臺灣博碩士論文加值系統

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.

口試委員會審定書 i
致謝 ii
中文摘要 iii
Abstract v
Chapter 1. Introduction 1
Chapter 2. Materials and Methods 6
2.1 Patients samples 6
2.2 Immunohistochemistry 6
2.3 Cell lines and cell culture 6
2.4 cDNA (complementary deoxyribonucleic acid) synthesis and RT-PCR (reverse transcriptase polymerase chain reaction) 7
2.5 Transfection and plasmid construction 7
2.6 Western blot analysis 8
2.7 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay 8
2.8 Transwell migration and Matrigel invasion assays 9
2.9 Phospho-receptor tyrosine kinase array assay 9
2.10 Lectin pull-down assay 9
2.11 Flow cytometry 10
2.12 cDNA microarray analysis 10
2.13 Knockout of C1GALT1 (Core 1 Synthase, Glycoprotein-N-Acetylgalactosamine 3-Beta-Galactosyltransferase 1) in MKN 45 cells using CRISPR/Cas9 system 11
2.14 In vivo xenograft animal models 11
2.15 Statistical analysis 11
Chapter 3. Results
3.1 C1GALT1 is overexpressed in gastric cancer 13
3.2 C1GALT1 expression correlates with poor survival in patients with gastric adenocarcinoma 13
3.3 C1GALT1 promotes malignant behaviors of gastric cancer cells 14
3.4 Effects of C1GALT1 on tumor growth and metastasis in NOD/SCID mice 15
3.5 Effects of C1GALT1 knockdown on multiple phospho(p)-RTKs (receptor tyrosine kinase) in gastric cancer cells 16
3.6 Impacts of C1GALT1 on Ephrin A1 (erythropoietin-producing human hepatocellular receptor-interacting protein A1) -triggered phosphorylation of EPHA2 (erythropoietin-producing human hepatocellular receptor A2) 17
3.7 Effects of EPHA2 knockdown on gastric cancer cells in vitro and in vivo 18
3.8 C1GALT1 mediates its pro-migratory effect on gastric cancer cells through EPHA2 19
3.9 Functional pathways affected by C1GALT1 knockdown 21
Chapter 4. Discussion 22
Chapter 5. Prospect 27
References 30
Tables 34
Table 1. Primers of PCR 34
Table 2. Baseline demographic and clinical characteristics of the 98 patients 35
Table 3. Correlation of C1GALT1 intensity and clinicopathologic features 36
Table 4. Univariate and multivariate Cox regression analysis for predictors of mortality 37
Table 5. Differential expression of selected genes in C1GALT1 knockdown AGS cells 38
Figures 39
Figure 1. C1GALT1 mRNA expression in normal and cancerous gastric tissues in the Oncomine database 39
Figure 2. C1GALT1 expression in paired gastric adenocarcinoma tumors. Immunohistochemical staining 40
Figure 3. Scoring of C1GALT1 expression analyzed using immunohistochemistry 41
Figure 4. Kaplan–Meier survival analysis according to the expression of C1GALT1 in gastric cancer patients 42
Figure 5. C1GALT1 expression in gastric cancer cells analyzed by Q-RT-PCR and western blot analysis 43
Figure 6. C1GALT1 modulates surface O-glycans on gastric cancer cells 44
Figure 7. Cell viability was analyzed using MTT assays 45
Figure 8. Cell migration was analyzed using transwell migration assays 46
Figure 9. Cell invasion was analyzed using Matrigel invasion assays 47
Figure 10. C1GALT1 knockdown enhanced 5-FU-induced apoptosis in AGS and MKN45 cells 48
Figure 11. (A) Western blots of stable C1GALT1 knockdown in AGS cells. (B) Tumor sizes and weights n NOD/SCID mice subcutaneously injected with AGS cells 49
Figure 12. C1GALT1 knockdown decreased lung metastasis in NOD/SCID mice intravenously injected with AGS cells 50
Figure 13. C1GALT1 knockout suppressed tumor growth in a NOD/SCID mouse model. 51
Figure 14. Tumor formation in NOD/SCID mice intraperitoneal injected with parental or C1GALT1 knockout MKN45 cell 52
Figure 15. Effects of C1GALT1 knockdown on multiple phospho-RTKs 53
Figure 16. C1GALT1 knockdown inhibited phosphorylation of EGFR, HER2, and AKT (western blot analysis) 54
Figure 17. HER2 was O-glycosylated 55
Figure 18. C1GALT1 knockdown modified O-glycans on HER2 and decreased phosphorylation of HER2 in N87 cells. (western blot analysis) 56
Figure 19. C1GALT1-mediated cell growth is suppressed by lapatinib 57
Figure 20. Soluble Ephrin A1 is present in conditioned medium of gastric cancer cells 58
Figure 21. Phospho-RTK array analysis showing effects of C1GALT1 knockdown on levels of phospho-RTKs in AGS cells treated with Ephrin A1-Fc 59
Figure 22. C1GALT1 knockdown-mediated decrease in phosphorylation of EGFR and MET is not the downstream of the Ephrin A1-EPHA2 signaling pathway. 60
Figure 23. EPHA2 was the predominant EPH receptor for Ephrin A1-Fc in AGS and MKN45 cells 61
Figure 24. C1GALT1 knockdown or knockout decreased phosphorylation of EPHA2 at Tyr588 in AGS and MKN45 cells (western blot analysis) 62
Figure 25. C1GALT1 could modify the Tn antigen expression on EPHA2 in AGS and MKN45 cells 63
Figure 26. C1GALT1 knockdown increases VVA binding to EPHA2 after removal of N-glycans 64
Figure 27. Mutations in O-glycosylation sites, S277 and T429, on EPHA2 modulated phosphorylation of EPHA2 65
Figure 28. Cell viability of EPHA2 knockdown in AGS and MKN45 cells analyzed by MTT assays 66
Figure 29. Cell migration of EPHA2 knockdown in AGS and MKN45 67
Figure 30. Tumor sizes and weights of EPHA2 knockdown in NOD/SCID mice subcutaneously injected with AGS cells 68
Figure 31. Lung metastasis of EPHA2 knockdown in NOD/SCID mice with AGS cells 69
Figure 32. C1GALT1 mediates its pro-migratory effect on gastric cancer cells through EPHA2 70
Figure 33. Quantification of band intensities in Figure 32 71
Figure 34. Impacts of C1GALT1 or EPHA2 knockdown on signaling in gastric cancer cells 72
Figure 35. C1GALT1 knockdown in stable EPHA2 knockdown AGS and MKN45 cells 73
Figure 36. C1GALT1 knockdown inhibited Ephrin A1-Fc-triggered migration in AGS cells 74
Figure 37. Human IgG Fc does not trigger EPHA2 migration in gastric cancer cells 75
Figure 38. C1GALT1 knockdown and C1GALT1 knockout decreased binding of Ephrin A1-Fc to AGS and MKN45 cells 76
Figure 39. Volcano plot analysis of microarray of the 1491 probes in the C1GALT1 knockdown AGS cells 77
Figure 40. Functional map of differentially expressed genes. 78
Figure 41. Quantitative RT-PCR validation of the microarray results 79
Figure 42. C1GALT1 knockdown causes cell cycle arrest in gastric cancer cells. 80
Appendix 81

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