2016-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658735摘要： 背景：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在癌症進展中的作用仍是未知。近日我們進行了卵巢癌組織檢體的初步研究發現，GALNT6的表現量在卵巢惡性檢體中有明顯增加，而且與病人較差的預後明顯相關。再者，研究發現抑制卵巢癌細胞株中GALNT6的表現可以降低細胞的惡性程度。這些結果強烈反映出GALNT6在卵巢癌癌化過程中佔有重要角色。此外，我們近期發表的研究發現MUC20 為一個O-醣基化蛋白，其同樣會在卵巢癌中過度表現，並且與不良預後密切相關。因此GALNT6 極有可能是影響MUC20 的O-醣基化及穩定性，從而提高了卵巢癌細胞的惡性特質。綜合而言，GALNT6 在卵巢癌中的作用以及與MUC20 之O-醣基化的關聯性都相當值得深入探討。 我們的假設：1. GALNT6的異常表現促進了卵巢癌細胞的惡性及侵襲能力。抑制GALNT6可以抑制腫瘤惡性程度，並增加卵巢癌細胞對癌症化療藥物的敏感性。2. GALNT6 的異常表現造成了MUC20 或其他接受器的異常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：第一年：1. 為了確定GALNT6在卵巢癌中的角色，本實驗將利用癌細胞株及動物模型來過量以及減少GALNT6 表現，進而研究GALNT6 所媒介之細胞的生長、活動和侵襲能力的變化，並且釐清GALNT6 促進卵巢癌癌化主要相關的分子機轉。並研究抑制GALNT6 是否可以提高卵巢癌細胞對化療的靈敏性，第二年：1. 為了確定GALNT6 是否以MUC20 為受體進行O-醣基化，我們將使用具活性的GALNT6以及不具活性的GALNT6 突變蛋白和正常MUC20 以及MUC20 串聯重複片段來進行體外半乳糖胺轉移酶試驗和細胞內免疫印跡試驗。2. 為了闡明GALNT6 與MUC20 的表現與卵巢癌惡性程度的相關性，將利用不同分期與不同組織型態的卵巢癌之組織晶片來確定GALNT6 與MUC20 的共同表現狀況。其後利用pRTKs 微陣列來研究GALNT6 與MUC20 的作用所影響的RTKs 訊息傳導與路徑。最後研究GALNT6 與MUC20 的交互作用在化療抗性中所扮演的角色。<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 cancer progression.Recently, our preliminary study found that GALNT6 was overexpressed in ovarian cancer (OVCA)samples and the GALNT6 expression significantly correlated with poor prognosis. Our findingsstrongly suggest a critical role of GALNT6 in OVCA progression.Furthermore, our previous study has shown that MUC20, as one O-glycosylated protein, isoverexpressed in most OVCAs, and the overexpression of MUC20 correlated with poor prognosis.Thus, altered expression of GALNT6 might contribute to aberrant O-glycosylation and stabilizationof MUC20, thereby increasing the malignant nature of human OVCA cells. Thus, the roles ofGALNT6 in OVCA and its role in O-glycosylation of MUC20 are worthy of evaluation. Our hypotheses:1. GALNT6 promotes the malignant nature of human OVCA cells. Knockdown of GALNT6 cansuppress tumor malignancy and increase cancer chemosensitivity.2. Altered expression of GALNT6 contributes to aberrant O-glycosylation and stabilization ofMUC20 and/or other receptors, thereby increasing the malignant nature of human OVCA cells. Our specific aims:First Year:1. To study the functional roles of GALNT6 in OVCA cells, the study will investigate theoverexpressed and knockdowned GALNT6-mediated growth, motility and invasive changes ofOVCA cells in vitro and in vivo; identify the target of GALNT6 and the underlyingmechanisms affecting OVCA malignant behaviors; and study whether knockdown of GALNT6can increase OVCA cells to chemotherapy- induced apoptosis.Second Year:1. To investigate whether GALNT6 O-glycosylates MUC20 as a substrate, we will use GALNT6as well as the inactive GALNT6 mutant proteins and performed in vitro GalNAc transferaseassay and in vivo Western blot assay using MUC20 corresponding to the tandem repeatfragment of MUC20 protein.2. To clarify the relationship of GALNT6 and MUC20 in OVCA neoplasms with their biologicalbehavior, the study will examine the co-expression profiles of GALNT6 and MUC20 invarious stages and/or histologic subtypes of human ovarian neoplasms using tissue arrays;analyze the effects of interaction between GALNT6 and MUC20 on RTKs signaling by usingthe pRTK arrays; and study the interaction of GALNT6 and MUC20 on chemoresistance inOVCA cells.