Ng et al., 2007). Hence, to investigate if degradation of N-CoR protein in HCC cells was due to misfolding, the effect of genistein on N-CoR was evaluated in SK Hep1 which was selected as a representative N-CoR negative HCC cell line. Our western blot data revealed that genistein promoted stabilization of N-CoR at a concentration of 25µM (Figure 3.3) which suggested that loss of N-CoR could be due to its misfolded conformation. 45 Figure 3.3. Genistein promotes stabilization of N-CoR protein in SK Hep1 cells Cells were treated with 25µM of genistein for 72 hours and the level of full length NCoR protein was determined by western blotting assay. 3.2.2 Inverse relationship between N-CoR and HBX expression levels Khan et al. has previously reported that PML-RARα, the transforming protein in APL, could induce N-CoR misfolding and its subsequent loss in APL (Khan et al., 2004). Based on that, we hypothesized that an oncogenic protein present in liver cancer cells could also contribute to the misfolding and eventual loss of N-CoR protein in HCC. HBX has been reported to be the most common oncogenic factor in hepatocarcinogenesis (David, 2006). Therefore, we analysed HBX transcript expression via semi-quantitative PCR analysis and found HBX transcripts to be inversely correlated with N-CoR protein level (Figure 3.4). The transcript level of HBX was significantly higher in cell lines that displayed loss of N-CoR protein. Western blot analysis of HBX was not done as the HBX protein was hardly detectable. 46 Figure 3.4. N-CoR protein level in HCC cells is inversely related to HBX transcript level Semi-quantitative RT-PCR analysis of HBX gene in various HCC cell lines revealed an inverse relationship to N-CoR protein. High level of N-CoR protein and negligible level of HBX transcripts was found in HepG2 cells, whereas the HBX positive cells displayed low level of N-CoR protein but high level of HBX transcripts. 3.2.3 Co-expression with HBX preferentially localizes N-CoR to the cytosol The data presented so far suggested that level of N-CoR protein might be regulated by HBX. To further characterize the role of HBX in HCC, we assessed whether ectopic expression of HBX could induce N-CoR localization to the cytosol. In this study, HBX was co-transfected with green fluorescent protein (GFP)-tagged NCoR expression vector in 293T cells and N-CoR localization was determined by the detection of green fluorescence signals. Fluorescence microscopy analysis showed that N-CoR was preferentially localized to the cytosol in the presence of HBX while N-CoR was localized mainly in the nucleus when co-expressed with the empty vector (Figure 3.5). It has been previously reported that functional N-CoR protein was localized in the nucleus while misfolded N-CoR to be localized in the cytosol (Khan et al., 2004; Ng et al., 2006, 2007), suggesting that aberrant sub-cellular distribution is an important hallmark of misfolded N-CoR. Taken together, HBX was thought to induce N-CoR misfolding. 47 Figure 3.5. HBX promotes cytosolic retention of N-CoR protein Sub-cellular distribution of N-CoR (Green Fluorescence) protein was determined by immunofluorescence staining. Preferential localization of N-CoR to the cytosol was observed after co-expression with HBX while N-CoR was localized mainly in the nucleus in absence of HBX. 48 3.2.4 N-CoR in HBX positive HCC cells is found to be co-localized with HBX in the cytosol To validate the finding observed in 293T cells, immunofluorescence (IF) analysis of the distribution of endogenous N-CoR and HBX was performed in HCC cells. Consistent with the observation in 293T cells, a majority of N-CoR protein was found to be localized in the cytosol of HBX positive HCC tumour cells while in HBX negative HepG2 cells, N-CoR displayed a predominantly nuclear localization (Figure 3.6). It was also observed that N-CoR co-localized with HBX in the cytosol of HBX positive HCC cells as indicated by the yellow signals (Figure 3.6, overlay). 3.2.5 N-CoR protein in liver tumour sections is also found to be localized in the cytosol To further determine if the misfolding of N-CoR might be prevalent in liver cancer tissue sections, sub-cellular distribution of N-CoR in human liver tissue sections (from tissue microarray) was determined by immunohistochemistry (IHC) assay with N-CoR antibody. Consistent with the findings in liver cancer cell lines, NCoR was observed to be predominantly distributed in the cytosol of liver cancer tissue sections. In contrast, N-CoR was found predominantly in the nucleus of normal liver tissue sections  (Figure 3.7). However, it should be highlighted that there is discrepancy of N-CoR detection between Western Blotting and immuno-blotting studies in Figure 3.1A and Figure 3.6 as well as in Figure 3.2 and Figure 3.7. This can be accounted by the fact that since N-CoR is degraded, there is no full length protein; however, the whole protein does not disappear with the fragments still present. Thus, in Western blot, the full length N-CoR protein was not detected but in IF/IHC, N-CoR protein fragments in the same cells were still detectable. 49 50 51 Figure 3.6. N-CoR of HBX positive HCC cells is found in the cytosol Sub-cellular distribution of endogenous N-CoR and HBX protein in HepG2 and HBX positive HCC cells was determined with N-CoR and HBX antibodies respectively, followed by fluorescence labelled secondary antibodies. Signals were acquired through fluorescence microscopy. Green signal represents N-CoR distribution while HBX is represented by the red signals. Nucleus is stained with DAPI and is represented by blue signals. In HepG2, majority of N-CoR signal was detected in the nucleus overlapping the DAPI signal. However, in HBX positive cells, a significant portion of N-CoR protein was found in the cytosol where it co-localized with HBX (yellow signals). Figure 3.7. N-CoR protein is mainly localized in the cytosol in tumour sections Human liver cancer tissue sections were stained with N-CoR antibody. The brown stains mark the N-CoR protein while the blue stains indicate nuclear location. In tumour tissue sections, N-CoR was localized in the cytosol while in normal tissue sections, N-CoR was mainly localized in the nucleus. 