臺灣博碩士論文加值系統

惡性的黑色素癌在人類的癌症中是發展最為快速的惡性腫瘤，尤其好發於年輕人肝癌衍生誘導生長因子(hepatoma-derived growth factor; HDGF)，為一個新發現的生長因子，由人類肝癌細胞株 Huh-7的培養液中所純化出，HDGF的過度表現在和許多種類的癌症的初期診斷相關連，包括了黑色素癌。然而，在黑色素癌化過程中，有哪些機制會影響HDGF過度表現至今尚未明瞭。在本論文中，外加HDGF蛋白質會刺激B16-F10黑色素癌細胞侵入和形成聚落的能力。利用腺病毒當載體攜帶HDGF和干擾型HDGF (HDGF-RNAi)，在B16-F10細胞中產生作用後，會改變HDGF的表現。由實驗中發現，HDGF的過度表現會刺激B16-F10細胞增生、侵入及形成非附著性聚落的能力，而HDGF-RNAi則會抑制而產生相反的結果。在轉移到肺部的動物實驗中，利用尾靜脈注射的方式，打入有過度表現HDGF的黑色素癌細胞至老鼠體內，HDGF的過度表現能增加轉移的能力及減少HDGF的表現能有效的抑制轉移。相同的，在原位的黑色素癌動物實驗中，利用皮下注射的方式打入有過度表現HDGF的黑色素癌細胞至老鼠體內，HDGF的過度表現會促進腫瘤的生長且HDGF的減少能有效的抑制響腫瘤生長的速度及大小。組織切片染色和免疫螢光染色的分析中，在HDGF的過度表現下，黑色素癌有細胞增生、血管新生及細胞凋亡的情形。更多的證據顯示，HDGF的過度表現會促進從原位腫瘤轉移到淋巴結及肺的能力。最後，由結果中得知，HDGF的過度表現會改變nuclear factor kappa B (NFκB)， Akt phosphorylation，及其上下游之轉錄因子，例如: PI3K, PTEN, IκB及其家族 IKKα, IKKβ, IKKγ在黑色素癌中的活性，此外，在基因傳送後，HDGF的過度表現也能增加MITF及HIF1α在黑色素癌中的含量。且改變EMT對於黑色素癌的影響。綜合許多結果，HDGF的增加會促進黑色素癌的生長及轉移，利用血管新生的方式來增加癌細胞的生存。此外，中斷HDGF的表現對於黑色素癌的治療在現今是個新穎的方式。

Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression during melanoma carcinogenesis remains unclear. In this study, adding exogenous HDGF stimulated the invasion and colonies formation of B16-F10 melanoma cells. Adenovirus vectors encoding HDGF and HDGF-RNAi were generated and characterized to up- and down-regulated HDGF expression in B16-F10 melanoma cells. It was found that HDGF overexpression stimulated the proliferation, invasiveness, anchorage-independent growth of B16-F10 melanoma cells whereas HDGF knockdown exerted opposite effects. In lung-metastasis model, intravenous injection of HDGF-overexpressing melanoma cells resulted in increased metastasis while HDGF-downregulated melanoma cells caused decreased metastasis. Similarly, in primary melanoma model, subcutaneous injection of HDGF-overexpressing melanoma cells enhanced while HDGF-downregulated melanoma cells reduced the tumor burden in mice. Histological analysis revealed increased tumor proliferation and neovascularization with concomitant reduction of apoptosis in HDGF-overexpressing melanoma. Moreover, HDGF-overexpressing melanoma also exhibited enhanced propensity to metastasize from the primary tumors to lymph node and lung. Finally, it was found that HDGF overexpression increased nuclear factor kappa B (NFκB) activities and Akt phosphorylation up and down stream alternation like PI3K, PTEN, IκB and it’s subunit IKKα, IKKβ, IKKγ in melanoma cells. It also found that HDGF overexpression influenced MITF and HIF1α in melanoma after gene delivery. HDGF also altered EMT changes like E,N-cadherin, vimentin, and β,γ-catenin. The present study provides conclusive evidence that HDGF upregulation promotes the growth and metastasis of melanoma by promoting the survival and vascularization. Besides, HDGF knockdown may constitute a novel strategy for melanoma control.

