Exploring Mechanisms of Multi-Drug Resistance in Cancer Cells Mediated by CCTβ Overexpression

在許多造成抗藥性的原因中，我們發現一個幫助細胞內蛋白折疊的Chaperonin containing t-complex polypeptide 1 (CCT)中的β次單元在具有抗藥性的癌細胞中有過量表現的情況，且在過度表現CCTβ的細胞中發現MDR1的表現量上升。然而，CCTβ過量表現造成抗藥性的機制卻不完全瞭解。因此在本論文中，首先，進一步探討是否除了MDR1的表現量上升之外，有其他ABC transporters也有表現，結果發現在臨床上被研究較多的MDR1、MRP1和BCRP在具有抗藥性的和CCTβ過量表現的MCF7細胞中都有增加表現的情形。而且，隨著抑制CCTβ在細胞中的表現，也發現到這些ABC transporters也有降低表現的情形。考慮到有些文獻顯示這些ABC transporters的蛋白質表現量和功能(其運輸藥物通過細胞膜的能力)並不一定成正比，因此進一步的去分析ABC transporters的功能在CCTβ表現量變化後的影響。發現到不論在具有抗藥性的細胞或是MCF7過量表現CCTβ的細胞中，這些ABC transporters的運輸藥物的能力都有增加，而且抑制CCTβ表現的細胞中也發現到ABC transporters在功能上也是有下降的。重要的是，一個在核內還未被確認出是哪種轉譯後修飾過的β-catenin被發現為CCTβ和ABC transporters表現量之間的調控分子，隨著CCTβ的表現量上升或下降，此種β-catenin的表現量也會跟著上升或下降。許多文獻也顯示β-catenin的活化確實會使ABC transporters的表現量上升 [1-3]。綜合這些結果顯示：CCTβ可能藉由調節β-catenin進而調控了ABC transporters的蛋白質表現量和功能而導致了多重抗藥性的產生。 除了ABC transporters之外，也有其他機制是會導致細胞具有抗藥性，包括：促進細胞存活和抗凋亡等等。抗凋亡Bcl2蛋白可以經由抑制cytocrome C從粒線體外膜釋出的機制而抑制細胞凋亡的進行，此蛋白在不論是在馴化後具有抗藥性的細胞或者是MCF7過量表現CCTβ的細胞中都可以發現到有表現量升高的情形。再者，在本篇研究中，XIAP和CCTβ之間的直接交互作用在被馴化後的7TR抗藥性細胞中被發現。更進一步發現到在MCF7中過量表現CCTβ的細胞中XIAP也有過量表現的情形，而且隨著抑制CCTβ表現也可以發現XIAP的表現量跟著下降。然而，在被馴化具有抗藥性的7TR細胞還有MCF7中過量表現CCTβ的細胞中，caspase7和活化態的caspase7的表現量卻是上升的。因此，綜合這些結果，CCTβ藉由穩定XIAP來和活化態的Caspase7形成XIAP:p12/p19-caspase7 複體以防止細胞凋亡，因此使得細胞具有抗藥性。 整體而言，這些研究闡述了CCTβ過量表現的抗藥性細胞用於產生抗藥性的可能機制，其中包括了：CCTβ藉由調控β-catenin而影響到ABC transporters的表現和功能增進、anti-apoptotic Bcl2的表現量增加而防止經由粒線體的胞內凋亡機制，還有提高XIAP表現量而使XIAP和活化的Caspase7結合而中止下游細胞凋亡的產生。這些結果增進了我們對多重機制共同造成抗藥性的了解，其中這些參與在形成抗藥性機制而且和CCTβ有交互作用的蛋白，在之後可以做為抗癌藥物設計的目標。Among the mechanisms causing drug resistance, the β subunit of chaperonin containing t-complex 1 (CCT), a molecular chaperonin that facilitates protein folding in eukaryotic cytosol, has been found up-regulated in drug resistant cancer cell lines as well as MDR1. However, the mechanisms underlying CCTβ expression and MDR1 expression are not fully understood. In this thesis, first of all, we examined the other ABC transporters besides MDR1 and found the expression levels of three clinically studied MDR1, MRP1, and BCRP have been increased by overexpressing CCTβ and decreased by knockdown of CCTβ. Considering that the pumping efficiency is not proportional to the expression level for these ABC transporters, we further examined their functional levels. We found the functional levels of three ABC transporters were elevated in drug resistant cell lines as well as in CCTβ-overexpression cell lines but reduced in CCTβ-knockdown cell lines. Importantly, a post-translationally modified form of β-catenin in nucleus was found to be mediated by the expression level of CCTβ and regulate the expressions of ABC transporters. In addition, the expression of ABC transporters has been related to transcription activity of β-catenin [1-3]. These results indicate that CCTβ can regulate protein expression and function of ABC transporters via β-catenin. Furthermore, apart from ABC transporters, increasing survival signaling and anti-apoptotic signaling cause increased survival rates and chemoresistance in cancer cells. First, anti-apoptotic protein Bcl2, which participates in preventing the release of cytochrome C from