胃癌是世界上最常見的惡性腫瘤之一，根據 Lauren分類，胃癌分為腸道型intestinal-type gastric cancers (IGC) 及瀰漫型diffuses-type gastric cancers (DGC)兩大類。 新興的胃癌全基因檢測根據基因特徵的差異把胃癌定義為四大類：艾伯斯坦-巴爾病毒型(Epstein–Barr virus)、微衛星不穩定型(microsatellite instability)、染色體不穩定型(chromosomal instability)及基因穩定型(genomically stable)。其中，瀰漫型胃癌大部分屬於基因穩定型，而當中約10%在RhoA基因上帶有點突變。RhoA是一個潛在性致癌基因，負責調控細胞生長及移動。但是，這個特別在瀰漫型胃癌發生突變的RhoA基因在胃癌進程中扮演的角色仍然十分不清楚。在這個研究中，我們著重在分析從臺灣林口長庚醫院的99個胃癌檢體中RhoA基因的點突變，而且其中兩個瀰漫型胃癌的病人在RhoA基因上都帶有Y42C點突變。我們發現在瀰漫型胃癌細胞株HGC-27、MKN45及SNU601中剔除RhoA基因的表現能抑制腫瘤細胞生長。此外，在瀰漫型胃癌細胞株AGS中剔除RhoA基因的表現更會抑制腫瘤細胞的遷移。相反，在AGS細胞中大量表現外來的RhoAY42C突變基因比起RhoAwt野生型基因更促進腫瘤細胞生長及移動。相較於ROCK激酶，剔除mDia1/2基因的表現才能抑制RhoAY42C突變基因引起的細胞生長及移動。整體而言，我們的研究結果提出RhoAY42C突變基因對於胃癌細胞生長及轉移扮演重要的角色。

Gastric cancer (GC) is one of the most common malignancies in the world. According to Lauren’s classification, gastric cancer divided into intestinal-type (IGC) and diffuses-type gastric cancers (DGC). Emerging genome-wide characterization of GCs reveals four subtypes of mutational signatures (Epstein–Barr virus-associated, microsatellite instability, chromosomal instability, and genomically stable signatures). Of those, DGC belongs to genomically stable signatures and ~10% of DGC cases carry mutation of RhoA, a potential driver gene that regulates the cell proliferation and/or cell migration in cancer cells. However, the underlying mechanisms of a specific RhoA mutation that contributes to gastric carcinogenesis remain largely unknown. In this study, we focus on characterization of SNPs of RhoA from DGC (n = 99) collected at CGMH. Of those, two were identified had the Y42C mutation. Knock down of RhoA in MKN45 significantly reduced the survival rate. Furthermore, knock down of RhoA in AGS cells had a significantly reduced level of migratory activity. Conversely, overexpression of exogenous RhoAY42C in AGS significantly increased the cell mobility compared with RhoAwt. Knock down the RhoA effector mDia1/2 perturbed the increased cell mobility mediated by RhoAY42C. Our results collectively point out a critical role of RhoAY42C in gastric cancer invasiveness.

Chapter 1 Introduction ........................................8
1.1 Gastric cancer .........................................8
1.2 Classification of gastric cancer .....................8
1.3 Recurrent mutation in diffuse-type gastric cancer–RhoA..8
1.4 Small Rho GTPase family RhoA ...........................9
1.5 Downstream effectors of RhoA ...........................10
Chapter 2 Materials and Methods ...............................12
2.1 Patients and tumor characteristics .....................12
2.2 Cell culture and antibodies ............................12
2.3 Genomic DNA extraction .................................12
2.4 Polymerase Chain Reaction, DNA purification of PCR product
and Sequencing ...............................................13
2.5 Real-time quantitative reverse transcription PCR .......13
2.6 Lentiviral shRNA knockdown and vector transfection .....14
2.7 MTT assay .............................................14
2.8 Wound healing assay/Scratch assay ......................15
2.9 Transwell migration and invasion assay .................15
2.10 Time-lapse cell imaging ...............................15
2.11 Soft-agar assay .......................................16
2.12 Western-blotting .....................................16
2.13 Statistics methods ....................................16
Chapter 3 Results .............................................18
3.1 Clinical relevance of RhoA ................................18
3.2 The presence of the RhoAY42C mutation in diffuse-type gastric
cancer in Taiwan ..............................................18
3.3 Knockdown of RhoA reduced proliferation and migratory
activity ......................................................19
3.4 RhoAY42C upregulated the migratory activity ...............20
3.5 RhoAY42C enhanced ability of anchorage-independent growth..21
3.6 RhoAY42C exhibited a significant portion of a blebbing
morphology ....................................................21
3.7 Depletion of mDia1/2 in AGS-mock/RhoAwt/ RhoAY42C cells
reduced proliferation, migration and invasion .................21
3.8 Down-regulation of mDia1/2 impaired ability of anchorage-
independent growth ............................................22
3.9 Down-regulation of ROCK1 impaired RhoAY42C -mediated membrane
blebbing ......................................................22
Chapter 4 Discussion ..........................................23
Chapter 5 Figures .............................................26
Chapter 6 Tables ..............................................48
Chapter 7 Reference ...........................................54

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