摘要:此計劃的目標是要了解肝癌的形成和其化療抗藥性的機制。腫瘤醣類抗原,包括Tn抗原,近年來引起了國際的注意,並將其發展成為新型的癌症診斷試劑和疫苗。然而,造成這些抗原表現的基因和這些基因在生理與病理上的角色仍不清楚。O-glycosylation的第一步驟是將UDP-GalNAc轉移至serine (S)或threonine (T)的羥基上,形成Tn 抗原(即:GalNAcα-S/T結構)。進行此反應的酵素是GALNT家族的成員,在哺乳類細胞中,目前已知至少有14種,命名為:GALNT1至14。不同的GALNT基因會在不同的組織及發育時間中表現。很多報告指出O-glycans 和GALNT基因在生物功能和人類疾病的重要性。然而,GALNT基因在肝癌中的表現和其所扮演的角色仍然不清楚。我們初步的實驗結果顯示,GALNT2可以修飾重要的細胞表面受體EGFR和MUC1,而且能調控肝癌細胞的生存、轉移、和入侵能力。很重要的,我們也發現GALNT2能夠調控許多訊息傳遞,包括ERK, AKT和FAK。因此,我們假設GALNT基因可以調控肝癌之生長與轉移能力,以及其化療的抗藥性我們的特定目標有以下幾點:1. 藉由real-time PCR,西方墨點,和免疫組織化學染色法,分析GALNT1和GALNT2在肝癌組織的表現。另外,我們會分析其表現量與肝癌臨床特性的關連性。2. 研究GALNT1和GALNT2對肝癌細胞株的影響,包括增生、凋亡、聚落形成能力、黏附、移動,和化療抗藥性。我們使用的肝癌細胞株將包括:PLC5、HepG2、Hep3B和Huh7。3. 在免疫缺陷小鼠實驗模式中研究GALNT1和GALNT2對腫瘤生長、血管新生,和轉移能力的影響。4. 了解經由GALNT1和GALNT2調控腫瘤惡性程度的分子機制,並分析GALNT1 和GALNT2所影響的訊息傳遞。5. 經由醣蛋白質體學鑑定因GALNT1或GALNT2 過量表現所作用的細胞表面分子為何,並研究這些分子在調控肝癌細胞行為的重要性。此結果將使我們更瞭解GALNT1和GALNT2如何影響肝癌細胞行為的分子機制。這將是第一個研究GALNT基因家族在肝癌中所扮演的生物和病理角色,此研究結果將有助於開發新的肝癌診斷試劑和治療的方式。
Abstract: The objective and long-term goal of this proposal is to understand the underlying mechanisms of hepatocellular carcinoma (HCC) development and chemoresistance. Tumor-associated carbohydrate antigens including Tn antigen have attracted international attention to develop diagnostic reagents and vaccines for cancer therapy. However, the genes responsible for the expression of these antigens and their pathophysiological roles in human cancers are largely unknown.O-glycosylation is initiated by the transfer of UDP-GalNAc to the hydroxyl group of a serine (S) or threonine (T) residue to form Tn antigen (GalNAcα-S/T). This reaction is catalyzed by a large family of polypeptide GalNAc transferases (GALNTs), which consists at least 14 members in humans, namely GALNT1~14. They are differentially expressed in various tissues in a spatiotemporal-dependent manner. Many reports have demonstrated the importance of O-glycans and GALNT genes in a variety of biological functions and human diseases. However, the expression patterns of GALNTs and their role in HCC are still unknown. Our preliminary data showed that GALNT2 can modify important cell surface receptors EGFR and MUC1 and regulate cancer cell viability, migration, and invasion. Importantly, signaling pathways including ERK, AKT, and FAK were significantly modulated by GALNT2. We therefore hypothesize that GALNT genes can regulate tumor growth and metastasis, and modify cancer chemoresistance of HCC.Our specific aims are:1. To analyze the expression patterns of GALNT1 and GALNT2 in primary HCC tissues by real-time PCR, Western blot, and immunohistochemistry. Furthermore, we will correlate the expression levels of GALNT1 and GALNT2 with clinical characteristics of HCC patients.2. To investigate the in vitro effects of GALNT1 and GALNT2 on HCC cell lines. We will analyze phenotypes including cell proliferation, apoptosis, colony-forming ability, adhesion, migration, invasion, and chemoresistance. The HCC cell lines we will use are PLC5, HepG2, Hep3B, and Huh7.3. To investigate the in vivo effects of GALNT1 and GALNT2 on tumor growth, angiogenesis, and metastasis in immunodeficient mice models.4. To investigate the molecular mechanisms by which GALNT1 and GALNT2 regulate tumor malignancy. Signaling pathways related to the phenotypic changes affected by GALNT1 and/or GALNT2 will be analyzed.5. To identify the cell surface molecules with increased Tn structure due to GALNT1 and/or GALNT2 overexpression by proteomics. The significance of the identified molecules with altered O-glycans in regulating cell behavior will be further investigated. The obtained information is essential for understanding the molecular mechanism by which GALNT1 and/or GALNT2 affects cancer phenotypes.This will be the first study to investigate the biologic and pathologic role of GALNT genes in HCC. The information gained from this study will provide pharmaceuticals to develop novel diagnostic and therapeutic reagents for HCC therapy.