Sorafenib一多功能激酶抑制劑(multi-kinase inhibitor)，是目前唯一通過美國食品藥物管理署(FDA)認證，為晚期肝細胞癌(advanced hepatocellular carcinoma, HCC)的一線用藥。過往文獻有指出，轉型生長因子β (transforming growth factor-β, TGF-β)誘發的細胞內訊息傳遞有助於HCC腫瘤發展，在腫瘤微環境中不論是自體分泌(autocrine)或是旁分泌(paracrine)的TGF-β，都能促使晚期腫瘤細胞增生以及產生上皮轉間質細胞(epithelial-mesenchymal transition, EMT)現象，增強腫瘤細胞移動和侵入能力使得病情惡化。Sorafenib被認為可以抑制TGF-β誘發EMT，並藉此對抗腫瘤的發展。但目前sorafenib雖然可以延緩HCC病人的病情，但隨後會產生抗藥性並再次復發，而其原因目前還尚未瞭解。因此探究sorafenib抑制TGF-β訊息傳遞的詳細機制，或許對治療HCC能有所幫助。在本篇論文中，我們發現sorafenib可以在肝癌細胞、肝細胞中促進細胞膜lipid-raft/caveolae胞吞，以減少整個細胞膜上的type II TGF-β receptor (TβRII)並隨之降解，並有效地影響細胞對於TGF-β的反應。然而上述sorafenib促進細胞TβRII降解的現象會被caveolae以及lysosome的抑制劑阻止；另一方面，sorafenib僅減少肝臟星狀細胞(hepatic stellate cells, HSCs)細胞膜上 lipid-raft的TβRII，而non-lipid raft的部分則不受影響，因此仍會對TGF-β產生反應。我們的結果顯示出，sorafenib主要藉由促進lipid-raft/caveolae介導TβRII的內吞並隨之降解，並降低肝臟上皮細胞對TGF-β誘發的smad2/3訊息傳遞。藉由促進TβRII聚集在lipid-raft/caveolae，或是尋找TGF-β receptor酵素活性的抑制劑等等抑制細胞對於TGF-β反應，或許對於sorafenib治療HCC有所幫助。

The multi-kinase inhibitor sorafenib is the FDA approved drug for the treatment of advanced hepatocellular carcinoma (HCC) and other solid tumors. Previous studies have showed that Transforming Growth Factor-β (TGF-β) signaling may help tumor progression in HCC. Both autocrine and paracrine TGF-β promote tumor growth, enhance metastasis ability and malignancy by inducing epithelial -mesenchymal transition (EMT). Sorafenib is thought to suppress tumor progression by inhibiting TGF-β induced EMT and tissue fibrosis. However HCC is resistant to sorafenib in patients and causes relapse, of which the detailed mechanism remains unknown. In this study, we found that sorafenib specific decreased cell surface TGF-β type II receptor in HCCs and hepatocytes (Hep-G2, Clone9) by increasing TβRII internalization through lipid-raft/caveolae-mediate endocytosis and then degrade in lysosome. Sorafenib-induced downregulation and degradation of TβRII can be protected by caveolae and lysosome inhibitor. On the other hands, sorafenib just affected TβRII localization in lipid-raft/caveolae but not in non-lipid raft on hepatic stellate cells (HSCs) so that TGF-β still induced smad2/3 signaling pathway in HSCs . Our result showed that sorafenib mainly induced TβRII internalization through lipid-raft/caveolae-mediate endocytosis and caused degradation of TGF-β type II receptor for suppression of TGF-β signaling. By gathering TβRII in lipid-raft or using TGF-β receptor kinase inhibitor may provide a direction of sorafenib treat with HCC and TGF-β related diseases.

論文審定書 i
致謝 ii
摘要 iii
Abstract iv
縮寫表 vi
前言 1
材料與方法 7
一、細胞株 7
二、細胞培養(cell culture) 7
三、質體製備 (Plasmids) 8
四、轉染(transfection) 9
五、螢光素酶冷光活性分析 (Luciferase assay) 9
六、西方墨點法 (Western blotting) 10
七、免疫螢光染色法 (Immunofluorescence) 11
八、蔗糖濃度梯度離心(Sucrose Gradient Centrifugation) 12
九、統計方法 13
結果 14
Sorafenib抑制TGF-β誘發上皮細胞promoter活性以及PAI-1蛋白的合成 14
Sorafenib抑制TGF-β引發的上皮細胞Smad2/3以及Stat3的磷酸化 15
Sorafenib 會誘發上皮細胞TβRII 的降解 16
Sorafenib促使TβRII經由lipid-raft/caveolae介導內吞而降解。 16
Sorafenib促使TβRII送往lysosome中降解 17
Sorafenib無法影響stellate cells位於non-lipid raft的TβRII 18
討論 38
參考文獻 42
補充圖表 47

1. Escudier B, Eisen T, Stadler WM, Szczylik C, Oudard S, Siebels M, et al. Sorafenib in advanced clear-cell renal-cell carcinoma. New England Journal of Medicine. 2007;356(2):125-34.
2. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc J-F, et al. Sorafenib in advanced hepatocellular carcinoma. New England journal of medicine. 2008;359(4):378-90.
3. Zhu Y-j, Zheng B, Wang H-y, Chen L. New knowledge of the mechanisms of sorafenib resistance in liver cancer. Acta Pharmacologica Sinica. 2017;38(5):614.
4. Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong H, et al. BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer research. 2004;64(19):7099-109.
5. Llovet JM, Bruix J. Molecular targeted therapies in hepatocellular carcinoma. Hepatology. 2008;48(4):1312-27.
6. Chen Y, Zhang X, Bai J, Gai L, Ye X, Zhang L, et al. Sorafenib ameliorates bleomycin-induced pulmonary fibrosis: potential roles in the inhibition of epithelial–mesenchymal transition and fibroblast activation. Cell death & disease. 2013;4(6):e665.
7. Jia L, Ma X, Gui B, Ge H, Wang L, Ou Y, et al. Sorafenib ameliorates renal fibrosis through inhibition of TGF-β-induced epithelial-mesenchymal transition. PLoS One. 2015;10(2):e0117757.
8. Wang Y, Gao J, Zhang D, Zhang J, Ma J, Jiang H. New insights into the antifibrotic effects of sorafenib on hepatic stellate cells and liver fibrosis. Journal of hepatology. 2010;53(1):132-44.
9. Grimminger F, Schermuly RT, Ghofrani HA. Targeting non-malignant disorders with tyrosine kinase inhibitors. Nature reviews Drug discovery. 2010;9(12):956.
10. Rosenbloom J, Mendoza FA, Jimenez SA. Strategies for anti-fibrotic therapies. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease. 2013;1832(7):1088-103.
11. Varga J, Pasche B. Transforming growth factor β as a therapeutic target in systemic sclerosis. Nature Reviews Rheumatology. 2009;5(4):200.
12. Annes JP, Munger JS, Rifkin DB. Making sense of latent TGFβ activation. Journal of cell science. 2003;116(2):217-24.
13. Massagué J, Blain SW, Lo RS. TGFβ signaling in growth control, cancer, and heritable disorders. Cell. 2000;103(2):295-309.
14. Principe DR, Doll JA, Bauer J, Jung B, Munshi HG, Bartholin L, et al. TGF-β: duality of function between tumor prevention and carcinogenesis. JNCI: Journal of the National Cancer Institute. 2014;106(2).
15. Chen YL, Lv J, Ye XL, Sun MY, Xu Q, Liu CH, et al. Sorafenib inhibits transforming growth factor β1‐Mediated Epithelial‐Mesenchymal Transition and apoptosis in mouse hepatocytes. Hepatology. 2011;53(5):1708-18.
16. Tai W-T, Chu P-Y, Shiau C-W, Chen Y-L, Li Y-S, Hung M-H, et al. STAT3 mediates regorafenib-induced apoptosis in hepatocellular carcinoma. Clinical Cancer Research. 2014;20(22):5768-76.
17. Chen Y-G. Endocytic regulation of TGF-β signaling. Cell research. 2009;19(1):58.
18. Zhao B, Chen Y-G. Regulation of TGF-β signal transduction. Scientifica. 2014;2014.
19. Chen C-L, Chen Y-P, Lin M-W, Huang Y-B, Chang F-R, Duh T-H, et al. Euphol from euphorbia tirucalli negatively modulates TGF-β responsiveness via TGF-β receptor segregation inside membrane rafts. PloS one. 2015;10(10):e0140249.
20. Chen C-L, Hou W-H, Liu I-H, Hsiao G, Huang SS, San Huang J. Inhibitors of clathrin-dependent endocytosis enhance TGFβ signaling and responses. Journal of Cell Science. 2009;122(11):1863-71.
21. Huang JJ, Blobe GC. Dichotomous roles of TGF-β in human cancer. Biochemical Society Transactions. 2016;44(5):1441-54.
