TLR3 INDUCES HEPATOCELLULAR CARCINOMA CELL DEATH AND INCREASES NATURAL KILLER CELL ACTIVITY TOW TINGTING CHARLENE NATIONAL UNIVERSITY OF SINGAPORE 2010 I TLR3 INDUCES HEPATOCELLULAR CARCINOMA CELL DEATH AND INCREASES NATURAL KILLER CELL ACTIVITY TOW TINGTING CHARLENE (B.Sc. (Hons),NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE II ACKNOWLEDGEMENTS My appreciation goes to Jean-Pierre Abastado, who has taken me patiently through my research in tumour immunology. His support and encouragement is what I would not be able to do without in the course of my studies. I would also like to thank Alessandra Nardin, who has been my supervisor, mentor and friend, for her help and guidance even when she has relinquished her duties in our lab. I would also like to express my appreciation to all the wonderful members of the Tumour Immunology lab who have made my duration in the lab both fun and memorable. Thank you especially to Valerie who have provided me with directions and who have contributed to this study, Xilei who have patiently taken me through the various techniques and been very approachable to all matters, and to Ben who have provided help and advice whenever possible. I would also like to express my thanks to the labs in SIgN who have helped me in one way or another. Next, I would like to extend my appreciation to our collaborators, Dr Irene Ng from the university of Hong Kong, Dr Toh Han Chong from the National Cancer Centre, Singapore and Adam Gehring from the Institute of Molecular Biology. Finally, I would like to express my gratitude to my family members and friends who have been my pillar of support in my studies. Thank you! I CONTENTS ACKNOWLEDGEMENTS ................................................................................................ I CONTENTS ....................................................................................................................... II SUMMARY ....................................................................................................................... V LIST OF FIGURES ........................................................................................................ VII LIST OF TABLES ......................................................................................................... VIII 1. INTRODUCTION ................................................................................................. 1 1.1 Hepatocellular Carcinoma: Incidence and Etiological factors............................... 1 1.2 Current management strategies for HCC 4 1.2 Current management strategies for HCC 5 1.3 Activation of the immune system has been associated with liver cirrhosis 8 Appropriate activation of the immune system can control tumour progression 10 Toll-Like Receptors 16 1.4 1.5 1.5.1 Role of TLR3 in anti-viral detection................................................................. 18 1.5.2 Localization holds the key to TLR3 activity ................................................... 19 1.5.3 Roles of TLR3 in antiviral and crosspriming responses. ............................... 20 1.6 TLR3 expression on tumour cells 22 1.7 The relevance of TLR3 in HCC tumours cells 24 1.8 The Role of TLR3 in Hepatic Cell Death. 26 1.9 Natural Killer cells and their relevance to HCC 28 1.10 Clinical use of TLR agonists 30 2. MATERIAL AND METHODS ............................................................................ 32 2.1 Reagents 32 2.2 Patient Samples 32 2.3 Cell Isolation 32 2.4 Cell Culture 33 II 2.5 Total RNA Extraction and cDNA Synthesis 2.6 Primer Design and quantitative PCR .................................................................... 34 2.7 Fluorescence-based Flow Cytometry 34 2.8 Silencing of TLR3 using siRNA 34 2.9 Cytotoxicity assay 35 2.10 Immunohistochemistry 36 2.11 ELISA 37 2.12 Statistical Analysis 38 3. RESULTS ............................................................................................................. 39 3.1 TLR3 expression within tumours correlates with prolonged HCC patient survival. 39 3.2 TLR3 is expressed by cancer cells and NK cells. 44 3.3 TLR3 expressed by both the cancer cells and NK cells are associated with longer patients survival in 2 cohorts. 46 HCC cells express functional TLR3 and triggering TLR3 augments its expression. 49 3.5 DsRNA triggers cell death in HCC cells that express TLR3. 52 3.6 DsRNA induced tumour cell death is mediated by TLR3. 56 3.7 Activated NK cells increases with dsRNA stimulation. 60 3.8 NK cells have higher TLR3 expression compared to HCC cells and dsRNA can increase the expression of TLR3 in NK cells. 