52 3.2.6 HBX induces N-CoR insolubility Apart from aberrant cytosolic localization, detergent insolubility is another hallmark of misfolded proteins. Ng et al. has previously reported that natively folded N-CoR protein was soluble in a buffer containing organic detergent NP-40 while misfolded protein was insoluble in the same buffer. Thus, based on this report, solubility assay was carried out to further validate the protein conformation defect observed earlier in IF and IHC. We co-transfected 293T cells with flag-tagged N-CoR and either HBX or empty vector and the solubility of ectopically expressed N-CoR protein was determined by immunoblotting of soluble and insoluble fractions with the Flag antibody. In contrast to the N-CoR expressed alone, a significant amount of NCoR protein co-expressed with HBX was found in the insoluble fraction (Figure 3.8). This result suggested that HBX could trigger N-CoR misfolding. As a control to determine cross contamination of the fractions, Coomassie Blue staining was done. Coomassie staining data showed that there was no cross contamination of the fractions which indicated that higher amount of insoluble fraction seen was not due to spill over from the soluble fraction. 53 Figure 3.8. HBX can induce N-CoR insolubility Relative solubility/insolubility of N-CoR protein in transfected 293T cells was determined by protein solubility assay. Soluble and insoluble fractions were separated by high speed centrifugation and N-CoR level in each fraction was determined by western blotting assay using Flag antibody. Actin was used as a loading control while Coomassie blue staining was used to determine the level of total protein. 3.2.7 HBX promotes degradation of ectopic N-CoR in transfected 293T cells The data obtained so far suggested that HBX could be directly linked to N- CoR misfolding in HCC. Since misfolding causes protein instability leading to its degradation, we decided to investigate whether HBX-induced N-CoR misfolding could lead to degradation. 293T cells were transfected with flag-tagged N-CoR alone or with HBX and level of flag-tagged N-CoR protein was detected by western blotting. As shown in Figure 3.9, the level of ectopic flag-tagged N-CoR in 293T cells co-transfected with the HBX plasmid was significantly lower than that of cells transfected with N-CoR-flag plasmid alone. This data suggested that HBX could trigger degradation of N-CoR protein. 54 Figure 3.9. HBX promotes N-CoR degradation Western blot analysis of flag-tagged N-CoR expressed in 293T cells with or without HBX showed N-CoR degradation in the presence of HBX. 3.2.8 HBX promotes ubiquitin-proteasome mediated degradation of N-CoR Poorly assembled and misfolded secretory proteins are known to be ubiquitinated and degraded by the ERAD-mediated protein quality control mechanism (Wickner et al., 1999; Pickart, 2001). This raises the question of whether N-CoR loss in HCC is ERAD-mediated. To test this, effect of MG132 (proteasome inhibitor) on HBX-induced N-CoR degradation was determined. Significant level of N-CoR stabilization after MG132 treatment of 293T cells was observed (Figure 3.10A). This result suggested the involvement of proteasome in HBX mediated N-CoR loss. In addition, misfolded proteins which are degraded via the proteasome usually exhibit high levels of ubiquitination. Therefore to test whether HBX-induced N-CoR degradation is preceded by ubiquitination, ubiquitination assay was performed. 293T 55 cells were co-transfected with N-CoR flag and ubiquitin-myc plasmids in the presence or absence of HBX. These transfected cells were then treated with proteasome inhibitor MG132 to block proteasome-mediated degradation. The results showed that level of ubiquitinated N-CoR (Ub-N-CoR) in HBX co-transfected cells was significantly higher when compared to cells lacking HBX (Figure 3.10B, upper panel). Ubiquitinated N-CoR was not detected in cells transfected with only N-CoR Flag, which indicated low levels of background ubiquitination (Figure 3.10B, lane 1). In addition, the amount of immunoprecipitated (IP) N-CoR was found to be similar in all experimental setups (Figure 3.10B, lower panel) despite the significant differences observed in the level of ubiquitinated N-CoR. Hence, the significantly higher levels of ubiquitinated N-CoR observed could be attributed to the presence of HBX and not to an unequal amount of N-CoR pulled down from the cells. 3.2.9 N-CoR loss in HBX positive HCC cells is mediated by the ubiquitinproteasome pathway To investigate if N-CoR is also ubiquitinated in liver cancer cell lines, SK Hep1 cells were treated with MG132 to determine the levels of ubiquitinated N-CoR. SK Hep1 cells were selected in this study as treatment of MG132 could restore the expression of N-CoR in these cells (Figure 3.11A). Extracts from MG132 treated tumour cells were prepared in ubiquitination buffer and the endogenous level of ubiquitinated N-CoR protein was detected after immunoprecipitation with N-CoR antibody. We observed that the level of ubiquitinated N-CoR was significantly higher in MG132 treated HBX positive SK Hep1 cells when compared to untreated cells (Figure 3.11B, upper panel). This finding suggested that similar to 293T cells transfected with HBX, N-CoR in the HBX positive SK Hep1 cells could have been degraded via the ubiquitin-proteasome mediated pathway. 56 It should be noted that the detection of N-CoR in input but not in IP was due to the low IP efficiency (usually around 1%-5%). N-CoR protein was still detectable in direct Western Blot, even if its level was low; however, since the level of N-CoR protein was already low, the amount of immunoprecipitated N-CoR was probably so low that it would not be detectable. A B Figure 3.10. HBX promotes ubiquitination of N-CoR protein (A) Treatment with 10µM MG132 for 12 hours stabilized N-CoR protein in HBX cotransfected 293T cells. (B) In this ubiquitination assay, transfection with various combinations of plasmids as mentioned on top of each lane was first done, then NCoR-flag protein was immunoprecipitated, and the immunocomplexes were detected with Myc antibody. A significant elevated level of ubiquitinated N-CoR in the HBX co-transfected 293T cells was observed (upper panel). The membrane was re-probed with Flag antibody to ensure that the immunoprecipitated (IP) protein was N-CoR in specific (lower panel). 57 A B Figure 3.11. Ubiquitin-proteasome mediated degradation of N-CoR protein in HBX positive HCC cells (A) SK Hep1, a HBX positive HCC cell, was treated with proteasome inhibitor MG132 at 10 µM for a duration of 12 hours and level of full length N-CoR protein was determined by western blotting assay. There was stabilization of endogenous NCoR protein by MG132. (B) Endogenous N-CoR protein in SK Hep1 cells treated with MG132 was immunoprecipitated with N-CoR antibody and level of ubiquitinated (Ub) N-CoR was determined by western blotting assay with Ub antibody (upper panel). The high level of Ub-N-CoR seen in MG132 treated cells indicated that N-CoR loss could be ubiquitin-proteasomal mediated. The membrane was then re-probed with N-CoR antibody to quantify the amount of immunoprecipitated (IP) N-CoR protein (middle panel). Level of N-CoR protein (input) in each starting sample was determined in western blotting assay with N-CoR antibody (lower panel). 