2016-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/642680摘要：本計畫的目標是探討特定位置的氧型醣化作用(O-glycosylation)在表皮生長因子受體(epidermal growth factor receptor, EGFR)與肝細胞生長因子受體(MET)上所扮演的角色，並建立尋找癌症標靶藥物的技術平台。長遠的目標是發展可應用於醣醫學研究、分子診斷造影、藥物傳輸、以及癌症的免疫治療藥物。已知Tn抗原(GalNAc-α-Ser/Thr)表現在許多癌症的腫瘤細胞表面而非正常細胞上。我們近期的研究證實Tn抗原表現於大多數的肝細胞腫瘤中，而且許多受體酪胺酸激酶(receptor tyrosine kinases, RTKs)，例如：EGFR與MET，都帶有此Tn抗原。但是特定位置的氧型醣化作用對於受體酪胺酸激酶的功能影響仍然不清楚。另外，由於醣類本身無法單獨引發T細胞調節的免疫反應。因此，由醣類與胜肽共同組成的抗原決定位(epitope)，具有開發診斷與治療藥物的巨大潛力。本計畫中，我們首先會以質譜技術，分析不同癌細胞EGFR和MET上的氧型醣類結構，並得知其確切的氧型醣化位置。更進一步，我們會以定點突變的方法將特定位置的氧型醣化位置去除，以研究其對EGFR和MET特性的影響，我們會以流式細胞儀、西方點漬法、螢光免疫顯微鏡法研究EGFR和MET的配體親和力、二聚作用、磷酸化作用、傳輸與降解等是否受影響。本計畫會延伸過去的研究發現，進一步探討兩個主要的氧型醣化酵素GALNT1和GALNT2在癌細胞中，如何合作和競爭性的調控EGFR和MET的特性以及細胞行為。另外，我們會以合成且含Tn抗原的EGFR和MET衍生胜肽作為標靶，建立以噬菌體展示與融合瘤技術尋找腫瘤標靶藥物的技術平台。所篩選出的標靶物質，會以及體內與體外方式確認其功效，並且將這些標靶物質應用於研究特定位置的氧型醣化作用在EGFR與MET上的功能。從這項研究中所獲得的訊息，將開拓GalNAc-type氧型醣化作用在受體酪胺酸激酶與癌症中之功能的新視野，並建立技術平台，用以提供生技製藥工業持續開發癌症診斷試劑和標靶藥物。<br> Abstract: The objective of this proposal is to investigate the roles of site-specific O-glycosylation in two critical cancer targets EGFR and MET and build a technology platform for the identification of tumor targeting agents. The long-term goal is to develop agents for glyco-medicine research, molecular imaging, drug delivery, and immunotherapy of various cancers. It is well known that Tn antigen (GalNAc-α-Ser/Thr), a short O-glycan structure, is expressed on cell surfaces of many cancer types but not on their corresponding normal cells. We recently demonstrated that Tn antigens are expressed in most hepatocellular carcinoma (HCC) tumors and that a number of important receptor tyrosine kinases (RTKs), including EGFR and MET, are decorated with the Tn antigens. However, the functional roles of site-specific O-glycosylation in RTKs remain unclear. Moreover, carbohydrate itself is insufficient to elicit T cell-mediated immune response. It is believed that the epitopes formed by sugars and peptide backbone hold great promise in developing diagnostic and therapeutic agents. In this proposal, we will first analyze the O-glycans and determine the precise O-glycosites on EGFR and MET in different cancer types by mass spectrometry. Furthermore, O-glycosites will be mutated by using site-directed mutagenesis and the roles of site-specific O-glycosylation in EGFR and MET properties including ligand binding affinity, dimerization, phosphorylation, trafficking, and degradation will be determined by flow cytometry, Western blotting, and immunofluorescence microscopy. To extend our previous findings, we will investigate the cooperative and competitive roles of two major O-glycosyltransferases, GALNT1 and GALNT2, in EGFR and MET properties as well as in cellular behaviors of cancer cells. Synthetic EGFR-and MET-derived glycopeptides containing Tn will be used as our model targets in establishing the technology platform for identifying the tumor targeting agents by phage display and hybridoma technology. The targeting agents will be analyzed in in vitro and in vivo mouse models. In addition, the targeting agents will be used as powerful tools to study the functional roles of site-specific O-glycosylation in EGFR and MET. The information gained from this study will open new insights into the functions of GalNAc-type O-glycosylation in RTKs and cancers. Moreover, the establishment of this technology platform will offer biopharmaceutical industry to continuously develop novel cancer diagnostic and therapeutic agents.