62 DsRNA augments the expression of NK cells effector molecules and increases cell mediated cytotoxicity towards HCC cells. 64 TLR3 expression in both cancer and NK cells is associated with tumour cells apoptosis and non-proliferating tumours. 65 TLR3 expression decreases in higher grade tumours and more advanced stage patients 69 3.4 3.9 3.10 3.11 33 4. DISCUSSION ....................................................................................................... 71 4.1 The expression of TLR3 is associated with prolonged HCC patient survival 71 What drives intratumoral expression of TLR3? 72 4.2 III 4.3 Possible mechanisms to explain the association of TLR3 with prolonged HCC patient survival 75 4.4 Other indirect effects of TLR3 in controlling tumour progression 78 4.5 Selective advantage for tumours to loose TLR3 79 4.6 Clinical significance of TLR3 in HCC 80 4.7 Conclusion and perspectives 82 REFERENCES ................................................................................................................. 83 IV SUMMARY Hepatocellular Carcinoma Cancer (HCC) is a highly aggressive cancer generally caused by chronic liver disease. A persistent, non-specific and ineffective activation of the immune system within the chronically infected liver has been proposed as the main cause of repeated cycles of tissue damage, repair and regeneration that eventually promote carcinogenesis. However, appropriate activation of the immune system can also prevent tumour progression. We hypothesized that the tumour immune microenvironment could affect the progression of established tumours and evaluated the potential immune mechanisms associated with clinical end point in HCC. Our results show that HCC patient survival is positively correlated with a proinflammatory innate immune gene signature within the tumours. This signature includes cytokines such as TNF/ IL-6 and LTA and two Toll-Like-Receptors(TLR): TLR3 and TLR4. Importantly, the expression of TLR3 is found to be positively associated with patient survival in two independent cohorts of patients. In addition, the survival-associated cytokines are mostly derived from tumour-infiltrating immune cells while TLR3 is expressed by both immune and tumour cells. We show that some HCC cell lines express TLR3 and stimulation of these cell lines with polyinosinic-polycytidilic acid (Poly I:C), a TLR3 ligand, results in an increase in TLR3 expression and subsequent tumour cell death by 24 hr. Proliferating immune cells, predominantly NK and T cells are also found in tumours of patients with longer survival. The density of these cells is correlated positively with cancer cell apoptosis and negatively with their proliferation. Our data identify TLR3-expressing leukocytes to be NK cells. NK cells express TLR3 and can upregulate TLR3 upon poly I:C V stimulation. Furthermore NK cells, even in the absence of antigen-presenting cells, upregulate IFN-γ secretion in response to poly I:C. Data from our lab showed that IFN-γ potentiates CXCL10 secretion by HCC cells and macrophages and that CXCL10 is attracting Th1 and NK cells. Therefore our finding suggest that poly I:C-induced expression of IFN-γ by NK cells can reinforce this paracrine loop, leading to increased activity towards tumours. NK cells isolated from PBMC of normal donors and treated with poly I:C showed increased expression of TLR3, granzyme B and perforin. They also display an increased killing of HCC cells. Finally, TLR3 expression decreases with the advancement of the disease in HCC patients. Patients with lower grade and early stages of HCC have higher TLR3 expression than patients with higher grades and more advanced stages. This suggests that low expression of TLR3 by cancer cells confers a selective advantage for the tumour, since TLR3 can result in cell death of the tumour, either directly or through the action of immune cells such as NK cells. Therefore, an inflammatory environment within the HCC tumour could be an important means to control tumour progression via immune cells activation and proliferation. Such activation may be promoted by danger signals sensed by TLR such as TLR3 on both NK and tumours. VI LIST OF FIGURES Figure 1. Odd ratios for the different etiological agents for the development of HCC. .... 4 Figure 2. Diagram illustrating staging classification and treatments available for HCC. . 7 Figure 3. Mechanism of Cytotoxic T- lymphocyte (CTL)-induced liver disease and viral clearance. ........................................................................................................................... 9 Figure 4. Postulated mechanism of immune mediated cancer rejection. ......................... 