58 3.3 The role of misfolded N-CoR in HBX-induced UPR 3.3.1 HBX positive HCC cells exhibit amplification of ER stress It was hypothesized that misfolded proteins can trigger ER stress and activate the UPR. Hence, to investigate the potential link between misfolded N-CoR protein and ER stress, levels of three ER stress markers in HCC cells were determined by western blotting assay. Amplification of ER stress is normally characterized by the presence of high molecular weight (HMW) PDI, GRP78 and phosphorylation of eIF2α (peIF-2α). As expected, the relative level of HMW PDI was higher in most HBX infected HCC cells when compared to HepG2 expressing native N-CoR (Figure 3.12A). In parallel, Figure 3.12B displayed slightly diminished levels of peIF-2α in HepG2 when compared to other HBX positive cells. The equal levels of eIF-2α   detected in all the cells proved that lower level of peIF-2α  observed  in  HepG2  could be attributed to lower ER stress. Additionally, there was a considerably high level of GRP78 in all HCC cell lines which implied the existence of high ER stress (Figure 3.12C). There was no marked decrease in GRP78 level in HepG2 though. Overall, Figures 3.12A-C illustrated evidences of amplification of ER stress in HBX positive HCC cells exhibiting N-CoR loss. Immunostaining of selected HBX positive HCC cells was performed to verify the western blot data. PDI displays cytosolic localization as it is an ER-resident protein. Sub-cellular distribution of N-CoR, together with PDI, was determined by costaining HCC cells with N-CoR and PDI antibodies. Figure 3.12D showed that NCoR and PDI signals were localized in the nucleus and cytosol respectively in HepG2 cells. In contrast, there was a significant co-localization between N-CoR and PDI in the cytosol of HBX positive cells as indicated by the yellow signals (Figure 3.12D, overlay). 59 A B C 60 D 61 Figure 3.12. HBX positive cells exhibit high level of ER stress Level of ER stress in HepG2 and HBX positive HCC cells was determined by the levels of high molecular weight (HMW) PDI protein (A), phospho-eIF2α  (B)  as  well   as GRP78 protein (C). Overall, most HBX positive cells exhibited high level of ER stress. (D) Sub-cellular localization of N-CoR and ER resident protein PDI in HCC cells was evaluated by fluorescence microscopy after co-staining the cells with NCoR antibody (green signal) and PDI antibody (red signal). DNA was stained with DAPI (blue signal). Significant co-localization (yellow signal) between N-CoR and PDI was observed in the HBX positive cells. 62 3.3.2 HBX promotes ATF6 activation Published reports have shown that upon ER stress, unfolded protein response (UPR) can initiate proteolytic processing of ATF6, creating a cleaved N terminal fragment of 50 kDa which translocates to the nucleus and then activates the transcriptional activity of the genes encoding ER chaperones and pro-apoptotic machinery (Ma and Hendershot, 2004). In view of this information, we investigated the status of ATF6 activation in HCC cells by determining the level of cleaved 50 kDa fragment of ATF6 (N) in western blotting with ATF6 antibody. A processed form of ATF6 (N) was detected in all HBX positive HCC cells which indicated that these cells were in a state of elevated ER stress and that UPR was activated (Figure 3.13A). Based on this observation, we hypothesized that UPR might be triggered by the accumulation of HBX-induced misfolded N-CoR protein. To test this hypothesis, HeLa cells were co-transfected with HA-tagged ATF6 (HA-ATF6), in which HA was tagged in frame to ATF6 sequence, with N-CoR or HBX plasmids. Since dithiothreitol (DTT) treatment has been reported to induce ATF6 activation, HeLa cells transfected with HA-ATF6 alone and treated with DTT was included as a control (Ng et al., 2006). This experiment was performed in HeLa cells because these cells have been frequently used in studies of the ER stress-induced activation of ATF6. As expected, ATF6 processing was observed in HeLa cells co-transfected with N-CoR, HBX and HA-ATF6; in contrast no ATF6 processing was observed when ATF6 was expressed alone (Figure 3.13B). Simultaneously, IF assay was carried out to further confirm that HBX-induced N-CoR misfolding triggers the UPR event. Transfection of HeLa cells was done in a similar way to the experiment mentioned above. Cells were then stained with HA 63 antibody for ATF6 detection and DAPI for nuclear staining. Intensity of ATF6 (red) signals in the nucleus of HeLa cells increased significantly when ATF6 was cotransfected with N-CoR and HBX (Figure 3.13C). Conversely, intensity of ATF6 signals appeared to be weaker in the other sets of transfected cells. The increased nuclear staining of ATF6 in N-CoR and HBX transfected cells could be due to the nuclear localization of cleaved 50 kDa HA tagged ATF6 fragment. This data corroborated with the findings of western blot analysis. 3.3.3 HBX abrogates N-CoR mediated repression of the ATF6 promoter To further investigate the regulation of UPR by HBX and N-CoR, we determined ATF6 activity by studying the relative induction of a reporter containing 5XATF6 response element through luciferase assay in HeLa cells. As presented in Figure 3.14, dose dependent reduction in ATF6 luciferase activity was achieved when N-CoR was ectopically introduced in HeLa cells. This trend was reversed when HBX was ectopically introduced in a dose dependent manner. Consistent with N-CoR’s  role   as a transcriptional repressor, our data suggested that N-CoR could block ATF6 activation, leading to UPR inhibition. On the other hand, HBX abrogated the N-CoR mediated repression of ATF6 as ATF6 activity was restored to its original level in the presence of HBX. This finding further supports the role of HBX-induced misfolded N-CoR in UPR activation. 