EB病毒感染與多種人類惡性疾病有關，如何杰金氏症和鼻咽癌，先前我們的研究指出，EB病毒在約96.4%的鼻咽癌組織中被發現，可知EB病毒在鼻咽癌的發生過程扮演重要的角色。EB病毒在NPC中以第二潛伏期存在，只有少數基因在這時期被表現，如EBNA-1, LMP1, LMP2和EBERs，LMP1為潛伏期表現的基因中首先被發現與細胞轉型和致癌能力相關。在人類表皮細胞中，LMP1可改變許多與癌細胞生長及侵犯能力有關的基因功能表現。另一方面，integrin αvβ6在所有integrin內具有表皮專一性，它能與其他細胞膜外蛋白質結合，像是fibronectin, vitronectin, tenascin和TGF-β。特別的是，integrin αvβ6並不表現於正常口腔表皮組織，而會在傷口復原和惡性細胞生長等組織重組時才會表現，因此integrin αvβ6的表現被認為會影響腫瘤的遷移和侵犯能力。目前的研究指出，在β6 promoter上可能有Ets-1及STAT3的結合位，而LMP1已被證實可調控Ets-1及STAT3的表現；並且，在B cell中LMP1也被證實可活化integrin αv promoter。依據上述，我們推測LMP1可能同時活化Integrin αv和β6的表現，進而影響NPC 細胞的遷移和侵犯能力。由我們的實驗結果顯示，雖然LMP1蛋白質在鼻咽癌細胞中能同時活化JNK、Ets-1及NF-κB 路徑的活化，但主要還是透過NF-κB 路徑而使得integrin αv和β6表現上升。我們也預測了β6 promoter上有NF-κB結合位，而Ets-1在鼻咽癌細胞中並不參與integrin β6活化，STAT3則在LMP1表現時磷酸化程度下降。除此之外，我們正在釐清integrin αv和β6的表現對鼻咽癌細胞的功能性影響。

Epstein-Barr virus (EBV) infection is associated with several human malignancies, including nasopharyngeal carcinoma (NPC). In our previous study, EBV was found in 96.4% primary NPC specimens, implying a role of EBV in tumorigenesis of NPC. EBV resides in NPC cells in a status of type II latency, with only limited genes being expressed, namely EBNA-1, LMP1, LMP2 and EBERs. LMP1 was the first EBV latent gene found to be able to transform cell lines and alter the phenotype of cells due to its oncogenic potential. In human epithelial cells, LMP1 alters many functional properties in tumor progression and invasions. Moreover, Integrins are αβ heterodimeric type I membrane proteins which are formed of 18 α and 8 β subunits to assemble into 24 distinct integrins. Integrin αvβ6 is epithelial-specific and binds to the ECM proteins, like fibronectin, vitronectin and tenascin, and also to the latency associated peptide of TGF-β. Especially, integrin αvβ6 is not expressed by healthy oral epithelia, but is upregulated during tissue remodeling, including that accompanying wound healing and inducing carcinogenesis. Integrin αvβ6 has been proposed to play an important role in tumor migration and invasion. Some studies have shown that Ets-1 and STAT3 could be regulated by LMP1, and putative Ets-1 and STAT3 binding sites on β6-promoter were considered to be important for transcriptional activation of β6 subunit. And also, LMP1 was proved to transactivate integrin αv promoter in human B cells. Taken together, we hypothesized that LMP1 can activate both αv and β6 genes to potentiate the invasion and migration abilities of NPC cells. We currently found that although LMP1 could activate Ets-1, JNK and NFκB pathways in NPC cells, the main pathway which regulated integrin αv and β6 was via NFκB pathway. Ets-1 was not involved in regulation of integrin β6 promoter, and STAT3 was downregulated in NPC cells. Furthermore, the cell functional effects of LMP1-mediated integrin αv and β6 expression will be elucidated.

