肝癌是世界上常見的惡性腫瘤之一，手術切除、肝臟移植及化療是早期肝癌患者的典型治療策略。不幸的是約有70%的病人會在治療後五年復發。蕾莎瓦為一種多重激酶抑制劑，已被核可用於臨床治療中晚期的肝癌病人，提高病人整體的存活率。然而，一旦病人對蕾莎瓦產生抗藥性則會導致預後效果不佳。因此，探討病人對蕾莎瓦產生抗藥性的分子機制，不僅可提供未來蕾莎瓦用藥的依據還可提升蕾莎瓦治療效用，並促進個人化醫學的發展。此篇研究中，我們發現兩種調控蕾莎瓦抗藥性的機制：(1) 已知Angiopoietin-like protein 1 (ANGPTL1)可以抑制血管新生及癌症轉移，且被認為是腫瘤抑制因子。我們在此篇研究中觀察到ANGPTL1的表現量與病人對蕾莎瓦治療的成效呈現正相關。此外，我們證實ANGPTL1會與MET受體結合並抑制其活化，進而阻斷下游AKT/ERK/Egr-1的訊息傳遞，抑制Slug表現。ANGPTL1藉由阻斷MET/AKT/ERK/Egr-1/Slug的訊息傳遞進而抑制肝癌上皮細胞間質化(EMT)，並降低肝癌細胞對蕾莎瓦產生之抗藥性及癌幹細胞特性。(2) Dicer在微核糖核酸(MicroRNA)的生合成過程中扮演著重要的角色，而在多種癌症中也發現Dicer的異常表現會導致病人預後狀況不佳及增加對藥物的抗藥性。此篇研究中，我們發現肝癌病人的Dicer表現量與Enhancer of zeste homolog 2 (EZH2)及長鏈非編碼RNA (lncRNA) HOXB-AS3的表現量呈現負相關。同時，在肝癌細胞中大量表現Dicer也會降低細胞對蕾莎瓦的抗藥性。而在機制層面上，我們證實HOXB-AS3會與EZH2結合，並誘導EZH2結合至Dicer的基因啟動子上，使其組蛋白H3之27號位的離胺酸(H3K27me3) 產生甲基化，從而抑制Dicer基因轉錄且導致細胞對蕾莎瓦產生抗藥性。此外，以上所述之機制皆透過體外和體內試驗及臨床的數據分析驗證。總而言之，我們的研究成果闡述蕾莎瓦產生抗藥性的機制，並提供研究人員進一步研究肝癌腫瘤惡化的依據，進而為肝癌患者開發更好的治療策略。

Liver cancer is one of the most common malignant tumors worldwide. Surgical resection, liver transplantation and chemotherapy are typical treatments for early stage of liver cancer. Unfortunately, the successful approach is limited because approximately 70% of these patients develop recurrent tumors within five years. Sorafenib is a multi-kinase inhibitor which is clinically approved for treatment with advanced liver cancer and shows an overall survival benefit. However, acquired resistance to sorafenib is responsible for a poor prognosis. Thus, uncovering the molecular mechanisms underlying sorafenib resistance can potential target for sorafenib treatment and improve sorafenib efficacy, as so called “precision medicine”. Herein, we discover the two distinct mechanisms which affect the activity of sorafenib: (1) Angiopoietin-like protein 1 (ANGPTL1) is considered as a tumor suppressor that blocks angiogenesis and cancer metastasis. In our study, we showed the expression of ANGPTL1 positively correlates with sensitivity to sorafenib of liver cancer cells and liver tumor tissues. Furthermore, we found that ANGPTL1 binds to and blocks the activation of MET receptor, which results in Slug suppression via inhibition of the extracellular receptor kinase/protein kinase B (ERK/AKT)-dependent early growth response protein 1 (Egr-1) pathway. We also found that ANGPTL1 inhibits sorafenib resistance and cancer stemness in liver cancer cells by repressing EMT through inhibition of the MET receptor/AKT/ERK2/Egr-1/Slug signaling cascade. (2) Dicer plays a key role of microRNA (miRNA) maturation and deregulation of Dicer is connected with a poor prognosis and drug resistance in various cancer types. Here, we found the expression of Dicer is inversely correlated with the expression of Enhancer of zeste homolog 2 (EZH2) and long non-coding RNA (lncRNA) HOXB-AS3 in liver cancer patients. Moreover, ectopic expression of Dicer declined the sorafenib resistance in liver cancer cells. Mechanistically, we found HOXB-AS3 acts as a recruiter that physically interacts with EZH2 and promotes EZH2 binding to Dicer promoter, which is followed by trimethylation on Lys 27 of histone H3, thereby transcriptional repressing of Dicer and driving sorafenib resistance. The above proposed mechanisms are strongly supported by in vitro and in vivo experiments and clinical analyses. In conclusion, our findings could provide important insights in sorafenib resistance and offer useful information for researchers on studying tumor progression of liver cancer, as well as development of better cancer therapies for liver cancer patients.