mitochondria, was shown to be overexpressed in drug resistant 7TR and CCT-β overexpressed MCF-7. Moreover, in this study, the interaction between XIAP and CCTβ was identified in 7TR, and XIAP overexprssion was identified in MCF7 overexpressing CCTβ cell line. On the other hand, knockdown of CCTβ decreased the expression of XIAP. However, caspase7 and cleaved caspase7 were found up-regulated in 7TR drug resistant cells and CCTβ-overexpressed MCF7 cells. These results indicate that CCTβ interacts and stabilizes XIAP from degradation, leading to the formation of XIAP: p12/p19-caspase7 complex to prevent cell death. In summary, these studies explored the possible mechanisms of drug resistance originated from CCTβ overexpression, which include β-catenin-mediated expressional and functional up-regulation of ABC transporters, increase of anti-apoptotic Bcl2 to prevent cytochrome c-mediated apoptosis, and raise of XIAP to form complexes with caspase 7 to prevent apoptosis. These results enhance our understanding in drug resistance caused by multiple causes. The proteins involved in the drug resistance or those interacting with CCTβ could be valuable targets for cancer drug design.TABLE of CONTENTS 中文摘要 4 ABSTRACT 6 ABBREVIATIONS 8 (1) INTRODUCTION 11 1.1 Chaperonin containing t-complex polypeptide 1(CCT) 11 1.1.1 Sequence divergence 11 1.1.2 ATP-driven chaperonin 12 1.1.3 Structure and the content of subunits 12 1.1.4 Substrate binding specificity 13 1.1.5 Tumorigenesis 14 1.2 Multi-drug resistance (MDR) 16 1.2.1 Classification of ABC transporters 17 1.2.2 Structures of ABC transporters 18 1.2.3 Three efflux pumps in cancers 19 1.3 Wnt signaling and β-catenin 22 1.3.1 The canonical Wnt signaling 23 1.3.1.1 Wnt-off: β-catenin degradation 23 1.3.1.2 Wnt-On: β-catenin stabilization 24 1.3.2 Lymphoid-enhancing factor/T-cell factors (LEF/TCFs) 26 1.3.3 β-catenin associated co-repressors and co-activators 27 1.3.4 β-catenin in drug resistance and tumor 28 1.4 The present work 30 (2)MATERIALS AND METHODS 32 2.1 Cell culture and cell lines 32 2.2 Stable cell line generation and infection 33 2.3 Total protein lysate preparation and the isolation of cytoplasmic and nuclear fraction 33 2.4 Western bloting and antibodies 35 2.5 Co-immunoprecipitation (Co-IP) 36 2.6 Drug-exporting efficiency of ABC transporters 37 2.7 Drug sensitivity assay 39 2.8 mRNA expression analysis using RT-PCR 39 (3) RESULTS 41 3.1 Overexpression of CCTβ confers drug resistance in cancer cell lines and patients 41 3.2 CCTb regulates protein expression and function of the drug efflux pumps 41 3.3 CCTβ regulates ABC pumps via β-catenin 43 3.4 CCTβ prevents β-catenin from degradation in cytosol, leading to its translocation into nucleus during drug resistance development 45 3.5 CCTb-dependent multidrug resistance is only minimally caused by Akt signaling 46 3.6 CCTb-dependent multidrug resistance is driven by Bcl2 anti-apoptotic signaling mediated by β-catenin or phosphatases 47 3.7 CCTb regulates XIAP-dependent anti-apoptotic signaling 48 3.8 Balance of TRiC/CCT from altering expression of CCTb 49 (4) DISCUSSIONS 51 REFERENCE 57 SUPPLEMENTARY 101 APPENDIX 10