64 A C B 65 Figure 3.13. Regulation of UPR by HBX and N-CoR (A) Whole cell lysates were used for western blotting with the ATF6 antibody. P and N indicated the position of precursor and nuclear (activated) forms of ATF6 protein. ATF6 precursor was cleaved in all the HCC cells. (B) Processing of ATF6 was determined in HeLa cells transfected with HA-ATF6 along with plasmids mentioned on top of each lane. In the first lane, extract of cells treated with DTT was used as positive control. Precursor and processed ATF6 proteins were detected with HA antibody. Proteolytic cleavage of ATF6 protein was seen in HeLa cells co-transfected with N-CoR and HBX. (C) HeLa cells were transfected with the HA-ATF6 plasmid, together with the various combinations of plasmids as shown on the left of each row. Sub-cellular localization as well as intensity of ATF6 (red) signal was determined using fluorescence microscopy. Strong intensity of red signals was detected in the presence of N-CoR and HBX, suggesting that HBX-induced misfolded N-CoR could activate ATF6 processing. Figure 3.14. Ectopic expression of HBX in HeLa cells repressed ATF6 promoter activity Effect of ectopic N-CoR on the activity of ATF6 promoter in HeLa cells transfected with varying concentrations of N-CoR-flag plasmid was determined in standardized luciferase assay. The value presented in each bar represents the average of three independent experiments. HBX blocked the N-CoR-induced repression of ATF6 promoter in a dose dependent manner. 66 3.4 Investigating the molecular mechanism underlying the misfolded N-CoR induced transformation of HCC cells Several mechanisms have been implicated in the pathogenesis of HCC. In particular, some pathways, for example chronic inflammation, EMT and autophagy are known to be regulated by normal N-CoR. Based on this; we tested which of these mechanisms would be activated due to N-CoR misfolding. A. N-CoR misfolding and Chronic inflammation 3.4.1 Upregulation of APR gene transcript in HCC cells In hepatic cells, N-CoR has recently been identified as a negative regulator of a set of pro-inflammatory genes known as acute phase response (APR) genes which include haptoglobin, Serum Amyloid   A   (SAA)   fibrinogen   β   and   PLAT (PAA-1) (Nicolas et al., 2010). Based on this report, we hypothesized that natively folded NCoR could play a role in the repression of APR genes in normal liver cells whereas HBX-induced misfolded N-CoR in HBX-positive HCC cells could have led to derepression of these genes. To test this hypothesis, we determined the level of transcript of these 3 APR genes in HCC cells through semi-quantitative RT-PCR. The result showed that expression levels of the 3 APR genes across all HBX-positive HCC cells was considerably high (Figure 3.15). This suggested that HBX-induced N-CoR misfolding and subsequent loss may have abrogated its transcriptional ability to repress the APR target genes. 3.4.2 Pro-inflammatory cytokine abrogates N-CoR function The inverse correlation between N-CoR and APR genes suggests that pro- inflammatory oncogenic signal could drive liver transformation by overriding the NCoR mediated transcriptional control of pro-inflammatory APR genes. To verify this 67 hypothesis, the induction of degradation of N-CoR upon stimulation with an oncogenic pro-inflammatory signal was determined. Since IL-1β  has  been  reported to induce the dissociation of N-CoR or SMRT co-repressor complexes from APR promoters (Nicolas et al., 2010), we stimulated HepG2 cells (has intact N-CoR) with 2.5nM and 5.0nM 1L-1β   for   4   hours   and   determined the status of N-CoR protein through western blot. Figure 3.16 showed that in comparison to the untreated cells, the level of N-CoR protein was greatly diminished upon induction with IL-1β.  Taken together, we thought that N-CoR loss could contribute to the transformation of HCC cells through a change of cellular response to inflammation. Figure 3.15. N-CoR loss in HCC cells might be associated with an up-regulation of APR genes Semi-quantitative PCR analysis of selected APR genes in HCC cells. The APR genes are highly expressed across all the cell lines used. 68 Figure 3.16. Pro-inflammatory cytokine promotes degradation of N-CoR Endogenous N-CoR protein in HepG2 was lost after treatment with 5.0nM IL-1β  for  4 hours. 3.4.3 APR genes are not repressed by N-CoR Next, to confirm if N-CoR was really involved in the repression of APR genes, we used genistein which had previously been shown to restore N-CoR in SK Hep1 cells (Figure 3.3) to analyse the effects of N-CoR restoration on APR gene expression. RT-PCR analysis of APR gene expression after genistein treatment revealed an up-regulation of mRNA expression levels for all the 3 APR genes in a dose dependent manner (Figure 3.17). This was in contrast to the hypothesized role of N-CoR in the repression of APR genes. Thus, we ruled out the possibility of misfolded N-CoR in the activation pro-inflammatory APR response in HCC cells. 69 Figure 3.17. APR genes were up-regulated in a dose dependent manner after genistein treatment APR gene expression levels in SK Hep1 cells after genistein treatment was determined via semi-quantitative RT-PCR. B. N-CoR misfolding and Epithelial Mesechymal Transition (EMT) 3.4.4 The levels of EMT gene transcripts in HCC cells are high N-CoR has been implicated in the activation of phosphatidylinositol 3' kinase (PI3K)/AKT pathway, a central feature of epithelial-mesenchymal transition (EMT). Thus, we wanted to investigate if N-CoR misfolding was linked to the activation of EMT pathway. Four characteristic EMT genes (Slug, Twist-1, Snail and Vimentin) were selected and their expression levels were screened through semi-quantitative RT-PCR. Based on similar hypothesis as described in 3.4.1, we wanted to identify the genes whose expression would reflect an inverse correlation to N-CoR status. Figure 3.18 showed that there was no distinct inverse correlation between N-CoR and the EMT genes with the exception for Slug. 70 Figure 3.18. Expression level of EMT genes Semi-quantitative PCR analysis of selected EMT genes in N-CoR expressing HepG2 cells and HBX positive HCC cells where N-CoR is lost. 3.4.5 EMT genes are not repressed by N-CoR To gain further insight if EMT may be a functional consequence of N-CoR loss, similar experiment as described in 3.4.3 was carried out. SK Hep1 cells were treated with 25µM genistein for 72 hours to restore N-CoR protein and the level of various EMT genes was screened via RT-PCR. This screening did not reveal any down-regulation of EMT genes after N-CoR restoration, indicating that none of these genes were possible N-CoR transcriptional targets (Figure 3.19). Hence, we eliminated any possibility of EMT’s  involvement  in  the transformation of HCC cells. 