肝癌衍生生長因子 (Hepatoma-Derived Growth Factor；HDGF)可以刺激肝癌細胞 (Hepatocellular carcinoma；HCC)有絲分裂，它由240個胺基酸組成，並且有2個核定位訊息序列(Nuclear Localization Signal；NLS)，分別位於C140 domain及PWWP domain。對於HDGF的有絲分裂活性來說，磷酸位點Ser103的磷酸化是非常重要的。過去的研究指出，在肝癌患者中，HDGF和活性氧 (Reactive Oxygen Species；ROS)的表現量會隨著癌化的進程增加，此外，HDGF和nucleolin之間的交互作用也會促進肝癌癌化。因此，在本研究中我們探討了在肝癌細胞中HDGF與ROS生成的關聯性。研究結果發現，HDGF及其C140片段和HDGF S103E蛋白能夠顯著的刺激SK-HEP1肝癌細胞的粒線體ROS生成及促進癌細胞的增殖和侵犯能力。而PWWP片段和HDGF S103A蛋白在促進ROS生成和癌化的功能是較弱的。另外，我們發現使用ROS抑制劑NAC (N-acetyl-L-cysteine) 阻斷ROS生成時HDGF促進癌細胞生長和侵犯的能力也跟著被抑制。接著我們使用抗體中和了HDGF的表面受體Nucleolin (NCL)，發現HDGF蛋白和C140片段的功能幾乎完全被阻斷，但HDGF S103E蛋白的功能只有些許的降低。由於HDGF誘導產生的ROS會使得 Superoxide dismutase 1、2 (SOD1、SOD2 )、 Mitofusin-2 (MFN2)、Peroxisome proliferator activated receptor gamma co-activator 1 (αPGC1α ) 表現量上升，Mitochondrial fission 1 protein (FIS1)、Dynamin-related protein 1(DRP1)表現量下降，因此我們推測肝癌細胞為了保護自身不受氧化壓力的影響，會增加抗氧化蛋白質並促進粒線體合成。總結以上結果，此研究證明了HDGF會刺激肝癌細胞的粒線體產生ROS並促進細胞的癌化。

Hepatoma-derived growth factor (HDGF) is a mitogen of 240 residues involved in hepatocarcinogenesis. HDGF consists of a highly conserved PWWP domain in the N-terminal 100 residues and the C140 domain with a nuclear localization signal (NLS) that located in C140 domain and PWWP domain respectively. Moreover, the Ser-103 phosphorylation site is necessary for mitogenic activity of HDGF. Studies have shown that HDGF expression and ROS levels increase as HCC progression from Grade Ι to Grade III in HCC patients. Additionally, the interaction of HDGF and nucleolin also promotes liver carcinogenesis. Therefore, we want to investigate the correlation between HDGF and ROS generation in hepatocellular carcinoma. In this study, we found that wild type HDGF protein, C140 domain and S103E protein enhanced ROS production, proliferation and invasion of SK-Hep-1 hepatoma cells. However, the effect of PWWP domain and S103A mutant of HDGF on inducing cell proliferation, invasion and ROS generation was weaker than C140 domain and S103E mutant of HDGF. Blocking ROS generation by NAC attenuated HDGF-induced cell growth and invasion. We further clarified that the function of HDGF protein and C140 domain could be blocked by neutralizing the cell surface dominant receptor nucleolin (NCL), but the capacity for blockage of S103E activity was diminished. Up-regulation of SOD1, SOD2, MFN2 and PGC1α as well as down-regulation of FIS-1 and DRP-1 suggested that HDGF-induced ROS might increase antioxidant proteins to detoxify from the elevated ROS and stimulate mitochondrial biogenesis in HCC. In conclusion, we demonstrated that HDGF-induced mitochondrial ROS generation provoked liver cancer progression.

論文審定書 i
誌謝 ii
中文摘要 iii
Abstract v
Figures and Legends viii
Introduction 1
Materials and methods 9
Results 15
Discussion 25
Figures and legends 30
References 57
Appendix 62

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7 Wu, Q. et al. Chemoresistance to gemcitabine in hepatoma cells induces epithelial-mesenchymal transition and involves activation of PDGF-D pathway. Oncotarget 4, 1999-2009, doi:1471 [pii]10.18632/oncotarget.1471 (2013).
8 Hu, T. H. et al. Expression of hepatoma-derived growth factor in hepatocellular carcinoma. Cancer 98, 1444-1456, doi:10.1002/cncr.11653 (2003).
9 Kishima, Y. et al. Hepatoma-derived growth factor stimulates cell growth after translocation to the nucleus by nuclear localization signals. J Biol Chem 277, 10315-10322, doi:10.1074/jbc.M111122200M111122200 [pii] (2002).
10 Wang, C. H. et al. Cell surface heparan sulfates mediate internalization of the PWWP/HATH domain of HDGF via macropinocytosis to fine-tune cell signalling processes involved in fibroblast cell migration. Biochem J 433, 127-138, doi:10.1042/BJ20100589BJ20100589 [pii] (2011).
11 Zhang, J. et al. Down-regulation of hepatoma-derived growth factor inhibits anchorage-independent growth and invasion of non-small cell lung cancer cells. Cancer Res 66, 18-23, doi:66/1/18 [pii]10.1158/0008-5472.CAN-04-3905 (2006).
12 Nakamura, H. et al. Molecular cloning of complementary DNA for a novel human hepatoma-derived growth factor. Its homology with high mobility group-1 protein. J Biol Chem 269, 25143-25149 (1994).
13 Everett, A. D., Yang, J., Rahman, M., Dulloor, P. & Brautigan, D. L. Mitotic phosphorylation activates hepatoma-derived growth factor as a mitogen. BMC Cell Biol 12, 15, doi:10.1186/1471-2121-12-151471-2121-12-15 [pii] (2011).
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