2017-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658880摘要： 背景及重要性：細胞醣基化(glycosylation)異常是癌症的一項重要特徵。包括黏液蛋白(mucin)在內的許多不同種類蛋白進行O-醣基化的起始步驟皆為藉由N-乙醯半乳糖氨基轉移酶(GALNTs)家族的作用來將乙醯半乳糖胺連接到serine或threonine殘基的羥基上。目前已經發現GALNTs參與了許多癌症發展。GALNT6雖為GALNTs家族的一員，然而對於GALNT6在卵巢癌中的作用仍是未知。我們目前正在進行的2016 年科技部計畫「研究N-乙醯半乳糖氨基轉移酶第6 型(GALNT6)引發之異常O-醣基化在卵巢癌癌化過程中所扮演的角色」已經建立了過量及減量表現GALNT6 的卵巢癌細胞株，並確定了下列結果：a. GALNT6 的表現量在卵巢惡性檢體中有明顯增加。b. GALNT6 過度表現與病人較差的預後存有相關的趨勢。c. 卵巢癌細胞減量表現 GALNT6 會下降細胞的惡性程度，包括生長、移動和侵襲能力。d. 卵巢癌細胞過量表現 GALNT6 會增加細胞的惡性程度。e. GALNT6 在卵巢癌細胞中會調節EGFR 的活性。研究仍然持續進行中，而目前結果強烈建議GALNT6能夠調節EGFR的O-醣基化因而參與卵巢癌的癌化過程，這些結果也刺激了我們想要在接下來一年進一步剖析GALNT6對於卵巢癌癌化的調控機制。 我們的假設：GALNT6 的異常表現造成了EGFR 的異常O-醣基化及穩定性，從而提高了卵巢癌細胞的惡性特質。 研究目的與方法：1. 為了研究GALNT6 對EGFR 訊息傳導路徑的影響。本實驗將使用Western blots 來驗證過量及減量GALNT6 表現對於EGFR 及其下游信號傳導活性的影響。再者，本實驗將使用EGFR 抑製劑來確認EGFR 活性在調節卵巢癌細胞的惡性表型中的意義。2. 為了研究GALNT6 對EGFR 的O-聚醣表現的影響。本實驗將在存在或不存在GALNT6的癌細胞中利用VVA 凝集素(檢測Tn 抗原表現)來進行流式細胞儀分析。再者，本實驗還將利用細胞裂解物來進行VVA 凝集素的Western blots 實驗來確定特異性糖蛋白是否可以被GALNT6 修飾。3. 為了研究GALNT6 對卵巢癌細胞化學敏感性的影響。本實驗將利用過量及減量表現GALNT6 的癌細胞來進行抗癌藥物處理。利用MTT assays 分析細胞生長，利用流式細胞儀試驗分析細胞凋亡和細胞週期，及利用球體形成試驗分析幹細胞特性。<br> Abstract:  Background and significance:Altered glycosylation is a hallmark of cancer. The N-acetylgalactosaminyltransferases(GALNTs) family conducts the transfer of N-acetyl galactosamine (GalNAc) to the hydroxyl groupof a serine or threonine residue in the first step of O-glycosylation of different proteins, includingmucins. GALNTs are involved in multiple cancer developments. The GALNT6 is a member of theGALNTs family. However, little is known on the function of GALNT6 in ovarian cancer (OVCA).Our ongoing 2016 MOST’s project “Analyzing the role of aberrant O-glycosylation bypolypeptide N-acetylgalactosaminyltransferase 6 (GALNT6) in ovarian cancer progression” hasestablished several GALNT6-overexpressed and GALNT6-knockdowned OVCA cell lines and hasfound that:a. GALNT6 expression is upregulated in OVCA tissues.b. GALNT6 overexpression is associated with poor survival of OVCA patients.c. GALNT6 knockdown could decrease malignant phenotypes, including viability, migration andinvasion.d. GALNT6 overexpression could enhance malignant phenotypes of OVCA cells.e. GALNT6 regulates EGFR activity in OVCA cells.Although the project is still ongoing, our results strongly suggest that GALNT6 could modifyO-glycosylation of EGFR and participate in the OVCA progression. Thus, these data encourage us tofurther dissect the regulatory mechanism of GALNT6 in the ovarian carcinogenesis in the followingyear. Our hypotheses:Altered expression of GALNT6 contributes to aberrant O-glycosylation and stabilization ofEGFR, thereby increasing the malignant nature of human OVCA cells. Our specific aims:1. To investigate the effect of GALNT6 on EGFR signaling pathways. Western blots will be usedto validate the effect of GALNT6 knockdown on activation of EGFR and its downstreamsignalings. EGFR inhibitor will be used to confirm the significance of EGFR activity in regulatingthe malignant phenotype of ovarian cancer cells.2. To investigate the effect of GALNT6 on O-glycan expression of EGFR. We will perform flowcytometry with VVA lectin, which detects Tn antigen expression in the presence or absence ofGALNT6 knockdown. We will also investigate whether specific glycoproteins could be modifiedby GALNT6, cell lysates are Western blotted with VVA lectin.3. To investigate the effect of GALNT6 on chemosensitivity in OVCA cells. We will analyze theGALNT6 overexpressed and knockdowned OVCA cells treated with anticancer drugs. Cellviability will be analyzed by MTT assays. Apoptosis and cell cycle will be analyzed by flowcytometry. Stem-like potential will be analyzed by sphere formation assay.N-乙醯半乳糖氨基轉移酶第6 型卵巢癌上皮生長因子接受器醣基化GANLT6ovarian cancerEGFRglycosylation