71 Figure 3.19. N-CoR did not repress EMT gene expression after genistein treatment EMT gene expression levels in SK Hep1 cells after genistein treatment were determined via semi-quantitative RT-PCR. The EMT genes were not repressed upon genistein treatment. C. N-CoR misfolding and Autophagy 3.4.6 Autophagy is activated in HCC cells Autophagy has recently been reported to be a cellular survival mechanism activated in mammalian cells in response to nutrient deprivation and stress. Several laboratories have also reported that autophagy could be induced by ER stress (Kawakami et al., 2009; Yorimitsu et al., 2006). Based on these reports, we decided to investigate if there was activation of autophagy in HCC cells where ER stress level was found to be high (Figure 3.12). The relative levels of LC3-II in the HCC cells were determined through western blotting analysis. LC3-II, the processed form of LC3, has a reported molecular weight of 14 kDa while LC3-I, the unprocessed form, was reported to be a 16 kDa protein. The LC3-II/LC3-I ratio was commonly used as an indicator for autophagy activity as reported earlier in several publications. This 72 however was debatable due to the dynamic nature of autophagic processes. Thus, a better approach of autophagy assessment would be to determine the expression level of LC3-II alone. We observed higher levels of LC3-II in majority of the HBX positive cell lines when compared to HepG2, suggesting a selective up-regulation of autophagy in HBX positive cells (Figure 3.20). 3.4.7 Formation of autophagosomes in HBX positive cells Autophagy is also characterised by the formation of autophagosomes. The hallmark of autophagosomes is a punctated dot-like distribution of LC3. We then conducted immunofluorescence staining to check the presence of autophagosomes by determining the distribution of LC3 protein. Their presence in HBX positive cells was evidenced by the numerous small peri-nuclear punctated red signals (Figure 3.21). We also observed partial co-localization of N-CoR (green signals) with LC3 as indicated by the speckled orange-yellow signals (Figure 3.21, overlay). This incomplete overlapping of N-CoR with LC3 could be due to the rapid degradation of N-CoR protein in the autophagosomes. In contrast, the red LC3 signal was greatly diminished in HepG2 cells with no co-localization of N-CoR and LC3 (Figure 3.21). 73 Figure 3.20. Level of LC3-II in HBX positive cells is elevated Level of LC3 in HCC cells (upper panel) was determined through western blot with LC3 antibody. The higher level of LC3-II seen in the HBX positive cells indicated an activated level of autophagy. Coomassie staining (lower panel) served as an experimental control. 74 75 Figure 3.21. Autophagosomes are seen in HBX positive cells Cells were fixed and stained for endogenous N-CoR (green) and LC3 (red) with NCoR and LC3 antibodies respectively. DNA was stained with DAPI (blue). Colocalization of N-CoR and LC3 was observed as orange-yellow signals in the HBX positive cells. There was no co-localization observed in HepG2 cells. In addition, the punctuated dot like pattern observed in HBX positive cells indicated the presence of autophagosomes. No dot like pattern was observed in HepG2. 76 3.4.8 Loss of N-CoR may be linked to autophagy Next, to study if N-CoR was degraded via autophagy in HCC cells, we tested the effect of bafilomycin A1 (BA-1), a chemical inhibitor of autophagy, on N-CoR loss in SK Hep1 cells. Western blot data showed that BA-1 promoted stabilization of N-CoR at a concentration of 0.5nM, suggesting that N-CoR loss in SK Hep1 cells could be due to autophagy. Figure 3.22. Bafilomycin A1 promotes stabilization of N-CoR protein in SK Hep1 cells SK Hep1 cells were treated with BA-1 for 72 hours and the level of full length N-CoR protein was determined by western blotting assay. BA-1 can restore N-CoR protein at 0.5nM. 77 3.4.9 HBX activates autophagy Our finding demonstrated that misfolded N-CoR could be degraded via autophagy (Figure 3.22). Since N-CoR misfolding is a consequence of HBX engagement (as discussed in the earlier sections), therefore we next investigated whether ectopic HBX could activate autophagy in an N-CoR dependent manner. The effect of HBX on autophagy was assayed by determining the level and distribution of LC3-II, the processed form of LC3 linked to GFP. Our western blot data revealed that in the presence of HBX and N-CoR, LC3-II level was slightly elevated when compared to its level in HeLa cells transfected with HBX alone (Figure 3.23A). For immunofluorescence (IF) assay, HeLa cells were transfected in a similar way as above and the presence of autophagosomes was detected using fluorescence signals of LC3 protein (green signals). The intensity of green florescence signals derived from LC3 was significantly higher when cells were transfected with HBX and N-CoR (Figure 3.23B), suggesting an increased formation of autophagosome when NCoR and HBX were expressed together. In contrast, HBX or N-CoR alone failed to induce a similar level of autophagic response. These findings collectively suggest that HBX-induced misfolded N-CoR may play a role in the induction of autophagy. 3.4.10 Autophagy supports the growth of HCC cells Based on the findings presented so far, we thought that activation of autophagy-mediated N-CoR degradation could confer pro-survival advantage to tumour cells during stress and starvation. Thus, to investigate if N-CoR was involved in sustaining the growth and survival of HCC cells, we determined the growth and survival of HCC cells before and after siRNA mediated N-CoR ablation in SK Hep1 cells. 78 A B 79 Figure 3.23. HBX induced activation of autophagy is mediated by misfolded NCoR (A) Processing of LC3 was determined in HeLa cells transfected with GFP-LC3 expression plasmid along with plasmids mentioned on top of each lane. LC 3-I and LC3-II indicated the position of unprocessed and processed (activated) forms of LC3 protein respectively. LC3 protein was detected using the GFP antibody. Higher level of LC3-II was observed in HeLa cells transfected with HBX and N-CoR. (B) HeLa cells were transfected with the GFP-LC3 plasmid, together with the various combinations of plasmids as shown on the left of each row. Sub-cellular localization as well as intensity of LC3 (green) signal was determined using fluorescence microscopy. Punctuated dot like structures appeared to be more prominent in the presence of HBX and N-CoR. First, efficiency of N-CoR ablation by N-CoR siRNA in SK Hep1 cells was confirmed by RT-PCR (Figure 3.24A). Next, the survival and growth of N-CoR ablated cells was compared to non-ablated cells through morphological analysis. After N-CoR ablation in SK Hep1 cells, there was a significant reduction of growth along with a noticeable increase in the number of dead cells, suggesting that N-CoR catabolism might be directly linked to the growth and survival of these cells (Figure 3.24B). Next, to examine if ATP generated from the proposed N-CoR catabolism was involved in maintaining the growth and survival of SK Hep1, the ATP levels in these cells was determined before and after N-CoR knockdown. A significant reduction (around 50%) in the intra-cellular ATP level was observed after N-CoR ablation, suggesting that autophagy-mediated degradation of N-CoR could act as a source of ATP generation in SK Hep1 cells (Figure 3.24C). 