摘要:神經母細胞瘤是幼兒期最常見的惡性腫瘤。大約60%的神經母細胞瘤病患在診斷時已經是第四期,他們的預後非常差,五年存活率不到30%。目前神經母細胞瘤的病理成因仍然不明,多數人認為有可能是胚胎神經母細胞無法分化或無法凋亡所造成。蛋白質正常的醣化對於神經系統的發育十分重要。胚胎神經母細胞醣化異常有可能導致細胞的異常發展及神經母細胞瘤的產生。β1,4-N-aceylgalactosaminyltransferaseIII (B4GALNT3)具有形成GalNAcβ1,4GlcNAc (LDN)結構的能力,過去已有文獻証明果蠅的B4GALNTA (等同人類B4GALNT3)可以調控神經發育。藉由免疫染色,我們發現B4GALNT3 的表現和神經母細胞瘤的組織分化程度成正比,且B4GALNT3 在腫瘤組織中的陽性表現可以預測較好的預後,而B4GALNT3 對神經母細胞瘤細胞分化的影響可能部份透過醣化β1-integrin 達成。這些證據顯示醣類轉移酵素可能在神經母細胞的腫瘤形成扮演重要的角色。為了進一步探討醣類轉移酵素在神經母細胞腫瘤形成的角色,本計劃將提出三個重要的工作目標:一、確認B4GALNT3 在生物體內體外對神經母細胞的影響。神經母細胞在過度表達或抑制B4GALNT3 後細胞行為的改變將加以記錄。並用這些細胞株建立小鼠腫瘤模型,以檢測B4GALNT3 對腫瘤生長的影響。二、確認被B4GALNT3 所醣化的蛋白質及其下游相關的訊息傳導路徑。控制組及過度表達B4GALNT3 的細胞株將以醣化蛋白質體學比較其差異醣化蛋白,以找出B4GALNT3 的目標醣化蛋白質。當找到可能的目標蛋白質後,其下游的訊息傳遞路徑也會加以檢測。在β1-integrin 部分也將進ㄧ步確認是否受到B4GALNT3 所醣化,並產生功能性影響。三、確認B4GALNT3 在細胞株腫瘤組織中基因表現的調控。我們將檢測B4GALNT3 在腫瘤組織中基因的甲基化情況。另外B4GALNT3 表現量不同的組織將以micro RNA array 加以評估其中microRNA 的差異。總結而言,我們的研究不僅可以進一步確定醣類轉移酵素B4GALNT3 對神經母細胞行為的影響,並且可以了解B4GALNT3 在神經母細胞中的表現是如何被調控,而B4GALNT3 又是如何來影響神經母細胞的細胞行為的。我們的研究將有助於對神經母細胞腫瘤形成原因的了解,同時也可能有助於對神經母細胞瘤標靶治療的發展。
Abstract: Neuroblastoma (NB) is the most common malignant tumor of infancy. The mechanismunderlying the tumorigenesis of NB remains largely unclear. It has been suggested that thepathogenesis of NB is due to a failure of differentiation or apoptosis of the embryonic NBcells. Well-regulated glycosylation is essential for the normal development of the nervoussystem. Altered expression of glycosyltransferases with resulting dysregulated glycosylationof neuroblastic cells might lead to the development of NB. Theβ1,4-N-acetylgalactosaminyltransferase III (B4GALNT3) exhibits GalNAc transferaseactivity to form the GalNAcβ1,4GlcNAc (LacdiNAc or LDN) structure. The DrosophilaB4GALNTA, homolog of human B4GALNT3, has been suggested to regulate the neuronaldevelopment. By immunohistochemical studies, we demonstrated that the expression ofB4GALNT3 correlated well with histological grade of differentiation in 87 NB tumor samples.In addition, positive B4GALNT3 expression predicted a favorable patient’s outcome.Furthermore, B4GALNT3 could possibly regulate NB cell differentiation throughglycosylation of β1-integrin. These lines of evidence suggest that the regulation ofglycosyltransferases is critical for the development of NB.To further explore the role of B4GALNT3 in the differentiation and development of NB,we propose a 3-year project with the following 3 major aims:Aim Ⅰ: Clarifying the effects of B4GALNT3 on NB cell behavior in vitro and invivo. For further understanding the effects of B4GALNT3 on NB cells, NB cells with stableoverexpression of B4GALNT3 are to be selected. Then NB cell phenotype and behaviorchanges after overexpression of B4GALNT3 are evaluated by in vitro assays as well as by anude mice xenograft model. In addition, the expression of B4GALNT3 will be suppressed bysiRNA, then the response of NB cells to ATRA-induced differentiation is evaluated.Aim Ⅱ: Clarifying the target proteins glycosylated by B4GALNT3 as well as theirassociated downstream pathways changes. The possible proteins glycosylated byB4GALNT3 are evaluated by comparing differential protein expressions betweenB4GALNT3-transfected and mock-transfected NB cells using glycoproteomics analysis.After identifying the target proteins modified by B4GALNT3, the associated downstreampathways to affect NB cell differentiation will also be evaluated. In addition, whetherβ1-integrin is glycosylated by B4GALNT3 with downstream functional changes will also beverified.Aim Ⅲ: Clarifying how the expression of B4GALNT3 is controlled epigeneticallyin human NB cell lines and tumor samples. The methylation status of the promoter sites ofB4GALNT3 is examined in various NB cell lines as well as in tumor samples. Furthermore,NB tumor samples exhibiting high and low B4GALNT3 levels are subjected to microRNAarray.Altogether, our studies will not only establish the functional role of B4GALNT3 in the cellbehavior of NB, but also illustrate how the expression of B4GALNT3 is regulatedepigenetically and how the B4GALNT3 affects NB cell behavior. Therefore, our resultsmight shed light to the oncogenesis of NB as well as target therapy of NB.