摘要 I
Abstract III
致謝 VI
目錄 VII
Chapter 1: Introduction 1
1.1 Liver cancer 1
1.2 Sorafenib resistance 2
1.3 Epithelial-mesenchymal transition (EMT) and Cancer stemness 3
1.4 Angiopoietin-like protein 1 (ANGPTL1) 4
1.5 Dicer 6
1.6 Enhancer of zeste homolog 2 (EZH2) 7
1.7 Aims of this study 9
Chapter 2: Materials and Methods 10
2.1 Cell lines 10
2.2 Western blotting 11
2.3 RNA isolation, reverse transcription PCR and qRT-PCR. 11
2.4 Flow cytometry analysis. 12
2.5 Sphere Formation Assay. 12
2.6 MTT assay. 13
2.7 In vitro binding assay. 13
2.8 Immunoprecipitation assay. 14
2.9 Animal Studies. 15
2.10 Specimens. 15
2.11 Luciferase reporter assay. 16
2.12 Chromatin immunoprecipitation assay. 17
2.13 RNA-immunoprecipitation assay. 17
2.14 Statistical analysis. 17
Chapter 3: Results and Discussions 19
3.1 Angiopoietin-like protein 1 antagonizes MET receptor activity to repress sorafenib resistance and cancer stemness in liver cancer. 19
3.1.1 The expression of ANGPTL1 enhances the sorafenib sensitivity of liver cancer in vivo and in vitro. 19
3.1.2 Suppression of Slug is involved in ANGPTL1-mediated sorafenib sensitization. 20
3.1.3 Slug rescues ANGPTL1-mediated repression of cancer stemness in liver cancer cells. 22
3.1.4 Inhibition of MET receptor/AKT/ERK signaling pathway is involved in ANGPTL1-mediate Slug suppression and sorafenib sensitization. 24
3.1.5 ANGPTL1 represses Slug-induced sorafenib resistance in an Egr-1 dependent manner. 26
3.1.6 ANGPTL1 blocks the interaction between HGF and the MET receptor by direct binding with MET. 27
3.1.7 Discussion 27
3.2 Transcriptional suppression of Dicer by HOXB- AS3/EZH2 complex dictates sorafenib resistance and cancer stemness. 31
3.2.1 Dicer enhances sorafenib sensitivity and suppresses the cancer stemness of liver cancer. 31
3.2.2 The expression of Dicer is transcriptional repressed by EZH2. 33
3.2.3 EZH2 enhances the sorafenib resistance and increases cancer stemness by suppressing the transcription of Dicer in live cancer. 35
3.2.4 LncRNA HOXB-AS3 acts as a recruiter that promotes EZH2 binds to Dicer promoter. 37
3.2.5 HOXB-AS3 suppresses the transcription of Dicer results in sorafenib resistance and cancer stemness. 38
3.2.6 Discussion. 39
Chapter 4: Conclusion and Future Perspectives 44
References 45
List of Figures and Tables 61
Figure 1. Decreased expression of ANGPTL1 reduces sorafenib sensitization in vitro. 61
Figure 2. Decreased expression of ANGPTL1 reduces sorafenib sensitization in vivo. 63
Figure 3. The expression of EMT markers in Huh7 and Huh7/SR cells. 65
Figure 4. The expression of Slug is suppressed by ANGPTL1. 66
Figure 5. ANGPTL1 suppresses the expression of Slug and involves in sorafenib sensitization in vitro. 67
Figure 6. ANGPTL1 suppresses the expression of Slug and involves in sorafenib sensitization in clinical data. 68
Figure 7. ANGPTL1 suppresses the Slug expression that represses the sphere formation ability. 69
