膀胱癌是國人相當常見之腫瘤，鈣網酪蛋白calreticulin為一多功能蛋白並參與細胞內多種機制，臨床上calreticulin已被使用來檢測膀胱癌。於本研究中，我們成功構築表達低量鈣網酪蛋白之穩定膀胱癌細胞株(J82 CRT-RNAi)顯示降低鈣網酪蛋白之表達會抑制膀胱癌細胞株之移行，並對細胞附著於基質之能力有重要之影響；反之，表達大量鈣網酪蛋白會促進膀胱癌細胞之移行與貼覆基質之能力。更重要的是，降低鈣網酪蛋白之表達亦會抑制膀胱癌細胞株於裸鼠中形成腫瘤，並明顯降低肺臟及肝臟之轉移，這些結果證明了鈣網酪蛋白可能是影響膀胱癌細胞癌化之重要調控因子。藉由核酸晶片比較J82 CRT-RNAi 細胞株與未轉染之細胞間基因表達之差異後，我們發現岩藻醣轉移酵素FUT-1 為其中表達差異性極大的一種蛋白質。岩藻醣化作用為細胞常見之蛋白質修飾，報告指出許多病症與高度岩藻醣化極具相關性。FUT-1 為細胞內負責蛋白質醣基修飾的重要酵素，本研究結果顯示CRT透過調控整合蛋白β1-integrin由FUT1所修飾之岩藻醣化作用，進而影響細胞附著能力。透過大量表達岩藻醣轉移酵素可增加細胞整合蛋白β1-integrin之岩藻醣化程度並有效提升膀胱癌細胞貼附能力。 我們的研究結果更進一步證實，整合蛋白β1-integrin之岩藻醣化作用影響細胞貼附能力是經由調控其整合蛋白本身之活性。 此外，我們也進一步釐清J82 CRT-RNAi 細胞株之FUT-1 表達降低之原因，是由於FUT1 RNA 穩定性下降之結果。根據本研究成果之驗證CRT透過穩定FUT1 mRNA，進而提升β1-integrin岩藻醣化作用藉此促進其活性，影響膀胱癌細胞轉移之能力。

Bladder cancer is a common urothelial cancer. Through proteomic approaches, calreticulin (CRT) was identified and proposed as a urinary marker for bladder cancer. CRT is a multifunctional molecular chaperone that regulates various cellular functions such as Ca2+ homeostasis and cell adhesion. CRT was reported to be overexpressed in various cancers; however, the mechanisms of CRT in bladder tumor development remain unclear. To clarify the roles of CRT in bladder cancer, J82 bladder cancer cells stably overexpressed or knockdown of CRT were generated to investigate the physiological effects of CRT on bladder tumors. Compared to the transfected control vector cells, the knockdown of CRT suppressed cell proliferation, migration, and attachment; on the contrary, overexpression of CRT enhanced cell migration and attachment. Most importantly, we observed that tumors derived from J82 CRT-RNAi cells were significantly smaller and had fewer metastatic sites in the lung and liver in vivo than did transfected control vector cells. To further investigate the precise mechanism of tumor metastasis regulated by CRT, we used DNA array to identify fucosyltransferase-1 (FUT1) as a gene regulated by CRT expression levels. CRT regulated cell adhesion through α1,2-linked fucosylation on β1-integrin and this modification was catalyzed by FUT1. To clarify FUT1 roles in bladder cancer, we transfected the human FUT1 gene into CRT-RNAi stable cell lines. FUT1 overexpression in CRT-RNAi cells resulted in increased levels of β1-integrin fucosylation and rescued cell adhesion to type-I collagen. Treatment with Ulex europaeus agglutinin I (UEA-1), a lectin recognizes FUT1-modified glycosylation structures, did not affect cell adhesion. In contrast, a FUT1-specific fucosidase diminished the activation of β1-integrin. These results indicated that α1,2-fucosylation on β1-integrin were not involved in the integrin-collagen interaction but promoted β1-integrin activation. In addition, we demonstrated that CRT regulated FUT1 mRNA degradation in 3′-untranslated region (3′-UTR). In conclusion, our findings suggested that CRT stabilized FUT1 mRNA, thereby leading to increase in fucosylation of β1-integrin. Furthermore, increased fucosylation levels activate β1-integrin rather than directly modifying the integrin binding sites.

口試委員審定書
致謝………………………………………………………………………………….…i
中文摘要………………………………………………………………………………ii
Abstract……………………………………………………………..………………..iv
List of Tables………………………………………………...………………...……viii
List of Figures………………………………………………..………………………ix
Chapter I. Introduction………………………………………………………………1
1. Fundamental information of calreticulin……………………………………….2
1.1 Protein structure…………………………………………………………………...2
1.2 Location of calreticulin……………………………………………………………3
2. Biological functions of calreticulin……………………………………………....3
2.1 Protein chaperone………………………………………………………………….4
2.2 Calcium homeostasis………………………………………………………………4
2.3 Cell adhesion…………………………………………………………………........5
2.4 RNA stability…………………………………………………………………........5
3. Calreticulin in cancer…………………………………………….........................6
3.1 Clinical outcome of calreticulin levels………………………….............................6
3.2 Functions of CRT in the immune system…………………….................................7
3.3 Functions of CRT in cancer cell proliferation………………..................................8
3.4 Functions of CRT in cell migration and adhesion………………............................9
4. Functions of fucosylation in cancer biology………………...............................11
4.1 Fucosylation in cancer…………………………….…………...............................11
4.2 Fucosyltransferase 1…………………………………………...............................11
4.3 Glycan on integrins regulates its functions………………....................................12
5. Bladder cancer…………………………………...…………...............................13
6. Rationale…………………………………...…………........................................14
Chapter II. Materials and Methods…..………………...………….........................15
Chapter III. Results…………………………………...………….............................28
Chapter IV. Discussions……...…………………………...………….......................40
Chapter V. Concluding remarks and future perspectives…………......................50
References…………………………………...………………………........................53
Appendix I: Changes in tumor growth and metastatic capacities of J82 human bladder cancer cells suppressed by down-regulation of calreticulin expression………………………………...………….................................................97
Appendix II: Calreticulin activates β1-integrin through fucosylation modification by fucosyltransferase-1 in J82 human bladder cancer cells…………………….106

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