中文摘要 I
英文摘要 II
誌謝 IV
CONTENTS V
ABBREVIATIONS i
INTRODUCTION 1
Nasopharyngeal carcinoma 1
Epstein-Barr virus and NPC 1
Latent membrane protein 1 and its downstream signaling pathways 3
Integrins 4
Integrin αvβ6 5
Study purpose 6
METERIALS AND METHODS 7
Materials 7
Cell lines 7
Plasmids 7
Primary antibodies 7
Secondary antibodies 8
small interfering RNAs (siRNAs) 9
Inhibitors 9
PCR Primers 9
Labwares and reagents 10
Methods 11
Transfection and inhibitor treatment 11
Protein extraction and quantification 12
Western blotting 12
RNA extraction 12
RT-PCR 13
Immunohistochemistry (IHC) staining 13
Cell growth and proliferation 14
Migration and matrigel invasion assay 14
RESULTS 15
EBV LMP1 induces integrin αv and β6 expression in NPC cell line. 15
LMP1 can activate Ets-1 in HONE-1 cell line. 15
Ets-1 is not involved in LMP1-regulated αv and β6 expression. 16
Identification of LMP1 truncation effects on integrin αv and β6. 16
LMP1-mediated signaling is not via Erk, PI3K and p38 pathways. 17
STAT3 phosphorylation was downregulated in LMP1-induced HONE-1 cell line. 17
JNK pathway is not involved in LMP1-mediated signaling. 18
NF-κB pathway plays a critical role in LMP1-induced signaling. 18
LMP1-induced cell functions in HONE-1 cell line. 19
Protein expression profiles of integrin αv and β6 in various cancer cell lines. 19
Integrin αvβ6 is expressed on NPC specimens 20
DISCUSSION 21
CONCLUSION 23
REFERENCES 24
FIGURES 31

Azare J, Leslie K, Al-Ahmadie H, Gerald W, Weinreb PH, Violette SM et al (2007). Constitutively activated Stat3 induces tumorigenesis and enhances cell motility of prostate epithelial cells through integrin beta 6. Mol Cell Biol 27: 4444-53.

Baichwal VR, Sugden B (1988). Transformation of Balb 3T3 cells by the BNLF-1 gene of Epstein-Barr virus. Oncogene 2: 461-7.

Bates RC, Bellovin DI, Brown C, Maynard E, Wu B, Kawakatsu H et al (2005). Transcriptional activation of integrin beta6 during the epithelial-mesenchymal transition defines a novel prognostic indicator of aggressive colon carcinoma. J Clin Invest 115: 339-47.

Breuss JM, Gallo J, DeLisser HM, Klimanskaya IV, Folkesson HG, Pittet JF et al (1995). Expression of the beta 6 integrin subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling. J Cell Sci 108 ( Pt 6): 2241-51.

Brooks L, Yao QY, Rickinson AB, Young LS (1992). Epstein-Barr virus latent gene transcription in nasopharyngeal carcinoma cells: coexpression of EBNA1, LMP1, and LMP2 transcripts. J Virol 66: 2689-97.

Buettner M, Heussinger N, Niedobitek G (2006). Expression of Epstein-Barr virus (EBV)-encoded latent membrane proteins and STAT3 activation in nasopharyngeal carcinoma. Virchows Arch 449: 513-9.

Cai X, Schafer A, Lu S, Bilello JP, Desrosiers RC, Edwards R et al (2006). Epstein-Barr virus microRNAs are evolutionarily conserved and differentially expressed. PLoS Pathog 2: e23.

Chang SH, Chang HC, Hung WC (2008). Transcriptional repression of tissue inhibitor of metalloproteinase-3 by Epstein-Barr virus latent membrane protein 1 enhances invasiveness of nasopharyngeal carcinoma cells. Oral Oncol.

Chen H, Hutt-Fletcher L, Cao L, Hayward SD (2003). A positive autoregulatory loop of LMP1 expression and STAT activation in epithelial cells latently infected with Epstein-Barr virus. J Virol 77: 4139-48.

Chen H, Lee JM, Wang Y, Huang DP, Ambinder RF, Hayward SD (1999). The Epstein-Barr virus latency BamHI-Q promoter is positively regulated by STATs and Zta interference with JAK/STAT activation leads to loss of BamHI-Q promoter activity. Proc Natl Acad Sci U S A 96: 9339-44.