CONTENTS Page
Abstract in Chinese 5
Abstract in English 6
Abbreviations 8
Introduction 9
Specific Aims 15
Materials and Methods 17
Results 29
Discussion 38
Figures and Legends 43
Appendix 76
References 81

1.de Vries E, Coebergh JW. Cutaneous malignant melanoma in Europe. Eur J Cancer 2004;40(16):2355-66.
2.Rigel DS, Carucci JA. Malignant melanoma: prevention, early detection, and treatment in the 21st century. CA: a cancer journal for clinicians 2000;50(4):215-36; quiz 37-40.
3.Grabacka M, Plonka PM, Urbanska K, Reiss K. Peroxisome proliferator-activated receptor alpha activation decreases metastatic potential of melanoma cells in vitro via down-regulation of Akt. Clin Cancer Res 2006;12(10):3028-36.
4.Amiri KI, Richmond A. Role of nuclear factor-kappa B in melanoma. Cancer metastasis reviews 2005;24(2):301-13.
5.Finn GJ, Creaven BS, Egan DA. Activation of mitogen activated protein kinase pathways and melanogenesis by novel nitro-derivatives of 7-hydroxycomarin in human malignant melanoma cells. Eur J Pharm Sci 2005;26(1):16-25.
6.Voelter V, Pica A, Laurent J, et al. An unusual case of metastatic melanoma sensitive to chemotherapy and immunotherapy, with late immune escape in the brain. Cancer Immun 2008;8:6.
7.Guo RR, Liu Y, Lu WL, et al. A recombinant peptide, hirudin, potentiates the inhibitory effects of stealthy liposomal vinblastine on the growth and metastasis of melanoma. Biological & pharmaceutical bulletin 2008;31(4):696-702.
8.Lopez-Bergami P, Fitchman B, Ronai Z. Understanding signaling cascades in melanoma. Photochemistry and photobiology 2008;84(2):289-306.
9.Aaronson SA. Growth factors and cancer. Science 1991;254(5035):1146-53.
10.McNamara DA, Harmey JH, Walsh TN, Redmond HP, Bouchier-Hayes DJ. Significance of angiogenesis in cancer therapy. Br J Surg 1998;85(8):1044-55.
11.Schirmacher P, Odenthal M, Steinberg P, Dienes HP. [Growth factors in liver regeneration and hepatocarcinogenesis]. Verhandlungen der Deutschen Gesellschaft fur Pathologie 1995;79:55-60.
12.Nakamura H, Izumoto Y, Kambe H, et al. Molecular cloning of complementary DNA for a novel human hepatoma-derived growth factor. Its homology with high mobility group-1 protein. The Journal of biological chemistry 1994;269(40):25143-9.
13.Nakamura H, Kambe H, Egawa T, et al. Partial purification and characterization of human hepatoma-derived growth factor. Clin Chim Acta 1989;183(3):273-84.
14.Klagsbrun M, Sasse J, Sullivan R, Smith JA. Human tumor cells synthesize an endothelial cell growth factor that is structurally related to basic fibroblast growth factor. Proceedings of the National Academy of Sciences of the United States of America 1986;83(8):2448-52.
15.Everett AD, Stoops T, McNamara CA. Nuclear targeting is required for hepatoma-derived growth factor-stimulated mitogenesis in vascular smooth muscle cells. J Biol Chem 2001;276(40):37564-8.
16.Hu TH, Huang CC, Liu LF, et al. Expression of hepatoma-derived growth factor in hepatocellular carcinoma. Cancer 2003;98(7):1444-56.
17.Bernard K, Litman E, Fitzpatrick JL, et al. Functional proteomic analysis of melanoma progression. Cancer research 2003;63(20):6716-25.
18.Zhang J, Ren H, Yuan P, Lang W, Zhang L, Mao L. Down-regulation of hepatoma-derived growth factor inhibits anchorage-independent growth and invasion of non-small cell lung cancer cells. Cancer research 2006;66(1):18-23.
19.Chang KC, Tai MH, Lin JW, et al. Hepatoma-derived growth factor is a novel prognostic factor for gastrointestinal stromal tumors. International journal of cancer 2007;121(5):1059-65.
20.Uyama H, Tomita Y, Nakamura H, et al. Hepatoma-derived growth factor is a novel prognostic factor for patients with pancreatic cancer. Clin Cancer Res 2006;12(20 Pt 1):6043-8.
