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

The Best PhD Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize,2008-2009 / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Medical Sciences