22. Nacif M, Shaker O. Targeting Transforming Growth Factor-[beta](TGF-[beta]) in Cancer and Non-Neoplastic Diseases. Journal of Cancer Therapy. 2014;5(7):735.
23. Angadi PV, Kale AD. Epithelial-mesenchymal transition-A fundamental mechanism in cancer progression: An overview. Indian Journal of Health Sciences and Biomedical Research (KLEU). 2015;8(2):77.
24. Thuault S, Valcourt U, Petersen M, Manfioletti G, Heldin C-H, Moustakas A. Transforming growth factor-β employs HMGA2 to elicit epithelial–mesenchymal transition. J cell Biol. 2006;174(2):175-83.
25. Kimelman D, Kirschner M. Synergistic induction of mesoderm by FGF and TGF-β and the identification of an mRNA coding for FGF in the early Xenopus embryo. Cell. 1987;51(5):869-77.
26. Potts JD, Runyan RB. Epithelial-mesenchymal cell transformation in the embryonic heart can be mediated, in part, by transforming growth factor β. Developmental biology. 1989;134(2):392-401.
27. Kaartinen V, Voncken JW, Shuler C, Warburton D, Bu D, Heisterkamp N, et al. Abnormal lung development and cleft palate in mice lacking TGF–β3 indicates defects of epithelial–mesenchymal interaction. Nature genetics. 1995;11(4):415.
28. Piek E, Moustakas A, Kurisaki A, Heldin C-H, ten Dijke P. TGF-(beta) type I receptor/ALK-5 and Smad proteins mediate epithelial to mesenchymal transdifferentiation in NMuMG breast epithelial cells. J cell Sci. 1999;112(24):4557-68.
29. Moustakas A, Heldin CH. Signaling networks guiding epithelial–mesenchymal transitions during embryogenesis and cancer progression. Cancer science. 2007;98(10):1512-20.
30. Xu J, Lamouille S, Derynck R. TGF-β-induced epithelial to mesenchymal transition. Cell research. 2009;19(2):156.
31. Liu L, Cao Y, Chen C, Zhang X, McNabola A, Wilkie D, et al. Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer research. 2006;66(24):11851-8.
32. Azzariti A, Mancarella S, Porcelli L, Quatrale AE, Caligiuri A, Lupo L, et al. Hepatic stellate cells induce hepatocellular carcinoma cell resistance to sorafenib through the laminin‐332/α3 integrin axis recovery of focal adhesion kinase ubiquitination. Hepatology. 2016;64(6):2103-17.
33. Rahimi RA, Leof EB. TGF‐β signaling: A tale of two responses. Journal of cellular biochemistry. 2007;102(3):593-608.
34. Derynck R, Akhurst RJ, Balmain A. TGF-β signaling in tumor suppression and cancer progression. Nature genetics. 2001;29(2):117.
35. Chen CL, Kao YC, Yang PH, Sung PJ, Wen ZH, Chen JJ, et al. A Small Dibromotyrosine Derivative Purified From Pseudoceratina Sp. Suppresses TGF‐β Responsiveness by Inhibiting TGF‐β Type I Receptor Serine/Threonine Kinase Activity. Journal of cellular biochemistry. 2016;117(12):2800-14.
36. Huang SS, Liu IH, Chen CL, Chang JM, Johnson FE, Huang JS. 7‐Dehydrocholesterol (7‐DHC), But Not Cholesterol, Causes Suppression of Canonical TGF‐β Signaling and Is Likely Involved in the Development of Atherosclerotic Cardiovascular Disease (ASCVD). Journal of cellular biochemistry. 2017;118(6):1387-400.
37. Wang L-H, Rothberg KG, Anderson R. Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. The Journal of cell biology. 1993;123(5):1107-17.
38. Daniel JA, Chau N, Abdel‐Hamid MK, Hu L, von Kleist L, Whiting A, et al. Phenothiazine‐Derived Antipsychotic Drugs Inhibit Dynamin and Clathrin‐Mediated Endocytosis. Traffic. 2015;16(6):635-54.
39. Di Guglielmo GM, Le Roy C, Goodfellow AF, Wrana JL. Distinct endocytic pathways regulate TGF-β receptor signalling and turnover. Nature cell biology. 2003;5(5):410.
40. Ehrlich M, Shmuely A, Henis YI. A single internalization signal from the di-leucine family is critical for constitutive endocytosis of the type II TGF-(β) receptor. Journal of cell science. 2001;114(9):1777-86.