Figure 8. ANGPTL1 decreases cancer stemness by suppressing the expression of Slug. 70
Figure 9. ANGPTL1 suppresses the phosphorylation of MET receptor. 71
Figure 10. ANGPTL1 promotes sorafenib sensitization by inhibiting MET activation. 73
Figure 11. ANGPTL1 promotes sorafenib sensitization by inhibiting MET/AKT/ERK activation. 74
Figure 12. ANGPTL1 suppresses Slug-mediated sorafenib resistance by inactivation of Egr-1 signaling. 75
Figure 13. ANGPTL1 suppresses Slug-mediated sorafenib resistance by inactivation of MET/AKT/ERK-Egr-1 signaling. 77
Figure 14. ANGPTL1 binds to MET and inactivates MET by competing with HGF. 78
Figure 15. Schematic diagram depicts the effect of ANGPTL1 on MET phosphorylation and the downstream signaling pathway. 80
Figure 16. The expression of Dicer is decreased in sorafenib resistance liver cancer cells. 81
Figure 17. Dicer expression is crucial for sorafenib sensitization of liver cancer in vitro. 83
Figure 18. Dicer expression is involved in cancer stemness of liver cancer in vitro. 85
Figure 19. Dicer expression is involved in sorafenib resistance and cancer stemness of liver cancer in vivo. 86
Figure 20. The mRNA and protein stability of Dicer in Huh7 and Huh7/SR cells. 88
Figure 21. EZH2 binds to Dicer promoter and catalyzes H3K27me3. 90
Figure 22. Knock-down of EZH2 results in increasing Dicer and sorafenib sensitization. 92
Figure 23. Overexpression of EZH2 results in reducing Dicer and sorafenib resistance. 94
Figure 24. EZH2-mediated Dicer suppression is crucial for sorafenib resistance of liver cancer in vitro. 96
Figure 25. EZH2-mediated Dicer suppression is crucial for cancer stemness of liver cancer in vitro. 98
Figure 26. EZH2-mediated Dicer suppression is crucial for sorafenib resistance and cancer stemness of liver cancer in vivo. 100
Figure 27. EZH2 inhibitor promotes the effect of sorafenib. 102
Figure 28. Candidates lncRNA are identified by lncRNA microarray. 103
Figure 29. The expressions of lncRNAs are depleted by siRNA. 105
Figure 30. LncRNA HOXB-AS3 negatively regulates the expression of Dicer. 106
Figure 31. HOXB-AS3 is crucial for EZH2 to bind to Dicer promoter and triggers H3K27me3. 108
Figure 32. HOXB-AS3-suppressed Dicer expression is crucial for sorafenib resistance of liver cancer in vitro. 109
Figure 33. HOXB-AS3-suppressed Dicer expression is crucial for cancer stemness of liver cancer in vitro. 110
Figure 34. HOXB-AS3-suppressed Dicer expression is crucial for sorafenib resistance and cancer stemness of liver cancer patients. 112
Figure 35. A schematic model illustrats downregulation of Dicer via the HOXB- AS3/EZH2 complex. 113
Table 1. Clinicopathologic characteristics of patients with high and low expression of ANGPTL1 in liver cancer. 114
Table 2. Clinicopathological features of sorafenib responders and non-responders in liver cancer patients. 115
Appendix 116
Appendix 1. List of antibodies. 116
Appendix 2. Sequences and information of primers 117

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