臺灣博碩士論文加值系統

自噬作用為細胞藉由溶酶體降解內蛋白及胞器的過程。儘管過去幾年，科學界投入大量的心力在研究關於不同環境下細胞如何調控自噬作用及其背後的相關機制與功能，我們對於自噬作用中分子調控機轉的了解還是不甚完善。

存活素 (survivin) 是細胞抗凋亡蛋白 (IAPs) 家族中的其中一員。過去已證實存活素只會存在於大部分的癌細胞中而不會存在於以分化完全的正常組織中。存活素的表現可藉由不同層面來調控，在後轉譯修飾的層面中，熱休克蛋白 (heat shock protein 90) 可以直接結合存活素蛋白使其穩定；然而當存活素與熱休克蛋白分離，存活素被泛素（ubiquitin）標的現象增加，接著被蛋白質分解酶 (26S proteasome) 分解。一般認為存活素在細胞及分子層面上擁有兩種促腫瘤形成的功能。存活素身為染色體移動複合物 (CPC) 複合物的一員，此複合物會參與在細胞分裂過程促進細胞分裂，進一步促進癌細胞的分裂。此外存活素可直接或間接的抑制癌細胞中的半胱天冬酶-3 (caspase-3)抑制癌細胞凋亡。

本篇研究中，我們發現存活素之新穎功能─負調控細胞中的自噬作用。藉由顯微鏡及西方墨點法的觀察，外送大量存活素於乳癌細胞中， MDC 所染到的螢光亮點數量減少及 p62/SQSTM1 蛋白表現的上升，證實酸性液胞胞器 (AVOs) 形成減少與細胞中自噬通量 (autophagic flux) 的下降。藉由西方墨點法也同時觀察到在乳癌細胞中外送大量的存活素能抑制參與在細胞自噬體形成中的分子Atg5、Atg、Atg12 表現及Atg5-Atg12 複合體的形成。更進一步，我們發現在同樣的條件下，存活素也會抑制 cathepsin L 及經過切割的Atg5。根據近期研究結果指出經過切割的Atg5會去調控半胱天冬酶-3的活性。這樣的結果顯示存活素可能會透過抑制自噬體形成的分子進一步抑制細胞自噬，並藉由降低切割過的Atg5表現而間接抑制半胱天冬酶-3活性。

有趣的是，我們發現存活素同時也做為細胞自噬的目標蛋白。無論我們利用藥理性質的抑制劑，或使用siRNA 抑制LC3、Atg7來抑制細胞中的自噬作用，皆發現存活素的表現量有上升的趨勢。重要的是，我們進一步利用免疫沉澱法觀察到存活素與 p62/SQSTM1 蛋白有直接相互作用的現象在MDA-MB-231乳癌細胞中。此外，在乳癌細胞中藉由氯奎寧 (Chloroquine) 及 LC3 siRNA抑制自噬作用後也使得存活素蛋白較為穩定。相反地，使乳癌細胞暴露於饑餓環境、處理他莫昔芬 (tamoxifen) 及雷帕黴素 (rapamycin)，這些促進細胞自噬作用也能反過來觀察到存活素下降的情形。更值得一提的是，我們發現一已知會抑制存活素轉錄作用的小分子抑制劑 YM155，也會透過細胞自噬作用來調控存活素的表現。

總結上述結果都指向，在癌細胞中存活素和自噬作用可能有著互相調控的關係。本篇研究結果可提供關於存活素功能及調控其表現的新觀點。並進一步提供現今已試驗到臨床的用藥 YM155更詳細的分子機轉。

Autophagy is a lysosomal degradation pathway for the breakdown of intracellular proteins and organelles. Despite various efforts have been made in the past few years in dissecting the differential functions and the related underlying regulations of autophagy in different cellular situations; our understanding on the molecular regulatory mechanisms of autophagy is still far from complete.

Survivin is a member of the inhibitors-of-apoptosis proteins family. It is widely demonstrated that survivin is mainly expressed in tumors but not in the differentiated normal tissues. The expression of survivin can be transcriptionally and post-translationally regulated in cells. At the post-translational level, survivin protein can be stabilized by Heat shock protein 90 (Hsp90) through physical interactions. Dissociation of Hsp90 promotes survivin ubiquitination and subsequent protein degradation by the 26S proteasome. It is widely believed that survivin exhibits two important pro-tumorigenic functions. First, it promotes mitosis by forming the chromosomal passenger complex (CPC) and second, it inhibits apoptosis through direct and indirect caspases inhibition.

