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系統識別號 U0026-1701201418483600
論文名稱(中文) 細胞自噬經由選擇性降解cyclin D1蛋白調控癌症發展之進程
論文名稱(英文) Autophagy regulates tumorigenesis through selective degradation of cyclin D1
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 102
學期 1
出版年 103
研究生(中文) 吳珊瑩
研究生(英文) Shan-Ying Wu
學號 S58961222
學位類別 博士
語文別 英文
論文頁數 120頁
口試委員 指導教授-劉校生
召集委員-沈孟儒
口試委員-呂增宏
口試委員-林秋烽
口試委員-陳鴻震
口試委員-洪文俊
中文關鍵字 Ras致癌基因  細胞自噬  BNIP3  細胞週期蛋白 Cyclin D1  人類肝癌腫瘤 
英文關鍵字 H-rasval12  Autophagy  BNIP3  Cyclin D1  HCC 
學科別分類
中文摘要 H-rasval12致癌基因經由Raf-1/ERK訊息傳遞路徑及其下游訊息分子誘發細胞自噬現象已經被報導。我們證明H-rasval12經由Raf/ERK路徑下游分子Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) 啟動子活性並伴隨著蛋白表現量增加,進而誘發細胞自噬反應。H-rasval12 所誘發的BNIP3會與Beclin 1競爭結合Bcl-2,因此導致Beclin 1被釋放並誘發細胞自噬現象的產生。在小鼠腫瘤實驗中,增加BNIP3的表現量亦能增強細胞自噬反應,並能抑制細胞增生,進而抑制H-rasval12所誘發的小鼠腫瘤之生成。在膀胱癌病人之腫瘤檢體,亦發現Ras、BNIP3和LC3-II的蛋白表現量較高,我們進一步證實BNIP3所誘發的細胞自噬與Ras相關的腫瘤生成之間維持一種平衡關係。 這些證據顯示Ras、BNIP3和細胞自噬現象在腫瘤形成過程中同時扮演重要的角色。進一步研究發現,在誘發細胞自噬現象的情況下,細胞週期停滯在G1期,伴隨著cyclin D1、 LC3 和p62三分子之結合能力增強。在人類肝癌病人檢體中,cyclin D1表現量下降或細胞自噬功能受損等情形已有研究分別報導,顯示此二者可能相關,因此我們推論細胞自噬現象可能參與細胞週期調控進而影響腫瘤生成。我們首先發現於152成對 (腫瘤及鄰近腫瘤之組織) 肝癌病人檢體中,細胞自噬程度與cyclin D1表現量呈負相關,而且高量 cyclin D1、高量p62及低量Atg5表達與肝癌病人之整體低存活率呈顯著之相關性。進一步利用自噬反應誘發劑amiodarone (亦為心律不整用藥) 誘發肝癌細胞Hep 3B 產生細胞自噬過程中,揭示純化之自噬小體 (autophagosome) 中於電子顯微鏡下的確有較多之cyclin D1分子存在。我們也證實細胞自噬反應可使細胞週期停滯於G1期並抑制肝癌細胞之增生。我們也發現誘發細胞自噬後cyclin D1與LC3蛋白之間的結合增加,伴隨著泛素 (ubiquitin) 標定的cyclin D1 量也上升。我們也證實cyclin D1選擇性的與p62結合於細胞自噬小體中並被降解。動物實驗結果發現,無論是在癌細胞異體移植的小鼠實驗或是自體原位移植的大鼠實驗中均顯示,amiodarone誘發的細胞自噬反應可以藉由降解cyclin D1表現而抑制細胞之增生並抑制肝癌的生成。總言之,本研究創新發現自噬反應經由p62媒介將泛素標定之cyclin D1降解抑制肝癌之形成,此發現可進一步應用臨床用藥amiodarone以cyclin D1為標的做為肝癌治療之新策略。
英文摘要 H-rasval12 induces autophagy through multiple signaling pathways including Raf-1/ERK pathway, and various ERK downstream molecules of autophagy have been reported. We clarify that Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is a downstream molecule of the Ras/Raf/ERK signaling pathway to induce autophagy in H-rasval12-related tumorigenesis. BNIP3 is up-regulated by H-rasval12 at the transcriptional level and then competes with Beclin 1 for binding with Bcl-2. Expression of ectopic BNIP3 further enhanced autophagy and suppressed H-rasval12 induced tumorigenesis in mice through the inhibition of cell proliferation. Consistently, high expression of H-rasval12, BNIP3 and LC3-II was also detected in the human bladder cancer specimens indicating that Ras, BNIP3 and autophagy are involved in tumorigenesis. We demonstrate a homeostasis between BNIP3 mediated autophagy and H-rasval12-induced tumor formation. Further analysis revealed that the cell cycle was arrested at G1 phase, and the interaction among cyclin D1, LC3 and p62 (an adaptor protein) was increased when autophagy was induced. It indicates that autophagy may participate in cell cycle regulation. In hepatocelluar carcinoma (HCC), dysregulated expression of cyclin D1 and aberrant autophagy has been reported separately. However, the relationship between them has not been explored. In this study, we demonstrated that autophagy was inversely correlated with cyclin D1 expression in 152 paired HCC patient specimens. HCC specimens with highly expression of cyclin D1 shows correlation with poor overall survival rate. Furthermore, induction of autophagy by amiodarone (antiarrhythmic drug) in Hep 3B cells, cyclin D1 was recruited into autophagosomes