80 A C B Figure 3.24. N-CoR loss is linked to ATP mediated growth of SK Hep1 cells (A) Level of N-CoR transcript in SK Hep1 cells exposed to scrambled or N-CoR siRNA was determined via RT-PCR. (B) The growth and survival of SK Hep1 cells exposed to scrambled or N-CoR siRNA was determined by morphological analysis. (C) Intracellular ATP level in SK Hep1 cells exposed to scrambled or N-CoR siRNA was determined by ATP assay. Around 50% reduction in ATP level was observed after N-CoR knockdown. 81 Chapter 4. Discussion 4.1 Role of misfolded N-CoR in HCC pathogenesis Misfolded N-CoR was first implicated in the pathogenesis of APL when it was found that PML-RARα, the fusion oncoprotein linked to transformation in APL, could induce N-CoR misfolding and loss through aberrant post translational modification. The misfolded N-CoR protein accumulated in the ER and activated unfolded protein response (UPR), resulting in N-CoR degradation ultimately (Khan et al., 2004). Based on APL data, it was essential to establish a role of misfolded N-CoR in other tumour as well. Since then, role of misfolded N-CoR has been established in several tumours such as lung and gastric cancer cells (data not shown) and HCC cells (Figure 3.1A). As presented in this report, we then analyzed the status of N-CoR protein in human liver cancers. An APL-like loss of N-CoR protein was observed in human primary cancer cells (Figure 3.1B), suggesting a potential pathophysiological role of N-CoR in the pathogenesis of liver cancer. Hence, we investigated the role of misfolded N-CoR based on the criteria established in APL cells. 4.2 HBX induces a conformational change in N-CoR Previously, it has been demonstrated that PML-RARα-induced aberrant post- translational modification of N-CoR contributed to its misfolding and instability in APL cells (Ng et al., 2006; Khan et al., 2004). PML-RARα can trigger a conformational change in N-CoR by inducing its ubiquitination and phosphorylation. Therefore, it is likely that MCDL of N-CoR in HCC cells is an outcome of similar post-translational modifications triggered by a yet to be identified oncogenic alteration. The findings reported here strongly suggested that viral proteins like HBX can create a PML-RARα like effect on the conformation of N-CoR protein. A study 82 has shown that the integrity of PML oncogenic domain (POD), of which N-CoR is an important component, was altered in several cases of viral infections (Day et al., 2004). Therefore, N-CoR itself seems to play an important role in the structural and functional integrity of POD which the oncogenic viruses like Hepatitis B virus (HBV) and Hepatitis C virus (HCV) must overcome to invade the cells and promote transformation. Based on the data presented so far, it is hypothesized that HBX may disrupt POD structural integrity and N-CoR mediated transcriptional repression activity in HCC cells. The molecular mechanism underlying the oncogenic potential of HBX is still unclear but it is possible that cytoplasmic HBX may engage N-CoR just after translation in the cytosol. All in all, we hypothesized that N-CoR may have acquired an abnormal conformation due to this engagement by HBX. 4.3 HBX-induced conformational change leads to instability and N-CoR protein degradation There are protein quality control mechanisms in the cell which are activated to deal with the consequence of protein misfolding. The first effect of accumulation of misfolded proteins is the activation of unfolded protein response (UPR) which activates chaperones to correct misfolding. However, if misfolded proteins fail to be corrected, they will be removed through the ubiquitin proteosome pathway (UPP). Most misfolded proteins that are cleared via the UPP tend to be ubiquitinated. Consistent with this notion, misfolded N-CoR appears to be highly ubiquitinated (Figure 3.10B, 3.11B), suggesting that misfolded N-CoR might be a target of the UPP. Based on this data, we hypothesized that the ubiquitination observed in HCC is a form of post translational modification (PTM) that tags misfolded N-CoR, so that it will be recognized by the proteasome for degradation. This then raises the question of 83 how misfolded N-CoR is ubiquitinated. It was hypothesized in APL that PML-RARα functioned as E3 ligases in N-CoR ubiquitination (Khan et al., 2004). The exact role of HBX underlying the ubiquitination of N-CoR protein remains unclear but based on the hypothesis in APL and our preliminary findings (Figure 3.10B, 3.11B); we speculated that HBX, like PML-RARα, could have function as an E3 ligase. Although misfolded N-CoR is highly ubiquitinated, it may not be entirely degraded by UPP since MG132 treatment did not result in complete N-CoR stabilization (Figure 3.10A, 3.11A). It could be possible that the proteasome function is inhibited by the misfolded protein itself thus preventing its effective removal. There is also evidence that complete removal of misfolded proteins by UPP is not achieved due to a direct inhibitory effect of misfolded proteins on UPP. For example, this situation  has  been  reported  in  Parkinson’s  disease  whereby  the  toxic  accumulation  of   abnormal proteins (Lewy bodies) inhibited the function of UPP. This led us to speculate that misfolded proteins may be ubiquitinated but not necessarily degraded by the proteasome under pathological conditions. So under this stressful condition whereby degradation might be impaired, how do tumour cells remove these aggregated proteins? 4.4 Role of autophagy in the survival and growth of HCC cells Degradation of misfolded N-CoR has been implicated to be an outcome of autophagic response (Figure 3.22) in HCC cells. Reports of autophagy being involved in the removal of misfolded proteins and in the alleviation of ER stress to noncytotoxic levels have also been described in Huntington disease (Ravikumar et al., 2002),  Alzheimer’s  disease  (Boland  et al., 2008) and Parkinson disease (Dice, 2007). Therefore, it is likely that in situations whereby the proteasome is overwhelmed with 84 a deleterious level of misfolded proteins, autophagy might be activated to remove the excess misfolded protein load. This degradation serves as a way to offset the toxic insult of misfolded proteins, allowing tumour cells to gain survival advantage. In addition, autophagy has been reported to promote catabolic reactions to satisfy the energy requirements of cells for maintenance and protein synthesis by recycling metabolites under stress conditions (Kadowaki et al., 2006; Levine and Kroemer, 2008). Therefore, other than protecting