2012-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/642612摘要：肝癌在全世界癌症死亡率中是排名第三位，本研究的長程目標是希望了解肝癌發育的機制及尋找新的癌症治療標靶。醣基化的改變通常和人類腫瘤惡性程度以及癌症病人的預後有相關連性，然而，在肝癌發育中醣基化扮演的角色仍不清楚，T-synthase 是在醣蛋白上將galactose (Gal)加到N-acetylgalactosamine (GalNAc)而形成T antigen(Gal-GalNAc-serine/threonine)的一種O-glycosyltransferase，本實驗室的初步結果顯示T-synthase 在大部分人類肝癌組織中表現量高於正常組織，且抑制T-synthase 的表現會降低肝癌細胞的惡性行為，除此之外，上皮-間質轉化、c-MET 的醣基化、還有HGF引發的訊息都會被T-synthase 所調控，我們因此假設(1) T-synthase 可以調控肝癌的惡性程度和腫瘤的進程，(2) T-synthase 可以直接修飾受器的O-glycosylation 並且影響它們下游的訊息傳遞方式。我們的特定目標有以下幾點:1. 研究 T-synthase 在肝癌組織的表現量以及T-synthase 表現量和肝癌臨床表徵的關聯性。我們使用西方墨點法和免疫組織化學染色法去分析T-synthase 的表現量和表現型。2. 研究 T-synthase 對肝癌細胞惡性程度的影響。我們將在多種肝癌細胞中分析其增生、聚落形成、移動和入侵能力。3. 研究 T-synthase 對肝癌細胞上皮-間質轉化、癌症幹細胞特性以及藥物敏感性的影響。用一系列的分子標誌來確認肝癌細胞上皮間質轉化、癌症幹細胞特性，治療肝癌用的Sorafenib 和化療藥物將用來分析T-synthase 對藥物敏感性的影響。4. 在免疫缺陷小鼠實驗模式中分析 T-synthase 對腫瘤生長、轉移和血管新生能力的影響。5. 分析 T-synthase 調控癌細胞行為之機制。我們將分析並鑑定受改變的癌細胞行為的訊息傳導路徑。6. 鑑定 T-synthase 作用的標的和T-synthase 對其生化特性的影響。我們使用醣蛋白質體學來鑑定T-synthase 的標的。將分析生化特性包括受器dimerization、胞內運輸和降解。這將是第一個探討 T-synthase 影響肝癌惡性程度和藥物敏感性的研究，此外，T-synthase的標的和T-synthase 如何調控細胞行為的機制將被了解。此研究將會對O-glycosylation如何調控癌症發育的機制有嶄新的發現。此研究的結果將可提供未來開發新的肝癌抗癌藥物的參考。<br> Abstract: Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. Thelong-term goal of this proposal is to understand the mechanism of HCC progression andidentify a novel target for HCC therapy. Changes in glycosylation are often associated withtumor malignancy and prognosis in human cancers. However, the role of glycosylation inHCC development remains largely unknown. T-synthase, highly expressed in the liver, is anO-glycosyltransferase which adds galactose (Gal) to N-acetylgalactosamine (GalNAc) to formT antigen (Gal-GalNAc-serine/threonine) on a glycoprotein. Our preliminary data showed thatT-synthase was frequently up-regulated in human HCC tissues compared with theirnon-tumorous part. Knockdown of T-synthase decreased malignant phenotypes of HCC cells.In addition, epithelial mesenchymal transition (EMT), glycosylation of c-MET, andhepatocyte growth factor (HGF)-triggered signaling were modulated by T-synthase. Wetherefore hypothesize that (1) T-synthase can regulate malignant properties and tumorprogression of HCC; and (2) T-synthase can directly modify O-glycosylation of receptors andaffect their downstream signaling pathways..Our specific aims are:1. To investigate the expression of T-synthase in primary HCC tissues and the correlationbetween T-synthase expression with clinical characteristics of HCC. Western blot andimmunohistochemistry will be used to analyze the expression level and expressionpattern of T-synthase, respectively.2. To investigate effects of T-synthase on malignant phenotypes of HCC cells. We willanalyze cell proliferation, cell cycle, adhesion, colony formation, migration, and invasionin multiple HCC cell lines.3. To investigate effects of T-synthase on epithelial mesenchymal transition, cancerstemness properties, and drug sensitivity in HCC cells. A panel of makers will be used toassess for EMT and cancer stemness properties. Sorafenib and chemotherapeutic drugsfor HCC will be used to analyze effects of T-synthase on drug sensitivity.4. To analyze effects of T-synthase on tumor growth, metastasis, and angiogenesis inimmunodecificent mouse models.5. To analyze the underlying mechanisms that T-synthase regulates cancer cell behaviors.Signaling pathways related to phenotypic changes will be analyzed.6. To identify the targets of T-synthase and effects of T-synthase on their biochemicalproperties. Glycoproteomics will be used to identify targets of T-synthase. Biochemicalproperties including receptor dimerization, endocytic trafficking, and degradation will beanalyzed.This study will be the first to investigate effects of T-synthase on HCC malignancy and drugsensitivity. In addition, the targets of T-synthase and the mechanisms by which T-synthaseregulates cell behaviors will be identified. This study will open new insights into themechanism of how cancer progression is regulated by O-glycosylation. Furthermore, theinformation gained from this study will provide pharmaceuticals to develop novel drugs forHCC therapy in the future.