demonstrated by immune-gold labeling of cyclin D1 after extraction of autophagosomes. We further demonstrated that autophagy suppresses hepatoma Hep 3B cell proliferation, and further analysis revealed that cell cycle was arrested at G1 phase. The interaction between LC3 and cyclin D1 was increased after autophagy induction. In addition, ubiquitinated-cyclin D1 was also increased after autophagy induction, and then is selectively degraded by autophagosome through binding with SQSTM1/p62 (an adaptor protein). Our in vivo study showed that amiodarone induced autophagy suppresses liver tumor formation in xenograft mouse and orthotopic rat model through decreasing cyclin D1 expression and inhibition of cell proliferation. Altogether, we reveal a novel mechanism, by which ubiquitinated cyclin D1 was degraded by p62 mediated autophagic pathway and amiodarone is a promising drug for targeting cyclin D1 in liver cancer therapy.
論文目次 Abstract I
Chinese abstract .III
Acknowledgments .V
Contents VII
Abbreviations XIII
Chapter 1 Introduction 1
1.1 Autophagy 1
1.2 The molecular mechanism of autophagy 1
1.3 Autophagy signaling pathway 2
1.4 Ras and autophagy 4
1.5 BNIP3 and autophagy 6
1.6 Autophagy and tumor formation 6
1.7 Hepatocellular carcinoma and autophagy 8
1.8 Anti-cancer drug and autophagy 8
Chapter 2 Materials and Methods 10
2.1 Cell lines and reagents 10
2.2 Western blot analysis 10
2.3 Promoter activity assay 11
2.4 Cell transfection and RNA interference 11
2.5 Immunofluorescent staining 12
2.6 Immunoprecipitation 12
2.7 MTT assay 13
2.8 Flow cytometry analysis 13
2.9 BrdU incorporation assay 13
2.10 Immunohistochemical staining 14
2.11 Detection of message RNA expression by real-time PCR 14
2.12 Autophagosome extraction 14
2.13 Transmission electron microscopy 15
2.14 Immune-gold labeled protein and electron microscopy 16
2.15 Mice and tumors 16
2.16 Orthotopic rat model of liver tumor formation 17
2.17 Clinical specimens 17
2.18 Statistical analysis 17
Chapter 3 Results 19
3.1 H-rasval12 induces a dynamic, multi-step autophagic process 19
3.2 BNIP3 and Raf-1/ERK signaling pathway are required for H-rasval12 induced autophagy 20
3.3 H-rasval12-induced BNIP3 triggers the release of Beclin 1 from Beclin 1-Bcl-2 complex 21
3.4 Mutant Rasval12 together with BNIP3 and LC3-II overexpression are detected in the tumor parts of clinical bladder cancer specimens 22
3.5 H-rasval12-induced autophagy suppresses cell proliferation and causes cell cycle arres 23
3.6 H-rasval12-related tumorigenesis is suppressed when autophagy activity is ectopically induced 25
3.7 H-rasval12 regulates cell cycle related with autophagy 27
3.8 Ubiquitination of Cyclin D1 binds with LC3 through p62 and is degraded by autophagic pathway 28
3.9 Autophagy medicated cyclin D1 degradation in human cancer cell lines 29
3.10 Amiodarone is a potential drug for HCC therapy by degrading Cyclin D1 expression through autophagy 30
3.11 Cyclin D1 overexpression is correlated with low autophagic activity and poor overall survival rate in HCC 30
3.12 Autophagy induction decreases cyclin D1 expression 32
3.13 Cyclin D1 is recruited to the autophagosome in Hep 3B cell 33
3.14 Ubiquitinated cyclin D1 binds with LC3 through p62 and is selectively degraded by autophagic pathway to suppress cell proliferation 33