cells by clearing the misfolded protein load, we hypothesized that autophagy-mediated degradation of N-CoR can also sustain the growth of HCC cells by recycling the N-CoR end products into ATP. The inhibition of cell growth (Figure 3.24B) and ATP level reduction (Figure 3.24C) after N-CoR ablation suggested that this hypothesis may be true. 4.5 Role of HBX-induced misfolded N-CoR in the activation of UPR and autophagy We think that accumulation of HBX-induced misfolded N-CoR protein in the ER of normal hepatic cells induces ER stress which ultimately leads to UPR-induced apoptosis. This phenomenon is known as cytotoxic UPR. Indeed, we observed a shrinkage/condensation of nucleus in HBX transfected 293T cells (refer to arrows in Figure 3.5) which are indicative signs of cell death. Supposingly, this should eliminate the possibility of tumour formation. So, how do the tumours arise if the HBX infected hepatocytes are killed due to ER stress? Based on our findings and the APL model, we proposed a novel component of UPR (cytoprotective UPR) that might induce transformation in cancer cells. We hypothesized that in HCC cells, a bulk of misfolded N-CoR protein (induced by HBX) will first be transported to the ER and degraded via the UPP (Figure 3.10, 3.11). The degradation helps HCC cells to survive the initial cytotoxic effects of UPR 85 and may possibly be the precursor for transformation. Subsequently under prolonged ER stress induced by the misfolded proteins whereby proteasomal-mediated degradation becomes defective, misfolded proteins may be targeted to the autophagic vacuoles for further removal (Figure 3.20, 3.21). Since autophagy supports cell growth and survival, a clonal growth advantage will be conferred to each transformed cell that ultimately forms the tumour bulk. In conclusion, we hypothesized that accumulation of misfolded N-CoR protein probably triggers synergistic actions on the proteasome and autophagy, thereby contributing to the survival of HCC cells through neutralization of ER stress and protection from UPR-induced apoptosis. At the same time, growth of HCC cells in a stressful and nutrient depleted condition is sustained by ATP generated from the autophagy induced degradation of misfolded N-CoR protein. A schematic model for the role of HBX-induced misfolded N-CoR protein in the survival and growth of HCC cells is presented (Figure 4.1). This model highlights the multiple connections between mechanisms behind N-CoR loss, resistance to UPR-induced apoptosis and autophagy triggered survival and growth pathways. 86 A Cytotoxic UPR N-CoR + N-CoR HBX HBX Misfolded protein B ER Stress Cell death Elimination of seed Cytotoxicity Cytoprotective UPR/autophagy N-CoR + HBX N-CoR N-CoR HBX PML HBX RAR Neutralization of ER Stress Cytoprotection Cell survival Activation of cytoprotective UPR Malignant growth Figure 4.1. Schematic representation of the regulation of UPR and autophagy in normal and HCC cells. A. In normal hepatic cells, accumulation of HBX-induced misfolded N-CoR protein in the ER will stimulate ER stress which would ultimately lead to UPR-induced apoptosis, thus eliminating the possibility of transformation. B. In HCC cells, however, toxicity associated with misfolded HBX-N-CoR will initially be neutralized due to the ubiquitin-proteasome mediated degradation of misfolded N-CoR protein. With further accumulation of misfolded proteins, autophagy is activated and assists in the clearance of misfolded proteins. This results in reduction of ER stress and eventual protection of HCC cells from UPR-induced apoptosis. HBX containing cells remain viable and with the help of autophagy in recycling the catabolites, the energy generated favours their growth. 87 4.6 Future areas of research 4.6.1 Mechanism of autophagy mediated N-CoR loss in liver cancer pathogenesis Our recent data suggest that autophagy could be the major pathological mechanism in liver malignancy. Nevertheless, there is a need to interpret this data with care and more experiments should be done to verify the hypothesis. The induction of autophagy could be verified further with p62, a widely used autophagy marker (Bjørkøy et al., 2009). Also, given that an increase in autophagy would bring about a parallel increase in lysosomal proton pumping and lysosomal acidity, an acidotropic dye, for example, acridine orange could be used for staining lysosomes based on pH (Paglin et al., 2001; Kanzawa et al., 2003). In addition, the presence of autophagosomes should be further confirmed using advanced techniques like electron microscopy (Dunn, 1990). All in all, there appears to be a gap in knowledge between how the misfolded protein from the initiation of UPR contributes to the subsequent malignant growth in HCC cells. More work is required to establish this link. 4.6.2 Identification of misfolded N-CoR as a molecular target in HCC The ultimate goal of this study is to discover potential therapeutic targets to improve cancer therapy. The limitation in the development of safe and effective anticancer drugs and devices lies in our inability to selectively kill cancer cells with agents or process which will have minimum effect on normal cells. Unfortunately, despite significant progress in our understanding about the mechanisms underlying malignant growth and transformation of tumour cells, we still do not have a precise understanding of the difference between a cancer cell and its normal counterpart. During the past decade, efforts to characterize that difference was mostly focused on identifying cell surface markers that are uniquely present only on cancer cells. 88 However, despite the intense search, only a handful of antigens have been identified so far that are specifically associated with a particular type of cancer cell. The outer membrane of a cancer cell may not always have a well defined signature that could distinguish it from a normal cell, however, somewhere among the thousands of macromolecules packed within each cancer cell are one or more proteins which bear the footprints of the fateful events that led the cell on the path of transformation. Therefore, to a large extent, identification and characterization of these protein conformation based oncogenic hallmarks within an intact cancer cell will not only enable us to detect the earliest sign of disease event, but will also help in creating highly selective therapeutic tools. Protein products of oncogenes and tumour suppressor genes, as well as proteins whose structures and functions are affected due to direct or indirect association with these proteins may represent the best possible intracellular signature for selective targeting of cancer cells. Identification