Chen H, Lee JM, Zong Y, Borowitz M, Ng MH, Ambinder RF et al (2001). Linkage between STAT regulation and Epstein-Barr virus gene expression in tumors. J Virol 75: 2929-37.

Chen YR, Liu MT, Chang YT, Wu CC, Hu CY, Chen JY (2008). Epstein-Barr virus latent membrane protein 1 represses DNA repair through the PI3K/Akt/FOXO3a pathway in human epithelial cells. J Virol.

Cordes N, Meineke V (2004). Integrin signalling and the cellular response to ionizing radiation. J Mol Histol 35: 327-37.

Dawson CW, Laverick L, Morris MA, Tramoutanis G, Young LS (2008). Epstein-Barr virus-encoded LMP1 regulates epithelial cell motility and invasion via the ERK-MAPK pathway. J Virol 82: 3654-64.

Dawson CW, Tramountanis G, Eliopoulos AG, Young LS (2003). Epstein-Barr virus latent membrane protein 1 (LMP1) activates the phosphatidylinositol 3-kinase/Akt pathway to promote cell survival and induce actin filament remodeling. J Biol Chem 278: 3694-704.

Elayadi AN, Samli KN, Prudkin L, Liu YH, Bian A, Xie XJ et al (2007). A peptide selected by biopanning identifies the integrin alphavbeta6 as a prognostic biomarker for nonsmall cell lung cancer. Cancer Res 67: 5889-95.

Everly DN, Jr., Mainou BA, Raab-Traub N (2008). The ID proteins contribute to the growth of rodent fibroblasts during LMP1-mediated transformation. Virology.

Fendri A, Khabir A, Hadhri-Guiga B, Sellami-Boudawara T, Ghorbel A, Daoud J et al (2007). Overexpression of COX-2 and LMP1 are correlated with lymph node in Tunisian NPC patients. Oral Oncol.

Gires O, Kohlhuber F, Kilger E, Baumann M, Kieser A, Kaiser C et al (1999). Latent membrane protein 1 of Epstein-Barr virus interacts with JAK3 and activates STAT proteins. EMBO J 18: 3064-73.

Hamidi S, Salo T, Kainulainen T, Epstein J, Lerner K, Larjava H (2000). Expression of alpha(v)beta6 integrin in oral leukoplakia. Br J Cancer 82: 1433-40.

Hazelbag S, Kenter GG, Gorter A, Dreef EJ, Koopman LA, Violette SM et al (2007). Overexpression of the alpha v beta 6 integrin in cervical squamous cell carcinoma is a prognostic factor for decreased survival. J Pathol 212: 316-24.

Hildesheim A, Levine PH (1993). Etiology of nasopharyngeal carcinoma: a review. Epidemiol Rev 15: 466-85.

Horikawa T, Sheen TS, Takeshita H, Sato H, Furukawa M, Yoshizaki T (2001). Induction of c-Met proto-oncogene by Epstein-Barr virus latent membrane protein-1 and the correlation with cervical lymph node metastasis of nasopharyngeal carcinoma. Am J Pathol 159: 27-33.

Horikawa T, Yang J, Kondo S, Yoshizaki T, Joab I, Furukawa M et al (2007). Twist and epithelial-mesenchymal transition are induced by the EBV oncoprotein latent membrane protein 1 and are associated with metastatic nasopharyngeal carcinoma. Cancer Res 67: 1970-8.

Hsiao JR, Jin YT, Tsai ST, Shiau AL, Wu CL, Su WC (2003). Constitutive activation of STAT3 and STAT5 is present in the majority of nasopharyngeal carcinoma and correlates with better prognosis. Br J Cancer 89: 344-9.

Huang S, Stupack D, Liu A, Cheresh D, Nemerow GR (2000). Cell growth and matrix invasion of EBV-immortalized human B lymphocytes is regulated by expression of alpha(v) integrins. Oncogene 19: 1915-23.

