Hepatocellular carcinoma (HCC) is the major type of primary liver cancer which is well-known for its high heterogenicity and metastatic potential. Despite of the current advancement in surgical resection and the availability of targeted therapy, HCC remains a barely curable and fatal disease. We previously demonstrated that deregulation of epigenetic regulators is a common event in human HCC. Herein, we identified the frequent up-regulation of the prototype of H3K9 tri-methyltransferase SUV39H1 in clinical HCCs. SUV39H1 over-expression was also significantly associated with increased Ki67 expression and the presence of venous invasion. By using both SUV39H1 over-expression and knockdown model, we consistently demonstrated that SUV39H1 contributed to HCC tumor growth and migration. Most importantly, SUV39H1 knockdown drastically suppressed in vivo tumorigenicity and extra-hepatic metastasis of HCC cells in nude mice model. These findings evidently demonstrated the oncogenic role of SUV39H1 in HCC and implied potential therapeutic targeting of SUV39H1 for HCC treatment. Molecularly, SUV39H1 knockdown HCC cell underwent morphological changes and accompanied with increased lysosomal β-galactosidase activity and elevated p21 protein and γH2AX level. This data suggested senescence induction in SUV39H1 knockdown HCC cells. SUV39H1 has been implicated in telomere regulation and transcriptional control. However, neither telomere length nor expression of tumor suppressor genes was altered in SUV39H1 knockdown HCC cells. Interestingly, we demonstrated a novel observation that SUV39H1 may potentially methylate non-histone substrates that are yet to be identified, which may contribute to the pro-tumorigenic function of SUV39H1 in HCC. We also investigated the upstream regulation of SUV39H1 and identified miR-125b as the negative post-transcriptional regulator of SUV39H1. Ectopic expression of miR-125b abolished SUV39H1 3’UTR-coupled luciferase activity and suppressed endogenous SUV39H1 at both mRNA and protein level. Clinically, miR-125b level was found inversely correlated with SUV39H1 expression. We have previously reported the frequent under-expression of miR-125b in HCC. Collectively, our data suggested that SUV39H1 up-regulation in HCC may be the sequential outcome of miR-125b down-regulation. In conclusion, we demonstrated for the first time that SUV39H1 up-regulation contributed to HCC development and metastasis, potentially via senescence evasion. SUV39H1 elevation in HCC was attributed to the loss of its negative regulator, the tumor suppressive miR-125b. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biochemistry / Master / Master of Philosophy

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

The Best PhD Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize,2009-2010 / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Master / Master of Philosophy

