李明亭臺灣大學：生化科學研究所蔡沛勳Tsai, Pei-HsunPei-HsunTsai2007-11-262018-07-062007-11-262018-07-062007http://ntur.lib.ntu.edu.tw//handle/246246/52751癌細胞的轉移一直是癌症研究的重要領域。癌細胞在轉移前，會先藉由epithelial-mesenchymal transition (EMT)的轉變，讓細胞變的較細長，入侵和移動能力都會顯著地提升。EMT中最具代表性的變化就是E-cadherin的減少，造成細胞間附著能力的下降，同時亦造成E-cadherin adhesion complex的降解，使得β-catenin進入細胞核的機率上升，引發下游基因表現，促進細胞的增生與移動。另外，在EMT中E-cadherin的減少，伴隨著N-cadherin表現量增加，其與許多蛋白的交互作用，可啟動有關癌細胞轉移的下游訊息傳遞機制。 本實驗室利用in vitro invasion assay，從母代細胞A431 (A431 P )成功地篩選出一株具有較強入侵能力的細胞，命名為A431 III，並以此觀察細胞附著蛋白cadherin的變化，對癌細胞轉移的重要性。我們發現在A431 III中E-cadherin的表現量些微地下降，而β-catenin的表現量反而上升；在兩者的結合關係上，A431 III中部份的β-catenin已經從E-cadherin上脫離，但並沒有進入細胞核，仍然留在細胞膜上。另外，在A431 III中N-cadherin和FGFR1的表現量明顯上升，兩者間結合程度也較A431 P高，且在本實驗之前的研究已證實ERK磷酸化程度上升，MMP9分泌增加，與N-cadherin和FGFR-1協同作用的訊息傳遞路徑相當符合，故我們推測N-cadherin的大量表現，可能是循此訊息傳遞機制，使MMP9增加，最終導致A431 III的入侵能力增加，有利於癌細胞的轉移。另外，我們還發現β-catenin與N-cadherin結合量增加，顯示N-cadherin可能藉β-catenin而穩定，有助於其啟動A431 P和A431 III相關的訊息調控。 未來我們將針對N-cadherin及E-cadherin在兩株細胞中的調控方式，更進一步探討，以期為A431 III轉移能力較強的現象提出合理的解釋。Metastasis is still one of the important areas to study in cancer research fields. Normally, before metastasis, cancer cells exhibit longer shape, and increase their invasion, migration ability by epithelial-mesenchymal transition (EMT). The noticeable properties of EMT are decreasing in E-cadherin expression and cell-cell adhesion. Besides, EMT could induce the degradation of E-cadherin adhesion complex, and translocation of β-catenin into the nucleus as well. The entrance of β-catenin into nucleus will trigger the expression of downstream genes, promote cell proliferation and enhance cell movement. In addition, the decrease of expression of E-cadherin was found to accompany with the increasing expression of N-cadherin, which in turn interacts with many proteins, and switch on signal transduction of cancer cells. In this study, we use in vitro invasion assay to isolate higher invasive carcinoma cells from A431 parental cells (A431 P). The cells passed through Boyden chamber three times were designed as A431 III. We then used A431 III cells to explore the role of cadherins in cancer metastasis. We observed that the level of E-cadherin showed little change, but the level of β-catenin was slightly increased in the A431 III sub-line as compared to A431 P. In addition, the decrease in interaction of E-cadherin and β-catenin was found in A431 III sub-lines. Moreover, the expression of N-cadherin and FGFR1 was increased in A431 III, and the binding of N-cadherin and FGFR1 was higher than that of A431 P. We further confirmed that the