41. Doré JJ, Yao D, Edens M, Garamszegi N, Sholl EL, Leof EB. Mechanisms of transforming growth factor-β receptor endocytosis and intracellular sorting differ between fibroblasts and epithelial cells. Molecular biology of the cell. 2001;12(3):675-84.
42. Chen C-L, Yang P-H, Kao Y-C, Chen P-Y, Chung C-L, Wang S-W. Pentabromophenol suppresses TGF-β signaling by accelerating degradation of type II TGF-β receptors via caveolae-mediated endocytosis. Scientific reports. 2017;7:43206.
43. Tai W-T, Cheng A-L, Shiau C-W, Huang H-P, Huang J-W, Chen P-J, et al. Signal transducer and activator of transcription 3 is a major kinase-independent target of sorafenib in hepatocellular carcinoma. Journal of hepatology. 2011;55(5):1041-8.
44. Su T-H, Shiau C-W, Jao P, Liu C-H, Liu C-J, Tai W-T, et al. Sorafenib and its derivative SC-1 exhibit antifibrotic effects through signal transducer and activator of transcription 3 inhibition. Proceedings of the National Academy of Sciences. 2015;112(23):7243-8.
45. Le Roy C, Wrana JL. Clathrin-and non-clathrin-mediated endocytic regulation of cell signalling. Nature reviews Molecular cell biology. 2005;6(2):112.
46. Bourguignon LY, Singleton PA, Zhu H, Zhou B. Hyaluronan promotes signaling interaction between CD44 and the transforming growth factor β receptor I in metastatic breast tumor cells. Journal of Biological Chemistry. 2002;277(42):39703-12.
47. Ito T, Williams JD, Fraser DJ, Phillips AO. Hyaluronan regulates transforming growth factor-β1 receptor compartmentalization. Journal of Biological Chemistry. 2004;279(24):25326-32.
48. Ito T, Williams JD, Fraser D, Phillips AO. Hyaluronan attenuates transforming growth factor-β1-mediated signaling in renal proximal tubular epithelial cells. The American journal of pathology. 2004;164(6):1979-88.
49. Fernando J, Malfettone A, Cepeda EB, Vilarrasa‐Blasi R, Bertran E, Raimondi G, et al. A mesenchymal‐like phenotype and expression of CD44 predict lack of apoptotic response to sorafenib in liver tumor cells. International journal of cancer. 2015;136(4).
50. Chen C-L, Liu I-H, Fliesler SJ, Han X, Huang SS, San Huang J. Cholesterol suppresses cellular TGF-β responsiveness: implications in atherogenesis. Journal of cell science. 2007;120(20):3509-21.
51. Chen C-L, Huang SS, San Huang J. Cellular heparan sulfate negatively modulates transforming growth factor-β1 (TGF-β1) responsiveness in epithelial cells. Journal of Biological Chemistry. 2006;281(17):11506-14.
52. Chen CL, Huang SS, Huang JS. Cholesterol modulates cellular TGF‐β responsiveness by altering TGF‐β binding to TGF‐β receptors. Journal of cellular physiology. 2008;215(1):223-33.
53. Atfi A, Dumont E, Colland F, Bonnier D, L'Helgoualc'h A, Prunier C, et al. The disintegrin and metalloproteinase ADAM12 contributes to TGF-β signaling through interaction with the type II receptor. The Journal of cell biology. 2007;178(2):201-8.
54. Fabregat I, Moreno‐Càceres J, Sánchez A, Dooley S, Dewidar B, Giannelli G, et al. TGF‐β signalling and liver disease. The FEBS journal. 2016;283(12):2219-32.
55. Thompson AI, Conroy KP, Henderson NC. Hepatic stellate cells: central modulators of hepatic carcinogenesis. BMC gastroenterology. 2015;15(1):63.
56. Coulouarn C, Factor VM, Thorgeirsson SS. Transforming growth factor‐β gene expression signature in mouse hepatocytes predicts clinical outcome in human cancer. Hepatology. 2008;47(6):2059-67.
57. Lin T-H, Shao Y-Y, Chan S-Y, Huang C-Y, Hsu C-H, Cheng A-L. High serum transforming growth factor-β1 levels predict outcome in hepatocellular carcinoma patients treated with sorafenib. Clinical cancer research. 2015.
58. Cheng A-L, Kang Y-K, Chen Z, Tsao C-J, Qin S, Kim JS, et al. Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial. The lancet oncology. 2009;10(1):25-34.
59. Waidmann O, Trojan J. Novel drugs in clinical development for hepatocellular carcinoma. Expert opinion on investigational drugs. 2015;24(8):1075-82.