In this study, we found that survivin is a novel negative regulator of autophagy. Fluorescence microscopic analysis and Western blot analysis revealed that overexpression of survivin decreased acidic vesicular organelles (AVOs) formation and inhibited autophagic flux, as indicated by the decreased amount of MDC labeled fluorescence puncta present and the increased accumulation of p62/SQSTM1 in breast cancer cells, respectively. Western blot analysis also revealed that overexpression of survivin negatively regulated both the expression of Atg7, Atg5 monomer, Atg12 monomer, and the formation of Atg5-Atg12 conjugate, which are important molecules and protein complex for the formation of autophagosome in the cells. Interestingly, the same treatment also decreased the expression and activity of cathepsin L and the truncated Atg5, which is a molecule recently discovered to play a role in the positive regulation of caspase-3 activation. These results indicate that survivin may negatively regulate autophagy through down-regulation of the autophagosome formation and indirectly inhibit caspase-3 activation through reduction of the amount of truncated Atg5 present in breast cancer cells.

Interestingly, we also found that survivin is an autophagic substrate protein. Inhibiting autophagy by either pharmacological inhibitors or siRNA against LC3 and Atg7 increased survivin expression in breast cancer cells. Importantly, Western blot analysis and immunoprecipitation assay showed that survivin bound to p62/SQSTM1 in MDA-MB-231 cells. In addition, inhibition of autophagy by either chloroquine or LC3 siRNA increased the protein stability of survivin in breast cancer cells. In contrast, inducing autophagy by serum starvation, tamoxifen and rapamycin decreased survivin expression in breast cancer cells. Furthermore, mechanistic study revealed YM155, a survivin inhibitor that was originally developed to inhibit survivin gene transcription, also down-regulated survivin expression through autophagic protein degradation.

Taken together, findings of this study provide new mechanistic insights into both molecular functions and regulations of survivin and the molecular mechanism of actions of YM155, a drug currently undergoes clinical trials, in cancer cells.

中文摘要 I
ABSTRACT IV
誌謝 VIII
Abbreviation IX
List of Tables XI
List of Figures XII
List of Appendices XIV
INTRODUCTION 1
1.1. Inhibitor of apoptosis (IAPs) family 2
1.2. Survivin 3
1.2.1. Survivin and cancer 3
1.2.2. Structure of survivin 4
1.2.3. Regulation of survivin expression 4
1.2.4. Well known functions of survivin 5
1.3. Autophagy 7
1.3.1. Introduction 7
1.3.2. Initiation of autophagy and formation of autophagosome 7
1.3.3. Vesicle docking/fusion 11
1.3.4. Degradation of targeted proteins 12
1.3.5. The autophagy independent function of Atg5 13
1.3.6. LIR domain and selective autophagy 13
1.3.7. Autophagy and cancer 14
1.4. Aims of this study 15
MATERIALS AND METHODS 17
2.1. Materials 18
2.2. Recipes 21
2.3. Methods 23
2.3.1. Cells and culture 23
2.3.2. Western blot analysis 23
2.3.3. Gene silencing by siRNA 24
2.3.4. Plasmid 24
2.3.5. Electroporation 25
2.3.6. Monodansylcadaverine (MDC) staining 25
2.3.7. RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) assays 26
2.3.8. Immunofluorescence 26
2.3.9. Cathepsin L activity detection assay 27
2.3.10. Immunoprecipitation(IP) 27
2.3.11. Protein stability assay 28
2.3.12. Statistical analysis 28
RESULTS 29
3.1. Overexpression of survivin inhibits autophagy in breast cancer cells. 30
3.2. Survivin regulates the expression of Atg5, Atg7 and Atg12 through transcription-independent mechanisms in breast cancer cells. 31
3.3. Survivin negatively modulates the expression and activity of cathepsin L in breast cancer cells. 33
3.4. Autophagy negatively modulates survivin expression via transcription- and Hsp90-proteasomal protein degradation-independent mechanisms in breast cancer cells. 34
3.5. Survivin physically interacts with the autophagy cargo protein, p62/SQSTM1, in breast cancer cells. 36
3.6. Pharmacological and molecular inhibition of autophagy increases survivin protein stability in breast cancer cells. 37
3.7. Serum starvation, rapamycin, and tamoxifen induced autophagy down-regulates survivin expression in breast cancer cells. 38
3.8. YM155 suppresses survivin expression through autophagy in breast cancer cells. 39
DISCUSSION ＆ CONCLUSIONS 41
4.1. Discussion 42
4.2. Conclusions 46
REFERENCES 47
TABLES 67
FIGURES 70
APPENDICES 95

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