21.Yamamoto S, Tomita Y, Hoshida Y, et al. Expression of hepatoma-derived growth factor is correlated with lymph node metastasis and prognosis of gastric carcinoma. Clin Cancer Res 2006;12(1):117-22.
22.Everett AD, Bushweller J. Hepatoma derived growth factor is a nuclear targeted mitogen. Current drug targets 2003;4(5):367-71.
23.Wanschura S, Schoenmakers EF, Huysmans C, Bartnitzke S, Van de Ven WJ, Bullerdiek J. Mapping of the gene encoding the human hepatoma-derived growth factor (HDGF) with homology to the high-mobility group (HMG)-1 protein to Xq25. Genomics 1996;32(2):298-300.
24.Ikegame K, Yamamoto M, Kishima Y, et al. A new member of a hepatoma-derived growth factor gene family can translocate to the nucleus. Biochemical and biophysical research communications 1999;266(1):81-7.
25.Dietz F, Franken S, Yoshida K, Nakamura H, Kappler J, Gieselmann V. The family of hepatoma-derived growth factor proteins: characterization of a new member HRP-4 and classification of its subfamilies. The Biochemical journal 2002;366(Pt 2):491-500.
26.Lukasik SM, Cierpicki T, Borloz M, Grembecka J, Everett A, Bushweller JH. High resolution structure of the HDGF PWWP domain: a potential DNA binding domain. Protein Sci 2006;15(2):314-23.
27.Beasley RP. Hepatitis B virus. The major etiology of hepatocellular carcinoma. Cancer 1988;61(10):1942-56.
28.Sue SC, Lee WT, Tien SC, et al. PWWP module of human hepatoma-derived growth factor forms a domain-swapped dimer with much higher affinity for heparin. J Mol Biol 2007;367(2):456-72.
29.Yang J, Everett AD. Hepatoma derived growth factor binds DNA through the N-terminal PWWP domain. BMC Mol Biol 2007;8(1):101.
30.Yamamoto S, Tomita Y, Hoshida Y, et al. Expression level of hepatoma-derived growth factor correlates with tumor recurrence of esophageal carcinoma. Ann Surg Oncol 2007;14(7):2141-9.
31.Okuda Y, Nakamura H, Yoshida K, et al. Hepatoma-derived growth factor induces tumorigenesis in vivo through both direct angiogenic activity and induction of vascular endothelial growth factor. Cancer Sci 2003;94(12):1034-41.
32.Everett AD, Narron JV, Stoops T, Nakamura H, Tucker A. Hepatoma-derived growth factor is a pulmonary endothelial cell-expressed angiogenic factor. Am J Physiol Lung Cell Mol Physiol 2004;286(6):L1194-201.
33.Kishima Y, Yoshida K, Enomoto H, et al. Antisense oligonucleotides of hepatoma-derived growth factor (HDGF) suppress the proliferation of hepatoma cells. Hepatogastroenterology 2002;49(48):1639-44.
34.Grosjean J, Kiriakidis S, Reilly K, Feldmann M, Paleolog E. Vascular endothelial growth factor signalling in endothelial cell survival: a role for NFkappaB. Biochem Biophys Res Commun 2006;340(3):984-94.
35.Chiarugi V, Magnelli L, Chiarugi A, Gallo O. Hypoxia induces pivotal tumor angiogenesis control factors including p53, vascular endothelial growth factor and the NFkappaB-dependent inducible nitric oxide synthase and cyclooxygenase-2. J Cancer Res Clin Oncol 1999;125(8-9):525-8.
36.Yang J, Richmond A. Constitutive IkappaB kinase activity correlates with nuclear factor-kappaB activation in human melanoma cells. Cancer research 2001;61(12):4901-9.
37.Jones PF, Jakubowicz T, Pitossi FJ, Maurer F, Hemmings BA. Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily. Proc Natl Acad Sci U S A 1991;88(10):4171-5.
38.Hayakawa J, Ohmichi M, Kurachi H, et al. Inhibition of BAD phosphorylation either at serine 112 via extracellular signal-regulated protein kinase cascade or at serine 136 via Akt cascade sensitizes human ovarian cancer cells to cisplatin. Cancer Res 2000;60(21):5988-94.
39.Tian YC, Phillips AO. Interaction between the transforming growth factor-beta type II receptor/Smad pathway and beta-catenin during transforming growth factor-beta1-mediated adherens junction disassembly. The American journal of pathology 2002;160(5):1619-28.
