針對進行胃癌（advanced gastric cancer）的標靶治療是需要的。在這項研究中，我們專注於探討組蛋白賴胺酸去甲基酶（KDM）的在胃癌疾病進程的角色，該酶的功能為從組蛋白上已有甲基的賴胺酸中除去甲基，進而重塑染色質之構型。從OncomineTM數據庫分析顯示，在胃癌組織中組蛋白賴胺酸去甲基酶7B（KDM7B即PHF8）表現量相對於正常組織有明顯升高的情形，此外我們也從Kaplan-Meier Plotter的分析中發現，胃癌患者若有高表現的PHF8，則其總體存活率與疾病首次進程的表現都是較差的。PHF8的剔減顯著降低了胃癌細胞株和動物疾病模型的癌症進展。從基因微陣列分析結果中，我們發現PHF8主要調控的基因群大多屬於細胞遷移/移動的的類別，值得注意的是，PHF8會在PRKCA（PKCα蛋白的基因）啟動子上與轉錄因子c-Jun相互作用共同調控該基因的表現，而PHF8或PKCα的剔減會大幅度提升PTEN表達，而這個現象可以透過異位表達PKCα表達載體或激活的Src來回復，這也清楚的表明，PTEN表現量的調控是透過PKCα–Src影響。在針對胃癌患者的回溯性的免疫組織化學染色分析也發現PHF8、PKCα和PTEN之間存在顯著相關性：PHF8和PKCα間呈正相關；而PHF8與PTEN之間以及PKCα與PTEN間呈負相關，並且，PHF8的表現量與腫瘤分期呈顯著正相關。使用PKCα抑制劑Midostaurin （2017獲FDA批准）和Src抑制劑Bosutinib （2017年獲FDA批准）會導致PTEN表現量升高，並呈劑量相關性，可顯著抑制腫瘤在斑馬魚模型中侵襲轉移與在小鼠模型中生長。
綜上所述，我們的研究為晚期胃癌中仰賴PHF8的訊號途徑提供了新穎的見解，並對該患者的治療方針產生了潛在影響。

There is an urgent need for targeted treatments of advanced gastric cancer (GC). In this study, we focus on histone lysine demethylases that removes the methyl moiety from methyl-lysyl histones and thus engaging in chromatin remodeling. The OncomineTM database for GC patients has revealed an elevated level of PHD finger protein 8 (PHF8/KDM7B) expression in the tumor as compared to the normal tissue. In addition, through the Kaplan–Meier Plotter analyses, PHF8 is associated with a worse overall survival and first progression. In both in vitro and in vivo models, there is a significant reduction in the cancer progression by depleting PHF8. Based on microarray analysis, PHF8 was found to regulate genes that are involved in cell migration/motility. Noteworthily, PHF8 endogenously interacts with c-Jun on PRKCA promoter region and hence regulates of PKCα protein expression. Both the depletion of PHF8 or PKCα significantly upregulated PTEN expression, and this can be rescued by either the ectopic expression of PKCα or an active Src. These findings suggest that PTEN is destabilized via the PKCα–Src axis. IHC analysis of GC subjects showed that there was a significant correlation between PHF8, PKCα and PTEN: positive correlation between PHF8 and PKCα; negative correlation between PHF8 and PTEN and that between PKCα and PTEN. Additionally, PHF8 abundance was positively correlated with tumor stage. Moreover, the use of midostaurin, a PKCα inhibitor (FDA-approved since 2017), and bosutinib, a Src inhibitor (FDA-approved since 2017) led to an elevated level of PTEN in a dose-dependent manner in vitro and significantly curb tumor progression in zebrafish and murine models.
Altogether, our studies provide novel insights into the PHF8-mediated pathway in GC progression and show significant impacts in translational medicine in advanced GC patients.

致謝 i
中文摘要 iii
Abstract iv
Abbreviation v
List of Figures xi
List of Tables xiii
Chapter 1. Introduction 1
1.1 Epidemiology and risk factors of gastric cancer 1
1.2 Current treatments in gastric cancer 3
1.3 Classification of gastric cancer 5
1.4 Molecular characterization and genetic regulation of gastric cancer 7
1.5 Epigenetic regulation of gastric cancer 8
1.7 Protein Kinase C signaling 12
1.6 The objective of this study 14
Chapter 2. Material and methods 16
2.1 Antibodies, inhibitors and plasmids. 16
2.2 Cell culture 16
2.3 Establishment of stable cells 17
2.4 Immunoblotting and Immunoprecipitation assay 17
2.5 Cell proliferation assay and Trans-well migration assay 18
2.6 Microarray 19
2.7 RNA collection and quantitative real-time PCR (qRT-PCR) 19
2.8 Chromatin immunoprecipitation assay 20
2.9 Luciferase activity assay 21
2.10 Micro-western array 21
2.11 Mice xenograft and inhibitory experiment 22
2.12 Migration assay and chemoresponse assay in zebrafish 22
2.13 IHC and IHC scoring 23
2.14 Statistical evaluation 24
Chapter 3. Results 26
3.1 Elevated expression of PHF8 is associated with worse clinical outcomes in GC 26
3.2 Biological role of PHF8 in advanced GC cells in vitro and in vivo 26
3.3 PHF8 directly regulates PKCα and ICAM-1 in promoting GC progression 28
3.4 PHF8 binds to c-Jun and they are co-recruited to the promoter region of PRKCA 29
3.5 PHF8 promotes PTEN destabilization via PKCα-Src 31
3.6 Inhibition the PKCα-Src pathway in advanced GC 32
3.7 Characterization of PHF8, PKCα, and PTEN in human GC samples 33
Chapter 4. Conclusion and Discussion 35
4.1 PHF8-dependent epigenetic landscape in gastric cancer 35
4.2 PTEN deficiency in gastric cancer 38
4.3 Targeting PHF8-mediated PKCα-Src pathway in gastric cancer 39
4.4 PHF8-PKCα as prognostic biomarker in gastric cancer 41
4.5 Concluding remarks 42
References 43
Figures and Tables 60

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