摘要:此計劃的長遠目標是要了解肝癌的形成和其產生抗藥性的機制。腫瘤醣類抗原,包括Tn與T抗原,經常與癌症病人的預後有關連,因此引起了國際的注意,並將其發展成為新型的癌症診斷試劑和疫苗。然而,造成這些醣類抗原表現的基因以及這些基因在生理與病理上的角色仍然很不清楚。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,在人類細胞中,目前已知有20種,命名為:GALNT1至14以及GALNTL1至6。這些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。我們最近研究(Cancer Res. 2011 Dec 1;71:7270-9, IF 8.2)發現GALNT1和GALNT2為肝組織中表現最高的GALNT基因,GALNT2在肝癌組織中表現量是下降的,而且GALNT2藉由修飾EGF受體的O-glycosylation而抑制肝癌的惡性程度。我們最近的初步的實驗結果顯示,GALNT1卻和GALNT2有截然不同的特性,GALNT1在肝癌組織中表現量是上升的,GALNT1表現高的病人預後較差。以siRNA抑制GALNT1表現,可以抑制肝癌細胞的惡性程度並增強sorafenib藥物的效果。因此,我們假設 GALNT1可以調控肝癌發展的進程以及藥物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、上皮-間質轉化,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對腫瘤生長、血管新生,和轉移能力的影響。4. 研究經由GALNT1調控腫瘤惡性程度的分子機制,並分析GALNT1 所影響的訊息傳遞。5. 經由醣蛋白質體學鑑定因GALNT1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如何影響肝癌細胞特性的分子機制。這將是第一個探討GALNT1在肝癌中所扮演的生物和病理角色的研究,此研究結果將有助於開發新型的肝癌診斷試劑和治療的方式。
Abstract: The objective and long-term goal of this proposal are to understand the mechanisms of hepatocellular carcinoma (HCC) development and drug resistance. Tumor-associated carbohydrate antigens, such as Tn and T antigens, are often associated with poor prognosis of cancer patients and 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 unclear.Mucin-type 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 of 20 members in humans, namely GALNT1 to 14 and GALNTL1 to 6. 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 unclear. Very recently, we found that GALNT1 and GALNT2 are expressed at the highest level in liver tissues (Cancer Res. 2011 Dec 1;71:7270-9, IF 8.2). In addition, GALNT2 is down-regulated and its re-expression suppresses the malignant character of HCC by modifying the O-glycosylation and activity of EGF receptor. Surprisingly, our preliminary data showed that GALNT1 exhibited an opposite effect on HCC malignant phenotypes compared with GALNT2. We found that GALNT1 was up-regulated in primary HCC and high levels of GALNT1 mRNA expression predicted poor survival. Our preliminary data also showed that knockdown of GALNT1 inhibited malignant phenotypes and sensitized HCC cells to sorafenib, an approved drug for targeted therapy. We therefore hypothesize that GALNT1 can regulate HCC progression and drug sensitivity.Our specific aims are:1. To analyze the expression patterns of GALNT1 in primary HCC tissues by real-time PCR, Western blot, and immunohistochemistry. Furthermore, we will correlate the expression levels of GALNT1 with clinical characteristics of HCC patients.2. To investigate the in vitro effects of GALNT1 on multiple HCC cell lines. We will analyze phenotypes including cell proliferation, apoptosis, colony-forming ability, adhesion, migration, invasion, epithelial-mesenchymal transition (EMT), and drug sensitivity. The HCC cell lines include PLC5, HepG2, Hep3B, and Huh7.3. To investigate the in vivo effects of GALNT1 on tumor growth, angiogenesis, and metastasis in immunodeficient mice models.4. To investigate the molecular mechanisms by which GALNT1 regulate tumor malignant properties. Signaling pathways related to the phenotypic changes affected by GALNT1 will be analyzed.5. To identify the cell surface molecules with increased Tn structure due to GALNT1 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 affects cancer phenotypes.This will be the first study to investigate the biologic and pathologic role of GALNT1 in HCC. The information gained from this study will provide pharmaceuticals to develop novel diagnostic and therapeutic reagents for HCC therapy.