Hepatocellular carcinoma (HCC), which constitutes 75%-80% of primary liver cancer, is one of the most common malignancies worldwide. Hepatocarcinogenesis is a complicated and slow process accumulating multiple genetic and epigenetic alterations. In spite of its prolonged pre-malignant stage, HCC is usually diagnosed late and of high aggressiveness. A better understanding of the genetic and epigenetic changes during HCC development and progression is of great importance to early diagnosis and treatment of HCC. Gain of chromosome 1q21 is one of most frequent genetic alteration in HCC and chromodomain helicase DNA binding protein 1-like (CHD1L) was recently identified to be responsible for this amplification. As a family member of SNF-2 like transcription factors, CHD1L plays an important role in HCC development via regulation of various downstream targets. In this study, a novel oncogene, sparc/osteonectin, cwcv and kazal-like domains proteoglycan 1 (SPOCK1), was identified as a CHD1L target. CHD1L protein directly bound to the promoter region (nt -1662 to +34) of SPOCK1 and activated its transcription. Clinically, overexpression of SPOCK1 was detected in 60% of human HCC samples and was significantly associated with advanced clinical stage (P=0.020), shorter overall survival (P=0.011) and poorer disease-free survival of patients (P=0.039). Functionally, the ectopic expression of SPOCK1 in HCC cells conferred strong tumorigenic ability, while shRNA-mediated SPOCK1 silencing abolished this effect. Further study showed that the SPOCK1-enhanced cell survival could be attributed to its anti-apoptotic effects. SPOCK1 could suppress HCC cell apoptosis through the activation of AKT and subsequent inhibition of the (cytochrome c)-(caspase-9)-(caspase-3) pathway. In addition to its tumorigenic roles, the overexpression of SPOCK1 in HCC cells conferred strong metastatic ability via MMP9-mediated extracellular matrix remodeling. In addition to genetic alterations, epigenetic changes also get increasing attentions due to their profound effects on gene activity and expression. A-to-I RNA editing is a post-transcriptional epigenetic modification which converts a site-selective adenosine nucleotide into inosine. The importance of RNA editing has long been underestimated because most of RNA editing modifications occur in a subtle way. The next-generation sequencing provides enough depth to unravel this mystery. The transcriptome sequencing data obtained from this study identified an A-to-I RNA editing at codon 367 (Ser→Gly) of antizyme inhibitor 1 (AZIN1). A high modification rate of AZIN1 was found to be prevalent in HCC specimens and closely associated with HCC pathogenesis. Adenosine deaminase acting on RNA-1 (ADAR1), but not ADAR2 or ADAR3, was responsible for AZIN1 RNA editing. This recoding editing event conferred “gain-of-function” phenotypes as manifested by augmented tumorigenic capabilities and higher aggressive potentials. Compared with wild-type AZIN1 protein, the edited form possessed stronger affinity to antizyme. As a result, edited AZIN1 demonstrated higher protein stability and ensuing neutralization of the antizyme-mediated degradation of ODC and CCND1 oncoproteins. The rescued ODC and CCND1 robustly accelerated cell proliferation thereby promoting HCC development. In conclusion, two novel molecular mechanisms, (CHD1L)-(SPOCK1)-(AKT) and (ADAR1)-(edited AZIN1)-(ODC/CCND1), were delineated during HCC initiation and progression. Also, a causal link between RNA hyper-editing activity and cancer development was established for the first time in this study. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers worldwide. The development and progression of HCC is a multistep process which attributed to the accumulation of genetic alterations. Recently, mounting evidence has suggested the role of non-coding RNAs (ncRNAs) as the master driver of carcinogenesis, through their regulation on different oncogenes and tumor suppressive genes. Deregulation of ncRNAs was frequently observed in multiple types of cancers, including HCC. Herein, we demonstrated the aberrant expression pattern of miRNAs and lncRNAs in human HCC and investigated their functional roles in promoting hepatocarcinogenesis. Deregulation of miRNAs was previously demonstrated as a common event in human HCC, and miR-142-3p and miR-142-5pwereidentified as one of the significantly down-regulated miRNAs in HCC. Down-regulation of miR-142-3p and miR-142-5p was frequently observed in HCC patients and their expressions were progressively decreased along the multi-steps HCC development and progression. Functionally, overexpression of miR-142 has significantly inhibited HCC cell migration and invasion. Ectopic expression of miR-142 also markedly attenuated stress fiber formation and disrupted the cytoskeleton organization of HCC cells. Mature miR-142-3p and miR-142-5p, which derived from the same miRNA precursor were shown to collaboratively inhibited HCC cell migration through targeting different components of the key pathways regulating cell motility. On the other hand, we demonstrated the aberrant expression of lncRNAs in HCC by profiling of 88 well-annotated lncRNAs in 20 pairs of primary HCC and their corresponding non-tumorous liver. HOXA distal transcript antisense RNA (HOTTIP)was identified as the most frequently up-regulated lncRNA in HCC. Functionally, knock down of HOTTIP significantly attenuated cell proliferation in HCC cells and markedly abrogated tumorigenicity in nude mice. Knockdown of HOTTIP had lead to a global reduction in the HOXA genes expression, which are highly expressed in human HCC. Our data suggested that HOTTIP may regulate the expression of its neighboring protein-coding genes and contribute to the development of HCC. We also investigated the up-stream regulation of HOTTIP and identified miR-125b and miR-29a as regulators of HOTTIP in HCC. Clinically, miR-125b and miR-29a exhibited a reverse expression pattern to HOTTIP in HCC. Ectopic expression of miR-125b and miR-29a abolished HOTTIP-coupled luciferase activity and suppressed the endogenous level of HOTTIP. Intriguingly, we also identified a negative feedback relationship between HOTTIP and miR-125b. Taken together, our findings suggested the up-regulation of HOTTIP may be attributed to the down-regulation of miR-125b and miR-29a in HCC, and the sophisticated regulatory network between HOTTIP and miR-125b has further increased the complexity of gene regulation in HCC. In conclusion, we demonstrated the dysregulated expression pattern of miRNAs and lncRNAs in HCC and well illustrated their functional roles in promoting hepatocarcinogenesis. From the studies of miR-142 and lncRNA HOTTIP we appreciated the complex interactions and regulations between different ncRNAs. Taken together, our study has enriched the current knowledge on ncRNAs and their involvements in HCC development. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

abstract / Clinical Oncology / Doctoral / Doctor of Philosophy