- 學位論文
蛋白醣化酵素C1GALT1在胰臟癌的表現與功能
Expression And Function of O-glycosyltransferase C1GALT1 in Pancreatic Cancer
摘要
胰臟癌致死率極高，在其中有高達百分之九十五為胰管腺癌，死亡率居國人十大癌症死因之七、全球之第四、甚至被預測在二零三零年將會成為美國第二癌症死因，其增加率卻高居第一且近三十年來並無顯著進展；目前最有效治療方式仍為根除性手術，卻只有百分之十五至二十的病人屬於可切除之胰臟癌，且即便完整廓清其預後仍不盡理想，輔助性化學放射治療或標靶治療效果亦不彰，胰臟癌之致病機轉仍深陷迷霧。 醣化是蛋白質最為常見且最複雜的轉譯後修飾作用，大部分的腫瘤都被發現有異常的醣類表現，例如Tn抗原和T抗原。蛋白醣化作用主要分為氧型蛋白醣化作用及氮型蛋白醣化作用，最普遍的氧型蛋白醣化作用為黏液型，N-乙醯基半乳糖胺基轉移酵素負責開啟黏液型醣化的第一步驟，在絲胺酸或蘇胺酸加上N-乙醯半乳糖胺，形成Tn抗原。蛋白醣化酵素（core 1 β1,3-galactosyltransferase, C1GALT1）進一步加上半乳糖，形成T抗原。過去研究指出Tn抗原、T抗原以及蛋白醣化酵素C1GALT1的過度表現與乳癌、胃癌、大腸癌、肝癌、頭頸癌、卵巢癌、攝護腺癌等之臨床病生理、腫瘤惡性表現度、及病患預後存活有關，然而在胰臟癌中的表現和功能仍然不清楚。 我們的研究顯示，與鄰近的非腫瘤組織相比，蛋白醣化酵素C1GALT1在高達百分之八十五的胰臟癌腫瘤中及胰臟癌多株細胞株中都會過度表現。蛋白醣化酵素C1GALT1的高表現與較差之無疾病存活期及較差之整體存活期相關（九十九位病患）；抑制調控蛋白醣化酵素C1GALT1可抑制胰臟癌細胞惡性表徵如生長、移行、侵犯、侵襲，促進胰臟癌細胞凋亡並增加對化療藥物吉西他濱（Gemcitabine）之敏感性，降低胰臟癌細胞附著能力，阻礙胰臟癌細胞週期進行；相反的，蛋白醣化酵素C1GALT1的過度表現則增強了細胞的移行及侵犯；在皮下注射和胰腺原位注射的活體小鼠（NOD/SCID mice）模型實驗中，抑制蛋白醣化酵素C1GALT1可降低活體小鼠的胰臟癌腫瘤生長和減少胰臟癌細胞的轉移。 探討其機制，蛋白醣化酵素C1GALT1的降低顯著抑制了細胞與細胞外基質的粘附，這與局部粘附激化酵素（Focal adhesion kinase, FAK）在位點Y397、Y925兩處的磷酸化降低以及整合素（包括β1、αV、α5亞基）上的氧型聚醣變化有關。使用功能性阻斷抗體，我們確定整合素αV是蛋白醣化酵素C1GALT1調控胰臟癌細胞侵襲性的關鍵因素；蛋白醣化酵素C1GALT1調控抑制降低胰臟癌細胞中與凋亡相關因子的活性，並抑制多種受體酪氨酸激化酵素（RTK）的磷酸化。 結論，這項研究不僅揭示了蛋白醣化酵素C1GALT1可作為胰臟癌的潛在治療標的，而且還提供了對於氧型糖基化在整合素α亞基中的作用的新穎見解。
並列摘要
Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death. PDAC is the most common (>95%) malignancy in pancreas, stands the 7th of all cancer mortalities and remains the most in progress in Taiwan or also stands the 4th universally. PDAC is even projected to be the 2nd of all cancer mortalities in 2030 in US. It had adhered to stability for the past 30 years without major breakthrough (5-year survival 3.8% to 10.0%). Surgical resection remains the most and only potential curative management but only 15-20% of patients are candidates. Even though, many of them have worse prognosis. New insight into the biology and genetics of pancreatic cancer is a unique entity of investigation in the breakthroughs of early diagnosis and further treatment. Glycosylation is the most abundant and diverse post-translational modification of proteins. Aberrant expression of glycans, such as Tn antigen and T antigen, are hallmark of most human cancers. Two major types of protein glycosylation are N- glycosylation and O-glycosylation. Mucin-type O-glycosylation, the most common type of O-glycosylation, is initiated when N-acetylgalactosamine (GalNAC) is added to form the Tn antigen, by GalNAc-transferases (GALNTs). The human GALNT family genes are differentially expressed in cells and have unique functions and considerable redundancy. Core 1 β1,3-galactosyltransferase (C1GALT1, or T synthase) catalyzes the transfer of Galactose to the Tn antigen to form T antigen. Altered glycosylation contributes to tumor progression and chemoresistance in many cancers. C1GALT1 is the key enzyme controlling the elongation of GalNAc-type O-glycosylation. Several studies have shown that up-regulation of Tn, T, and C1GALT1 contributes to clinicopathological features and malignant phenotypes in breast cancer, gastric cancer, colon cancer, hepatocellular carcinoma, head and neck cancer, ovarian cancer, and prostate cancer. However, the expression and function of Tn, T, and C1GALT1 in pancreatic cancer remain unclear. Here we showed that C1GALT1 was overexpressed in 85% (107/126) of PDAC tumors compared with adjacent non-tumor tissues. High expression of C1GALT1 was associated with poor disease-free and overall survival (n = 99). C1GALT1 knockdown using siRNA suppressed cell viability, migration, and invasion as well as increased gemcitabine sensitivity and arrested the cell cycle in PDAC cells. In contrast, C1GALT1 overexpression enhanced cell migration and invasion. In subcutaneous and pancreatic orthotopic injection models, C1GALT1 knockdown decreased tumor growth and metastasis of PDAC cells in NOD/SCID mice. Mechanistically, C1GALT1 knockdown dramatically suppressed ECM adhesion, which was associated with decreased phosphorylation of FAK at Y397/Y925 and changes in O-glycans on integrins including the β1, αv, and α5 subunits. Using functional blocking antibodies, we identified integrin αv as a critical factor in C1GALT1-mediated invasiveness of PDAC cells. C1GALT1 knockdown inhibited apoptosis-related activities and inhibited phosphorylation of several RTKs in PDAC cells. In conclusion, this study not only reveals that C1GALT1 could be a potential therapeutic target for PDAC but also provides novel insights into the role of O-glycosylation in the α subunits of integrins.