and characterization of conformational defect of N-CoR protein in HCC may possibly be useful in the development of novel conformation based therapeutic agents. Previously, a role of genistein as a potent inhibitor of N-CoR misfolding has been demonstrated in APL (Ng et al., 2007). Interestingly, we found out that genistein could promote stabilization of N-CoR protein in HBX positive SK Hep1 cells (Figure 3.3). Genistein works by correcting the conformation of N-CoR protein and thus restores its tumour suppressive functions. We have also showed that MG132 (proteasome inhibitor) and BA-1 (autophagy inhibitor) could promote stabilization of N-CoR protein in HCC cells (Figure 3.11A, 3.22). Since degradation of misfolded N-CoR might confer stress tolerance and protect cells from ER stressinduced apoptosis, hence, by inhibiting N-CoR degradation, this escape route will be 89 blocked and tumour cells will be re-sensitized to ER stress-induced apoptosis. As such, there will not be malignant transformation. Encouraged by this, we intend to test the pro-apoptotic effect of a clinical grade proteasome inhibitor, bortizomib in HCC cells. All in all, investigation on the mechanisms behind N-CoR misfolding/loss provides useful information for therapeutic purposes. 4.6.3 Appropriate experimental controls By and large, the data obtained so far is undoubtedly incomplete and fragmented. One critical issue in this study is the unavailability of reliable and positive controls. Two different types of cell lines (HepG2 and HBX positive HCC cells) with differing HBX and N-CoR properties were used in this study. Although HepG2 is HBX negative and harbours intact N-CoR protein, it is not an appropriate control because of its inherent tumourigenic properties. This probably explains for the inconsistencies that we have observed in our experiments. For example, we did not expect to detect a high level of GRP78 in HepG2 (Figure 3.12C). 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J Cell Biol. 162, 59-69 [...]... classes of proteins in the ER assist nascent proteins to fold properly – molecular chaperones like glucose-regulated proteins (GRPs) GRP78, GRP94 and lectin-like proteins, folding enzymes like protein disulpide isomerase (PDI) and ERdegradation-enhancing  α-mannosidase-like protein (EDEM) (Schroder et al., 2005) One of the most abundant proteins in the ER is binding Ig protein (BiP) BiP/GRP78- a member of. .. loss of N-CoR protein in HCC cells 43 Figure 3.2 Evidence of N-CoR loss in primary human liver cancer samples 44 Figure 3.3 Genistein promotes stabilization of N-CoR protein in SK Hep1 cells 45 Figure 3.4 N-CoR protein level in HCC cells is inversely related to HBX transcript level 46 Figure 3.5 HBX promotes cytosolic retention of N-CoR protein 47 Figure 3.6 N-CoR of HBX positive HCC cells is found in. .. Figure 3.7 N-CoR protein is mainly localized in the cytosol in tumour sections 51 Figure 3.8 HBX can induce N-CoR insolubility 53 Figure 3.9 HBX promotes N-CoR degradation 54 Figure 3.10 HBX promotes ubiquitination of N-CoR protein 56 X Figure 3.11.A-B Ubiquitin-proteasome mediated degradation of N-CoR protein in HBX positive HCC cells 57 Figure 3.12.A-D HBX positive cells exhibit high level of ER stress... lectin like proteins Numerous cycles of binding to and release from calnexin and calreticulin alternates until the protein is correctly folded or is sentenced for degradation by EDEM by removing its mannose residue (Ellgaard et al., 2003) GRP94, another abundant chaperone protein has been reported to bind partially folded proteins that are unfolded by GRP78 or calnexin/calreticulin and retaining them in. .. 30 Table 2.9 Clinical characteristics of patient samples used in this study 31 IX LIST OF FIGURES Figure 1.1 Model of bifunctional role of nuclear receptor corepressor in acute promyelocytic leukemia pathogenesis 11 Figure 1.2 UPR signalling pathways in mammalian cells 16 Figure 1.3 ER stress pathways implicated in mediating cell apoptosis 18 Figure 1.4 Kinetics of N-CoR misfolding in APL 25 Figure... monoglucosylated N-linked glycans are bound to lectin like proteins - calnexin (transmembrane ER protein) and calreticulin (soluble luminal ER protein) (Bergeron et al., 1998) Glucosidase II then removes the glucose residue on the oligosaccharide and releases the bound glycoprotein If the glycoprotein remains unfolded, it is re-glucosylated by UDP-glucose:glycoprotein glucosyltransferase (UGGT) for interaction... bovine serum XII GFP green fluorescent protein GRP glucose-regulated protein HBV hepatitis B virus HBX hepatitis B virus X protein HCC hepatocellular carcinoma HCV hepatitis C virus HDACs histone deacetylase complexes HMW high molecular weight HRAS Harvey-ras proto-oncogene Hsp heat shock protein IF immunofluorescence IHC immunohistochemistry IRE1 inositol requiring kinase 1 JNK c-Jun N-terminal kinase... proliferation through protein- protein binding This mechanism allows HBX to undergo favourable alteration in the cellular environment for further viral replication (Tang et al., 2006) In the infected liver host cells, HBX appears to induce variety of responses such as genotoxic stress response, transcription modulation, protein degradation, cellular signalling pathways, cell cycle checkpoints and apoptosis... Unfolded protein response ER stress occurs when the processing capacity of the ER is overwhelmed The normal physiological state of the ER is thus perturbed Unfolded protein response (UPR) is then activated in response to restore a favourable physiological state UPR involves three main signalling pathways: the activating transcription factor 6 (ATF6) pathway, the inositol requiring kinase 1 (IRE1) pathway. .. ER resident transmembrane proteins are thought to sense ER stress through Grp78 binding/release via their respective luminal domains, although structural studies have also suggested that IRE1 may interact with infolded proteins directly The GADD34 protein, a protein phosphatase up-regulated by the PERK pathway, dephosphorylates eIF2α to restore global protein synthesis 17 In response to ER stress, .. .ROLE OF PROTEIN MISFOLDING PATHWAY IN HBX- INDUCED HEPATOCELLULAR CARCINOMA (HCC) TAN SU YIN (B.Sc.(Hons.), NTU) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF MEDICINE... consisting of the constitutive form of the heat shock 70 kDa protein (hsc70), the heat shock protein of 40 kDa (hsp40), the heat shock protein of 90 kDa (hsp90), the hsc70-interacting protein (hip),... catabolism, in HCC cell growth These findings identify an important role of autophagy -induced recycling of misfolded N-CoR protein in the selective activation of autonomous survival and growth in HCC