12 Figure 5. High levels of innate immune genes in HCC core tumours are associated with prolonged patient survival................................................................................................ 15 Figure 6. Schematic overview of TLR signaling pathways. ........................................... 17 Figure 7. TLR3 expression within tumours correlates with prolonged HCC patient survival ............................................................................................................................. 42 Figure 8. TLR3 is expressed by both cancer cells and NK cells. ................................... 47 Figure 9. TLR3 expressed by both the tumour cells and NK cells is associated with prolonged patient survival................................................................................................ 48 Figure 10.TLR3 in HCC cells is functional and triggering TLR3 with dsRNA augments its own expression. ........................................................................................................... 51 Figure 11. DsRNA stimulation on TLR3-positive HCC cell can lead to cell death. ...... 54 Figure 12. TLR3 knock down inhibits poly I:C-induced cell death. ............................... 58 Figure 13. NK cells are activated by poly I:C stimulation. ............................................. 61 Figure 14. Poly I:C enhances NK cell activity and cell-mediated cytotoxicity ............... 63 Figure 15. TLR3 is associated with tumour cell apoptosis and non-proliferating tumours . .......................................................................................................................................... 67 Figure 16.TLR3 expression decreases in higher grade and more advanced stage patients. .......................................................................................................................................... 70 VII LIST OF TABLES Table 1. Age standardized incidence rates for HCC ........................................................................ 2 Table 2. Comparison of clinical and demographic characteristics of HCC patients from Singapore and Hong Kong. ............................................................................................................................. 40 VIII 1. INTRODUCTION 1.1 Hepatocellular Carcinoma: Incidence and Etiological factors Hepatocellular Carcinoma (HCC) is a primary malignancy of the liver epithelial cell and accounts for 90% of primary liver cancer. 5% of which are cholangiocarcinoma and the rest are of indeterminate origin. Angiosarcoma is extremely rare [1]. HCC is the third leading cause of cancer-related death worldwide [2]. Due to its high mortality rate, the fatality ratio (overall ratio of mortality to incidence) of HCC is close to one, indicating that most patients who develop liver cancer will die. According to the recent statistics in 2008, there are 696 000 deaths per year from liver cancer worldwide [2,3]. HCC has long been one of the most important cancers in the world, not only because of its high fatality ratio but also because it ranks highly in cancer frequency, being the fifth most common cancer in men and eighth in women. The incidence of HCC varies by geographical region. The incidence of HCC is higher in countries such as Africa, Asia, China and Africa compared to Europe, America and Australia/New Zealand. Global variations in incidence rates of HCC closely reflect the variation in risk factors for HCC. Therefore countries with a high occurrence of chronic hepatitis B virus (HBV) and /or hepatitis C virus have a higher incidence of HCC. This is consistent with the fact that about three-quarters of HCC are attributed to chronic HBV and HCV infections [2-4]. 1 Countries Men Women Mongolia 116.6 74.8 Middle East 18.9 9.6 China 37.4 13.7 Caribbean 6.3 4.4 Europe 25.4 8.8 America 19.4 13.1 Asia 29.2 12.9 Africa 64.1 28.8 Australia/New Zealand 5.0 2.0 Korea 38.4 10.6 Table 1. Age standardized incidence rates for HCC All values expressed per 100 000 of the population in 2008 (data from globocan 2008 (adapted from[5]) HCC generally arises as a consequence of underlying liver disease. Viral causes such as HBV and HCV as well as non-viral causes such as chronic alcoholism, tobacco use or family history such as diabetes, obesity, non-alcoholic steatohepatitis can promote an inflammatory condition which when it becomes a chronic condition can lead to cirrhosis. Figure 1 shows the different etiological agents for the development of HCC in the Asia Pacific region [3,6]. Cirrhosis is the condition in which scar tissue replaces healthy liver tissues, impairing the function of the liver. In this chronic inflammatory condition, and together with accumulated mutations, patients with cirrhosis may develop HCC. Other causes such as water contamination with Aflatoxin B that can cause DNA mutations, which can result in oncogene activation or loss of tumour suppressor genes. 