Invasion and metastasis are the major causes of treatment failure and high mortality rate in hepatocellular carcinoma (HCC) patients. Cell motility is crucial to tumor invasion and metastasis, requiring the ability of tumor cells to interact with extracellular matrix, which is regulated by integrins and integrin-associated molecules at the focal adhesions. Recent studies have demonstrated the role of β1 integrin (CD29) overexpression in HCC and its correlation with cancer cell invasiveness and metastastic potential, as well as its protective role against cancer cells against chemotherapeutic drug-induced apoptosis, yet the mechanism is not fully known. Focal adhesion proteins serve as binding platforms for additional cytoskeletal and signaling molecules in the CD29 signaling pathway. Recently, Actopaxin has been demonstrated to form complex with numerous molecules at the focal adhesions, including ILK, which interacts with the cytoplasmic tail of CD29. Through these interactions, Actopaxin has been shown to regulate different cellular events, including cell survival, spreading and cell migration. In this study, the role of Actopaxin in HCC was investigated. In particular, its role in the regulation of tumor invasion and metastasis of HCC cells was demonstrated. This study showed that Actopaxin expression was overexpressed in HCC specimens when compared with the adjacent non-tumorous liver, and that its overexpression positively correlated with tumor size, stage and metastasis in HCC specimens. Actopaxin expression was also correlated with the metastatic potential in HCC cell-lines. Functional studies established that overexpression of Actopaxin conferred invasive phenotypes in primary, non-metastatic HCC cells, whereas down-regulation of Actopaxin could revert the invasive phenotypes and metastatic potential of metastatic HCC cells in vitro and in vivo. Suppression of Actopaxin expression was associated with reduced expression of ILK, PINCH, Paxillin and cdc42, whereas expressions of E-cadherin, β-catenin and GSK3β were induced, indicative of a less invasive and invasive phenotype. Conversely, overexpression of Actopaxin in primary, non-metastasis HCC cells accordingly up-regulated the expression of ILK, PINCH, Paxillin and cdc42, and down-regulation of of E-cadherin, β-catenin and GSK3β, suggestive of an enhanced invasive phenotype. The expression of Actopaxin was found to be correlated with CD29 level, indicating that Actopaxin is a CD29-associated protein and involved in CD29-regulated signaling. Finally, Actopaxin down-regulation enhanced chemosensitivity of of HCC cells towards chemotherapeutic treatment. Treatment with Oxaliplatin was enhanced in Actopaxin-deficient HCC cells, which showed a stronger inhibitory effect on cell proliferation and cell cycle progression, accompanied with induction on apoptosis. The enhanced chemosensitivity effect was a collective result of suppression of Survivin protein, β-catenin and mTOR pathways; and up-regulation of p53. To conclude, this study demonstrated for the first time that Actopaxin is involved in HCC invasion, metastasis and chemosensitization, providing the basis to further investigate the potential role of this protein or its downstream effectors as a therapeutic target for inhibiting the development of metastasis and enhancing chemotherapy efficacy to combat HCC, and perhaps other invasive cancers. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

 Hepatocellular carcinoma (HCC) is the primary cancer of liver. It is the fifth common malignant tumor in men while seventh common in women. Aetiology of HCC is complex; however, it is now believed that sustained chronic liver injury and fibrosis are critically involved in the development of HCC. Prevention and treatment of HCC is far from desirable and prognosis remains poor. Coptis is a Chinese herbal Medicine which has been used for more than thousands years for clearing heats, dampness and toxics. Recently, studies from our group reported the hepatoprotective effect of Coptis and its major active component, berberine, on acute liver injury and berberine was extensively studied for their anti-tumor effect. However, there’s no comprehensive investigation focusing on the preventive and curative potential of berberine on HCC. Hence, here we hypothesized Coptis and berberine exhibits both preventive and curative effects on HCC. The prevention of HCC by berberine and Coptis may rely on their effects on chronic liver damage and fibrosis, and the curative action may depend on their actions on the angiogenesis, tumor growth and invasion of HCC. Both in vitro cell models and in vivo animal system were used in our study and some molecular events were investigated. We found that berberine and Coptis could significantly