3.15 Amiodarone induced autophagy decreases cyclin D1 expression and suppresses liver tumor formation in xenografic mouse and orthotopic rat model 35
Chapter 4 Discussion 37
Chapter 5 Conclusion 48
References 49
Tables and Figures 64
Table 1. Characteristics of HCC patients 64
Figure 1. H-rasval12 induces autophagic flux 65
Figure 2. H-rasval12 induces autophagy through ERK pathway and regulated by BNIP3 67
Figure 3. H-rasval12-induced BNIP3 triggers the release of Beclin 1 from Beclin 1-Bcl-2 complex 70
Figure 4. Mutant Rasval12 together with BNIP3 and LC3-II overexpression are detected in the tumor parts of the bladder cancer specimens 72
Figure 5. H-rasval12 overexpression inhibits cell growth but not induces cell senescence, cell death and mitochondria membrane potential decreasing in NIH3T3 cells 73
Figure 6. Autophagy suppresses H-rasval12-induced cell proliferation 75
Figure 7. Autophagy switches to a promoting role in cell proliferation of long time ras overexpression cells 78
Figure 8. Autophagy inhibits cell proliferation to suppress Ras-induced tumor formation 79
Figure 9. Autophagy suppresses tumor formation through BNIP3 regulation 80
Figure 10. The correlation between H-rasval12 -induced autophagy and the expression of cell cycle related proteins 83
Figure 11. Cyclin D1 mRNA and ubiquitinated cyclin D1 are increased under H-rasval12 overexpression 84
Figure 12. Cyclin D1 is recruited into the autophagosomes when H-rasval12 overexpression 85
Figure 13. Cyclin D1 is interacted with LC3, p62 and lamp 1 under H-rasval12 overexpression 86
Figure 14. P62 is required for cyclin D1 to interact with LC3 90
Figure 15. Autophagy induces cyclin D1 reduction, cell growth inhibition, cell cycle arresting and cyclin D1-LC3 interaction in cancer cell lines 91
Figure 16. Cyclin D1 protein is overexpressed in HCC specimens 95
Figure 17. High cyclin D1 expression correlate with low autophagic activity and overall survival rate in HCC 96
Figure 18. Autophagy induction suppresses cyclin D1 expression is related to autophagic degradation pathway 98
Figure 19. The expression of cell cycle G1 phase related proteins are not regulated by autophagic degradation pathway 99
Figure 20. Cyclin D1 is accumulated in the autophagosome 100
Figure 21. Ubiquitinated-cyclin D1 is required for it recruitment into autophagosome 102
Figure 22. P62 is an adaptor protein for cyclin D1 degradation by autophagy 104
Figure 23. P62 is required for ubiquitinated-cyclin D1 to interact with LC3 105
Figure 24. Cyclin D1 interacts with p62 and LC3 in the autophagosome 107
Figure 25 .The involvement of autophagy in cell number, cell viability and DNA synthesis 108
Figure 26. Liver tumors are suppressed by manipulation level of autophagy expression in xenograft mouse and orthotopic rat model 110
Figure 27. Liver tumors are suppressed by manipulation level of autophagy expression in orthotopic rat model 113
Figure 28. The mTOR signaling pathway is activated by H-rasval12 115
Figure 29. H-rasval12 overexpression-induced autophagy suppresses cell proliferation in human bladder cancer cell line 116
Figure 30. Cyclin D1 expression is increased and autophagic activity is decreased during liver tumorigenesis 117
Figure 31. A hypothetical model of autophagy selectively regulation of cyclin D1 by p62 and reduction of autophagy associated with high cyclin D1 expression in HCC 119

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