2 Aflatoxin contamination can also act synergistically with HBV in the pathogenesis of HCC [7]. The integration of HBV genome after infection, and viral genes such as HBx, which can activate a variety of cellular promoters such as NK-κB and interfere with signal transduction pathways such as JaK1 and MAP kinases, may also account for the small percentage of cases which are associated with the development of HCC in the absence of cirrhosis [8]. The incidence of HCC is higher in males than in females. This striking gender disparity in the incidence of HCC suggests an important role of sex hormones in HCC pathogenesis. In a study by Naugler et al, the authors showed that estrogen could inhibit the secretion of IL-6 from Kuppfer cells and lead to a reduced chronic hepatitis [9]. Other effects such as the tumorigenic effect of androgens could also play a role in HCC. Inhibition of androgen in male mice leads to cell cycle arrest and induction of apoptosis due to increased synthesis of transforming growth factor-β 1(TGF-1)[10]. The expresson of VEGF, a target gene for androgen receptor in liver, have also reported to be enhance by androgen receptor and HCV infection [11]. Despite the associations of estrogen and androgen in HCC, hormonal treatments with HCC are still not very successful [12,13] and are not used as a therapy for HCC 3 Figure 1. Odd ratios for the different etiological agents for the development of HCC. (Yuen M.F et al [3]) . 4 1.2 Current management strategies for HCC The development of HCC in patients with cirrhosis is a stepwise process that starts with the development of nodules. As the disease progress, small vascular invasion leads to intrahepatic invasion and systemic dissemination. With further progression, the tumours can extend into larger hepatic vessels. Once this occurs, curative treatment is rarely possible. HCC has therefore been a difficult target for systemic therapies. Most of the available treatments are physical, which usually directly attack the tumour or its blood supply. The fact that most cases of HCC develop in the presence of cirrhosis with impaired liver function poses an additional challenge to HCC treatment. For the few patients with no cirrhosis, or for those with preserved liver functions and who develops HCC in one region of the liver, resection is the preferred option. However, only 20-30% of patients are eligible for such treatment because of advanced or multifocal disease. Liver transplantation is another preferred treatment options for HCC because it can firstly remove the tumour with any intrahepatic metastasis and secondly, cures the underlying cirrhosis. However, the lack of available organs have resulted in strict selection criteria so that only those patients with early HCC and the highest likelihood of survival after transplantation are chosen to undergo this procedure. Other non-surgical treatment for HCC includes percutaneous ablation (PEI) which works by directly injecting alcohol into the tumours and radiofrequency ablation, which involves inserting an electrode into the tumour and applying radiofrequency energy to destroy the tumours. Both methods are 5 effective in destroying small tumours. However in all of the above treatments, 70% of the patients usually relapse within the first 5 years. In addition, the major drawback of these techniques is that they do not work for larger tumours. For bigger tumours, TACE (transarterial chemoembolization ), a method where an angiogenic catherer is inserted into the branches of the hepatic artery and a chemotherapeutic agent such as deoxyrubin is injected, combined with a occluding reagent such as polyvinyl beads where they lodge the vessels, can cause tumour necrosis. For more advanced stage patients, Sorafenib, a tyrosine kinase inhibitor, has led to some positive results but the benefit of these treatments is only an additional 3 months disease-free survival after which the patients relapse again. Commonly administered chemotherapies, such as Tamoxifen, Octreotide and interferons showed no impact on patient survival [14]. In conclusion, while resection and transplantation remains the most successful treatments for HCC, there is no first line systemic therapy that has emerged for advanced HCC. 6 HCC Very early stage, single tumors [...]