attenuate the chronic liver injury and fibrosis by restoring the anti-oxidative enzyme SOD activity in CCl4-, bile duct ligation- and alcohol-induced liver injury and fibrosis model. Recovery of SOD activity prevents the hepatocytes from apoptosis by inhibiting the oxidative stress-induced Erk1/2 signaling activation. The prevention of berberine and Coptis on chronic liver injury and fibrosis may contribute to its preventive effect against HCC. Then we found that berberine (as representative to Coptis) could suppress the angiogenesis of HCC, in which berberine does not directly act on the blood vessel formation, but suppress the expression and secretion of pro-angiogenic factors VEGF in HCC cells, and Id-1 inhibition by berberine plays a central role in the suppression of HIF-1α/VEGF and NF-κB pathways. We also found that berberine could induce both apoptotic and autophagic cell death in HCC, and the mitochondria related-caspases activation confers the apoptosis while mTOR inhibition initiates autophagy in berberine treated- cells. We found that berberine could suppress the migration and invasion of HCC cells as well, and Rho-GTPases/ROCK signaling is the particular target in berberine’s anti-invasive action. Finally, to dig out some molecular events involved in berberine’s action on HCC, we studied critically the mechanism underlying berberine’s inhibition on Cyclin D1 in HCC. We found berberine may promote the IKKα-induced Cyclin D1 phosphorylation at T286, and this may initiate the ubiquitination-dependent proteasomal degradation of Cyclin D1 in berberine-treated HCC cells and contribute to berberine’s anti-HCC action. Critical clinical trials and OMICS techniques were planned to further our comprehensive study on Coptis and berberine’s effects on HCC. In all, we found that berberine targets on different stages and molecules and exerts preventive and curative potential against HCC. Our study sheds light on the clinical application of berberine in HCC treatment. / published_or_final_version / Chinese Medicine / Doctoral / Doctor of Philosophy

Hepatocellular carcinoma (HCC) is the seventh most prevalent cancer and the third leading cause of cancer-related mortality globally. Metastasis is a major cause of mortality. HCC is also highly chemoresistant which limits treatment options to patients. Understanding the molecular mechanisms involved in these two events is of crucial significance. Deregulation of Rho/ROCK signaling is common in HCC and regulates different cellular events including cell invasion and survival. In this study, we aimed to further investigate how members of the Rho/ROCK pathway regulate HCC cell invasion and chemoresistance. By screening 71 pairs of human HCC samples using real-time qPCR, we identified that RhoE was frequently downregulated in human HCC. RhoE serves as an antagonist of the Rho/ROCK pathway. Clinicopathologically, downregulation of RhoE associated with shorter patient disease-free survival. In virto assays showed that stable knockdown of RhoE enhanced both HCC cell migration and invasion. In vivo mouse models also demonstrated that knockdown of RhoE promoted HCC invasiveness and intra-hepatic metastasis. Mechanically, knockdown of RhoE increased ROCK activity By screening 71 pairs of human HCC samples using real-time qPCR, we identified that RhoE was frequently downregulated in human HCC. RhoE serves as an antagonist of the Rho/ROCK pathway. Clinicopathologically, downregulation of RhoE associated with shorter patient disease-free survival. In virto assays showed that stable knockdown of RhoE enhanced both HCC cell migration and invasion. In vivo mouse models also demonstrated that knockdown of RhoE promoted HCC invasiveness and intra-hepatic metastasis. Mechanically, knockdown of RhoE increased ROCK activity and inhibition of ROCK reversed the effect of RhoE knockdown on cell migration. RhoE overexpression induced disassembly of stress fibers while knockdown of RhoE enhanced formation of plasma membrane blebs. These findings suggested that RhoE acts as a metastatic suppressor in HCC via inhibiting Rho/ROCK signaling. Downregulation of RhoE can increase ROCK activity which is reported to regulate cell survival. Therefore we investigated if the frequent downregulation of RhoE contributes to the high chemoresistance in HCC cells. Stable knockdown of RhoE suppressed cell death/apoptosis induced by chemotherapeutic agents such as cisplatin and doxorubicin. This effect could be reversed by addition of ROCK inhibitor. In vivo mouse model also confirmed that RhoE knockdown augmented HCC chemoresistance. We also observed that combined treatment of cisplatin and ROCK inhibitor profoundly inhibited tumor growth in nude mice. This part of our findings indicated that RhoE/ROCK