醣類轉換酵素C1GALT1在胃癌之功能角色

Abstract

胃癌是全球重要的惡性腫瘤之一。在2018年，全球胃癌新診斷人數為一百萬名患者，排位在所有癌症的第五名。全球當年因為胃癌而死亡的人數高達78萬3千人，是因癌症導致死亡人數的第三位，僅次於肺癌與大腸癌，甚至高於眾所熟知的肝癌和乳癌。在台灣，胃癌也是十大致死癌症的第七位。 生物體基因體數量的多寡，並不足以解釋生物個體功能複雜的程度。蛋白質轉錄後修飾(post-translational modification)是蛋白質的表現及功能多樣化非常重要的關鍵步驟。醣基化 (glycosylation) 即是蛋白質轉錄後修飾中一種重要的方式。其中O-聚醣醣基化是醣基化五種重要的醣基化方式之一，影響了各種生長激素的調控與免疫系統的調節。 細胞醣基化 (glycosylation)的異常會造成許多腫瘤的發生並影響許多腫瘤細胞的特性。核心黏蛋白型O-聚醣合成的醣類轉換酵素core 1 β1,3-galactosyltransferase（C1GALT1）是控制GalNAc型 O-醣基化的關鍵步驟，影響及於許多生理及病理狀態，甚至癌症的惡性行為表現。 促紅細胞生成素產生肝癌細胞EPH (erythropoietin- producing human hepatocellular carcinoma)受體是受體蛋白酪胺酸激酶RTKs (receptor tyrosine kinases)最大的家族受體，調控著多種生理病理的發展過程，與許多疾病息息相關。然而，C1GALT1在EPH受體的訊號傳遞過程中所扮演的角色，過去卻未有學者深入研究探討。 在我們的研究中發現C1GALT1在胃癌細胞中的過度表現與多樣的臨床病理特徵有關，同時也是臨床上一個重要的不良預後因子。基因敲落 (knockdown)或基因剔除 (knockout) C1GALT1會抑制胃癌細胞株的活性、移行、侵襲性、腫瘤的生長及腫瘤遠端轉移。 藉由RTK磷酸化微陣列晶片以及西方墨點法分析，缺乏C1GALT1會抑制游離的Ephrin A1-Fc所誘發EPHA2的酪胺酸磷酸化表現。EPHA2上的O-聚醣分子會受到C1GALT1的調控；藉由EPHA2上兩個醣化位置S277A及T429A的突變，也顯著的提高了EPHA2的酪胺酸磷酸化表現。這樣的結果顯示，不僅全面的O-聚醣結構會調控著EPHA2的活性，單一的O-醣基化的特定位置也影響著EPHA2的活性。 此外，缺乏C1GALT1會降低因Ephrin A1-Fc所誘發的細胞移行並減少Ephrin A1與細胞表面受體結合。在活體內或是試管中的實驗中發現，藉著將胃癌細胞株上的EPHA2基因剔除，也會如同基因敲落 (knockdown)或基因剔除 (knockout) C1GALT1般，抑制胃癌細胞株細胞的侵襲性表現。 這些結果顯示C1GALT1可以藉由修飾EPHA2的-O醣基化作用，促進EPHA2的磷酸化以及提高EphrinA1所媒介的移行表現。我們的實驗結果突顯了在EPH受體調控的疾病上，GalNAc型 O-醣基化的重要性，更進一步地闡述了C1GALT1是未來研究胃癌治療的潛在目標。

Glycosylation is an important post-translational protein modification whereby sugar groups are added to target proteins. Based on the site of attachment, there are two major types of glycosylation: N-glycosylation and O-glycosylation. Mucin-type O-glycosylation is characterized by the initial addition of N-acetylgalactosamine (GalNAc) onto the hydroxyl group of serine or threonine residues. These glycans are abundant on mucins, proteins typified by repeating domains rich in proline, threonine, and serine (PTS domains). The extensive O-glycosylation occurring within these repeating domains serves to extend the protein backbone, transforming it from globular to extended rod-like structure. C1GALT1 (Core 1 synthase, glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase 1) controls a crucial step of GalNAc-type O-glycosylation in both physiological and pathological settings, including cancer. EPH (erythropoietin-producing human hepatocellular carcinoma) receptors comprise the largest family of receptor tyrosine kinases (RTKs) and modulate diverse developmental and pathological processes. However, the role of C1GALT1 in EPH signaling has been largely overlooked. In 2018, gastric adenocarcinoma was the third leading cause of cancer-related death worldwide. Here , we showed that C1GALT1 is highly expressed in gastric adenocarcinoma; its overexpression correlated with adverse clinicopathological features and was an independent prognostic factor for poor overall survival. Silencing or loss of C1GALT1 inhibited cell viability, migration, invasion, tumor growth, and metastasis. Phospho-RTK arrays and western blot analysis demonstrated that C1GALT1 depletion suppresses soluble ephrin A1-Fc -induced tyrosine phosphorylation of EPHA2. We also found that O- glycans on EPHA2 are modified by C1GALT1, and that EPHA2 variants with mutated S227 and T429 (known O-glycosites) display dramatically enhanced phosphorylation at Y588. These findings imply that not only overall O-glycan structures but also site-specific O-glycosylation can regulate EPHA2 activity. Furthermore, depletion of C1GALT1 decreased ephrin A1-Fc-induced cell migration and reduced the binding of ephrin A1 to the cell surface. The effects of C1GALT1 knockdown or knockout on gastric cancer cell invasiveness in vitro and in vivo were phenocopied by EPHA2 knockdown. Collectively, these results suggest that C1GALT1 promotes the phosphorylation of EPHA2 and enhances soluble ephrin A1-mediated cell migration, primarily by modifying O glycosylation of EPHA2. Our study highlights the importance of GalNAc-type O-glycosylation in EPH receptor-regulated diseases and identifies C1GALT1 as a potential therapeutic target for gastric cancer.