... 4, 5 and 6 are expressed on the cell surface and TLR3, 7, 8 and 9 are expressed on the endosome-lysosome membrane Figure 6 shows the respective ligands for TLR TLR 1, 2 and 6 recognise bacterial lipopeptides, TLR4 and 5 are the receptors for gram negative bacterial cell wall components, lipopolysaccharides (LPS), and flagellin respectively Intracellular TLR3, 7/8, and TLR9 detect viral derived and synthetic... activate NK-κB and activate IRF3/7 Furthermore apoptosis is induced not only by TLR3 but also RIG1 [57] 1.5.2 Localization holds the key to TLR3 activity The answer to this question might lie in the intracellular localization and cellular expression pattern of TLR3 as compared with that of RIG1 TLR3 localises to an intracellular compartment in dendritic cells and cannot be detected on the cell surface... fibrosis Firstly, poly I:C induces NK cell activation and activated NK cells then kill early activated or senescence activated HSC that have increased expression of NK cell activating ligands [84,85] Second, poly I:C induces NK cells to produce IFN-y and subsequently induces HSC apoptosis and cell cycle arrest [86] Recent data have also shown that poly I:C treatement on HSC can lead the cells to produce type... cross-presentation by dendritic cells and the induction of enhanced primary and memory CD8+ T cell responses [61,69] However, TLR3 have also been found to be expressed on nonimmune cells, such as keratinocytes or endothelial cells [70] More importantly, reports of TLR3 on tumour cells have increased in recent years TLR3 expression is found in human melanoma cancer cells and the triggering of this receptor... synthetic TLR3 agonist) and results show that only patients with TLR3- positive tumours 22 benefited from the treatment and showed increased overall survival [73] Similarly TLR3 was found to be over-expressed in 70% of primary and metastatic clear cell renal cell carcinoma (CCRCC) patients and a growth inhibitory effect is seen when TLR3 signaling is triggered [74] 23 1.7 The relevance of TLR3 in HCC...1 INTRODUCTION 1.1 Hepatocellular Carcinoma: Incidence and Etiological factors Hepatocellular Carcinoma (HCC) is a primary malignancy of the liver epithelial cell and accounts for 90% of primary liver cancer 5% of which are cholangiocarcinoma and the rest are of indeterminate origin Angiosarcoma is extremely rare [1] HCC is the third leading cause of cancer-related death worldwide [2] Due to... signaling molecules such as IRF3 and caspase 8 and finally apoptosis of tumour cells [88] 25 1.8 The Role of TLR3 in Hepatic Cell Death The liver is especially susceptible to injury and cell death due to its central role in metabolism including drugs, alcohol, lipid and fatty acid metabolism, enterohepatic circulation of bile acids and prevalence of hepatotropic viruses Apoptosis and necrosis are the most... cytoplasmic linker of TLR3 contains a sequence motif that is important for intracellular targeting of TLR3, since deleting this linker region interferes with normal intracellular targeting and causes cell surface expression of TLR3 This intracellular targeting of TLR3 leads the receptor to the same cytoplasmic membranes where TLR7 is localized and adjacent to phagosomes containing apoptotic cell particles... tumours cells Recent studies have shown that TLR3 plays an important role in the pathophysiology of a variety of liver diseases [20,47,75] This may be attributed to the wide expression of TLR3 on all types of liver cells, including hepatocytes [76,77], stellate cells [78], sinusoids endothelial cells [79], kupffer cells, billary epithelial cells [80], as well as immune cells such as NK cell, NKT cells... result in anti-tumour NK activation [62] TLR3 is also reported to be expressed on macrophages, mast cell and NK cells NK cells are 20 major players in anti-viral immunity As NK cells express TLR3, they can also be activated directly in response to poly I:C [63-65] 21 1.6 TLR3 expression on tumour cells In addition to mediating an antiviral host immune response, TLR3 has been implicated as a crucial danger .. .TLR3 INDUCES HEPATOCELLULAR CARCINOMA CELL DEATH AND INCREASES NATURAL KILLER CELL ACTIVITY TOW TINGTING CHARLENE (B.Sc (Hons),NUS) A THESIS... 1.6 TLR3 expression on tumour cells 22 1.7 The relevance of TLR3 in HCC tumours cells 24 1.8 The Role of TLR3 in Hepatic Cell Death 26 1.9 Natural Killer cells and their relevance to HCC 28 1.10... express functional TLR3 and triggering TLR3 augments its expression 49 3.5 DsRNA triggers cell death in HCC cells that express TLR3 52 3.6 DsRNA induced tumour cell death is mediated by TLR3 56 3.7