played an important role in regulating chemoresistance in HCC. We further identified two downstream molecular pathways which were involved in Rho/ROCK-induced chemoresistance. We found that STAT3 and JAK2 were activated by RhoE knockdown but inhibited by addition of ROCK inhibitor. Upon ROCK inhibition, expression of IL-6 and IL-6 receptor were suppressed and the transcription activating activity of STAT3 was also repressed. Finally, ROCK inhibition attenuated Erk1/2 activation. Literature searching suggested nuclear PTEN as a potential candidate for inactivating Erk1/2. We demonstrated that inhibition of ROCK increased the population of nuclear PTEN while overexpressing ROCK2 decreased it. Overexpression of nuclear PTEN alone could already reduce Erk activation in HCC cells. Our findings indicated that RhoE/ROCK may exert their effects on chemoresistance in HCC via regulating the IL-6/JAK2/STAT3 and PTEN/Erk pathways. In conclusion, our study demonstrated the important role of RhoE in HCC. First, aberrant underexpression of RhoE promoted HCC invasion and intra-hepatic metastasis through upregulating the Rho/ROCK signaling. Second, downregulation of RhoE increased activity of the pro-survival IL-6/JAK2/STAT3 and Erk signalings to enhance chemoresistance in HCC cells. Our findings also suggested the Rho/ROCK signaling to be potential therapeutic target in anti-metastatic and chemo-sensitizing therapy. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Hepatocellular carcinoma (HCC) is the major type of liver cancer. In Hong Kong, thousands of deaths are related to this disease every year. Hepatitis B virus (HBV) infection is one of the major risk factors of HCC development. The high prevalence of HBV carriers in Southeast Asia including Hong Kong can account for the particularly high HCC cases in these areas. HCC is often asymptomatic. The diagnosis and treatment are often delayed which lead to inapplicable of surgical resection. Meanwhile, conventional treatment regimes such as systemic chemotherapy were found to have limited responses. Hence, the case mortality rate of HCC is the second highest among all the cancers. Granulin-epithelin Precursor (GEP) is a glycoprotein growth factor which regulates multiple cellular functions. Our group has demonstrated that GEP is over-expressed in more than 70% of HCC cases and GEP expression is positively correlated to tumor malignancy. Our group has also verified that suppression of GEP by monoclonal antibody leads to significant inhibition of HCC growth and reduction of malignancy. Therefore, GEP has the potential to be a novel therapeutic target of HCC. MicroRNAs (miRNAs) are short non-coding RNAs that regulate mRNA translation. Previous studies showed that miRNA dys- regulation is closely associated with HCC progression and the high stability of miRNAs allows them to be cancer biomarkers or therapeutic targets. This project aims to investigate the miRNAs that regulate GEP and their functions in HCC. Potential GEP-regulating miRNAs were identified by literature review and in silico prediction by bioinformatics tools. MiR-615-5p, miR-588, miR-29b, miR-195, and miR-659 were identified as the potential candidates. Quantitative polymerase chain reaction (qPCR) was utilized to examine the miRNAs’ expressions in HCC clinical samples. Only miR-29b and miR-195 were detected and hence they were selected for further study. Our results showed that miR-29b and miR-195 expression levels were significantly decreased in HCC comparing to adjacent non‐tumor tissue (P<0.001) in more than 70% of cases. MiR‐195 and miR‐29b were over‐expressed in Hep3B HCC cell lines by miRNA mimics and GEP protein level was significantly suppressed after miR-29b mimic transfection. The transcript level of GEP was found to be unchanged after the miR‐29b over-expression. This suggests miR‐29b does not regulate GEP protein expression by mRNA degradation. The effects of miR‐195 and miR‐29b on HCC proliferation were also examined. The growths of HCC cells were suppressed notably after over-expression of miR‐195 (P<0.005) and miR‐29b (P<0.005) respectively. In conclusion, miR‐195 and miR‐29b are frequently down-regulated in HCC. MiR‐29b can negatively regulate GEP expression and does not interfere with GEP mRNA level. Furthermore, miR‐195 and miR-29b can function to inhibit HCC cell growth significantly. / published_or_final_version / Surgery / Master / Master of Philosophy

abstract / Pathology / Master / Master of Philosophy

published_or_final_version / abstract / Molecular Biology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Surgery / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / toc / Molecular Biology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Surgery / Master / Master of Philosophy