40.Wheelock MJ, Johnson KR. Cadherins as modulators of cellular phenotype. Annual review of cell and developmental biology 2003;19:207-35.
41.Christofori G. Changing neighbours, changing behaviour: cell adhesion molecule-mediated signalling during tumour progression. The EMBO journal 2003;22(10):2318-23.
42.Hazan RB, Qiao R, Keren R, Badano I, Suyama K. Cadherin switch in tumor progression. Annals of the New York Academy of Sciences 2004;1014:155-63.
43.Busca R, Berra E, Gaggioli C, et al. Hypoxia-inducible factor 1{alpha} is a new target of microphthalmia-associated transcription factor (MITF) in melanoma cells. The Journal of cell biology 2005;170(1):49-59.
44.Graham FL, Prevec L. Methods for construction of adenovirus vectors. Mol Biotechnol 1995;3(3):207-20.
45.Folkman. J. an organizing principle for drug discovery? Nature reviews 2007;6(4):273-86.
46.Dhawan P, Su Y, Thu YM, et al. The Lymphotoxin-{beta} Receptor Is an Upstream Activator of NF-{kappa}B-mediated Transcription in Melanoma Cells. The Journal of biological chemistry 2008;283(22):15399-408.
47.Marin YE, Wall BA, Wang S, et al. Curcumin downregulates the constitutive activity of NF-kappaB and induces apoptosis in novel mouse melanoma cells. Melanoma research 2007;17(5):274-83.
48.Li J, Yen C, Liaw D, et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 1997;275(5308):1943-7.
49.Luo Y, Radice GL. N-cadherin acts upstream of VE-cadherin in controlling vascular morphogenesis. The Journal of cell biology 2005;169(1):29-34.
50.Wang ML, Panasyuk G, Gwalter J, et al. Regulation of ribosomal protein S6 kinases by ubiquitination. Biochem Biophys Res Commun 2008;369(2):382-7.
51.Foo IT, Warren PM, Drummond GB. Influence of oral clonidine on the ventilatory response to acute and sustained isocapnic hypoxia in human males. British journal of anaesthesia 1996;76(2):214-20.
52.Pouyssegur J, Dayan F, Mazure NM. Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature 2006;441(7092):437-43.
53.Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med 1971;285(21):1182-6.
54.Mosevitsky MI, Novitskaya VA, Iogannsen MG, Zabezhinsky MA. Tissue specificity of nucleo-cytoplasmic distribution of HMG1 and HMG2 proteins and their probable functions. Eur J Biochem 1989;185(2):303-10.
55.Rigel DS, Friedman RJ, Kopf AW. The incidence of malignant melanoma in the United States: issues as we approach the 21st century. Journal of the American Academy of Dermatology 1996;34(5 Pt 1):839-47.
56.Zamora-Avila DE, Franco-Molina MA, Trejo-Avila LM, Rodriguez-Padilla C, Resendez-Perez D, Zapata-Benavides P. RNAi silencing of the WT1 gene inhibits cell proliferation and induces apoptosis in the B16F10 murine melanoma cell line. Melanoma research 2007;17(6):341-8.
57.Ren H, Tang X, Lee JJ, et al. Expression of hepatoma-derived growth factor is a strong prognostic predictor for patients with early-stage non-small-cell lung cancer. J Clin Oncol 2004;22(16):3230-7.
58.Folkman J, Merler E, Abernathy C, Williams G. Isolation of a tumor factor responsible for angiogenesis. The Journal of experimental medicine 1971;133(2):275-88.
59.Ryschich E, Lizdenis P, Ittrich C, et al. Molecular fingerprinting and autocrine growth regulation of endothelial cells in a murine model of hepatocellular carcinoma. Cancer research 2006;66(1):198-211.
60.Hay ED. An overview of epithelio-mesenchymal transformation. Acta anatomica 1995;154(1):8-20.
61.Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nature reviews 2002;2(6):442-54.
62.Merlino G. Cancer biology: the weakest link? Nature 2005;436(7047):33-5.
63.Waldmann V, Wacker J, Deichmann M. Mutations of the activation-associated phosphorylation sites at codons 308 and 473 of protein kinase B are absent in human melanoma. Archives of dermatological research 2001;293(7):368-72.