phosphorylation of MAPK/ERK was enhanced and MMP9 secretion was increased in A431 III. The aforementioned results were in agreement with the synergistic pathway of N-cadherin and FGFR-1. On the other hand, we observed that β-catenin didn’t translocate into the nucleus, but switched to bind N-cadherin. This switch may be responsible for stabilization of N-cadherin and induce some signal pathways related to N-cadherin. In summary, we showed important roles of E-cadherin andβ-catenin in tumor metastasis. The decrease in E-cadherin expression resulted in lose cell-cell adhesion, suggested the importance of E-cadherin as a therapeutic target in cancer metastasis.目錄 中文摘要 1 Abstract 2 引言 4 壹、癌細胞的轉移 4 貳、癌細胞爭取空間之機制 4 一、減弱細胞間結合力 (Disruption of cell-cell adhesion) 5 二、Extracellular matrix（ECM）的降解 5 参、Epithelial-Mesenchymal Transition（EMT） 6 一、EMT在生物學上的角色 6 二、EMT的型態特徵 6 肆、Cadherin 7 一、Cadherin在生物學上的角色 7 二、Cadherin之基本結構及大致分類 7 伍、E-cadherin與β-catenin 9 一、β-catenin的調控機制 9 二、β-catenin的磷酸化機制 10 陸、N-cadherin 12 一、E-cadherin/N-cadherin 的轉變 12 二. N-cadherin的構造及基本功能 12 三. N-cadherin參與的訊息傳遞路徑 13 柒、A431 P 與A431 III的挑選方式 14 捌、實驗目的 16 材料與方法 26 壹、Material 26 貳、Whole cell lysate和membrane protein萃取 26 参、SDS聚丙醯胺凝膠電泳分析（SDS polyacrylamine gel electrophoresis，SDS-PAGE） 27 肆、西方墨點法分析（Western blotting） 28 伍、Gelatin zymography 28 陸、In vitro invasion assay 29 柒、Migration assay 29 捌、Wound healing migration assay 29 玖、免疫沉澱分析Immunoprecipitation 30 拾、免疫螢光呈色Immunofluorescence 30 拾壹、反轉錄PCR(reverse transcription-PCR) 31 結果 32 壹、A431 P-->A431 III 屬於EMT的轉變 32 貳、A431 P與A431 III在E-cadherin和β-catenin蛋白上的差異 32 参、A431 III 在β-catenin與E cadherin結合程度較A431低 33 肆、A431P與A431 III在N-cadherin和FGFR1蛋白表現量及結合情形的差異 34 伍、A431 P與A431 III中N-cadherin和β-catenin蛋白結合情形的差異 35 討論及未來工作 48 引用文獻 52 圖表目錄 圖一：癌細胞轉移 17 圖二：EMT的訊息傳遞路徑 18 圖三：Cadherin的結構 19 圖四：cadherin surperfamily 20 圖五：E-cadherin所調節的 cell–cell adhesion 22 圖六：E-cadherin-deficient 可能出現的下游訊息傳遞機制 23 圖七：N-cadherinu在癌細胞中有關的調控機制 24 圖八：A431 sub-lines的挑選方式 25 圖九：A431 P 和A431 III細胞形態及群落聚集 36 圖十：A431 P 和A431 III的EMT marker在DNA microarray中的表現(I) 37 圖十一：A431 P 和A431 III的EMT marker在DNA microarray中的表現(II) 38 圖十二：A431 P和 A431 III中E-cadherin表現量 39 圖十三： A431 P和A431 III中β-catenin表現量 40 圖十四： A431 P和 A431 III中β-catenin與E-caderin結合情形 41 圖十五： A431 P和A431 III中β-catenin磷酸化程度 42 圖十六： A431 P和A431 III中β-catenin與E-caderin免疫螢光呈色圖 43 圖十七： A431 P 和 A431 III中N-cadherin表現量 44 圖十八：A431 P和A431 III中FGFR1表現量和FGFR1與N-cadherin結合情形 45 圖十九：A431 P和A431 III中β-catenin與N-caderin結合情形。 46 圖二十：A431 P與A431 III 之間，E-cadherin和N-cadherin相互調控機轉 47en-US癌症轉移附著蛋白A431invasionEMTcadherinβ-cateninFGFR1[SDGs]SDG3other