Huen DS, Henderson SA, Croom-Carter D, Rowe M (1995). The Epstein-Barr virus latent membrane protein-1 (LMP1) mediates activation of NF-kappa B and cell surface phenotype via two effector regions in its carboxy-terminal cytoplasmic domain. Oncogene 10: 549-60.

Hynes RO (1992). Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69: 11-25.

Hynes RO (2002). Integrins: bidirectional, allosteric signaling machines. Cell 110: 673-87.

IARC (1997). IARC monographs on the evaluation of carcinogenic risks to humans:
Epstein-Barr Virus and Kaposi's Sarcoma Herpesvirus/Human Herpesvirus 8. Lyon,
IARC.

Impola U, Uitto VJ, Hietanen J, Hakkinen L, Zhang L, Larjava H et al (2004). Differential expression of matrilysin-1 (MMP-7), 92 kD gelatinase (MMP-9), and metalloelastase (MMP-12) in oral verrucous and squamous cell cancer. J Pathol 202: 14-22.

Jeyakumar A, Brickman TM, Doerr T (2006). Review of nasopharyngeal carcinoma. Ear Nose Throat J 85: 168-70, 172-3, 184.

Jones J, Watt FM, Speight PM (1997). Changes in the expression of alpha v integrins in oral squamous cell carcinomas. J Oral Pathol Med 26: 63-8.

Kaye KM, Izumi KM, Kieff E (1993). Epstein-Barr virus latent membrane protein 1 is essential for B-lymphocyte growth transformation. Proc Natl Acad Sci U S A 90: 9150-4.

Kim KR, Yoshizaki T, Miyamori H, Hasegawa K, Horikawa T, Furukawa M et al (2000). Transformation of Madin-Darby canine kidney (MDCK) epithelial cells by Epstein-Barr virus latent membrane protein 1 (LMP1) induces expression of Ets1 and invasive growth. Oncogene 19: 1764-71.

Kondo S, Wakisaka N, Schell MJ, Horikawa T, Sheen TS, Sato H et al (2005). Epstein-Barr virus latent membrane protein 1 induces the matrix metalloproteinase-1 promoter via an Ets binding site formed by a single nucleotide polymorphism: enhanced susceptibility to nasopharyngeal carcinoma. Int J Cancer 115: 368-76.

Kung CP, Raab-Traub N (2008). Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor through effects on Bcl-3 and STAT3. J Virol 82: 5486-93.

Kutz H, Reisbach G, Schultheiss U, Kieser A (2008). The c-Jun N-terminal kinase pathway is critical for cell transformation by the latent membrane protein 1 of Epstein-Barr virus. Virology 371: 246-56.

Lee DC, Chua DT, Wei WI, Sham JS, Lau AS (2007). Induction of matrix metalloproteinases by Epstein-Barr virus latent membrane protein 1 isolated from nasopharyngeal carcinoma. Biomed Pharmacother 61: 520-6.

Liu LT, Peng JP, Chang HC, Hung WC (2003). RECK is a target of Epstein-Barr virus latent membrane protein 1. Oncogene 22: 8263-70.

Liu MT, Chang YT, Chen SC, Chuang YC, Chen YR, Lin CS et al (2005). Epstein-Barr virus latent membrane protein 1 represses p53-mediated DNA repair and transcriptional activity. Oncogene 24: 2635-46.

Lo HW, Hsu SC, Xia W, Cao X, Shih JY, Wei Y et al (2007). Epidermal growth factor receptor cooperates with signal transducer and activator of transcription 3 to induce epithelial-mesenchymal transition in cancer cells via up-regulation of TWIST gene expression. Cancer Res 67: 9066-76.

Lu J, Chua HH, Chen SY, Chen JY, Tsai CH (2003). Regulation of matrix metalloproteinase-1 by Epstein-Barr virus proteins. Cancer Res 63: 256-62.

Ma BB, Hui EP, Chan AT (2008). Systemic approach to improving treatment outcome in nasopharyngeal carcinoma: current and future directions. Cancer Sci 99: 1311-8.

Mitchell T, Sugden B (1995). Stimulation of NF-kappa B-mediated transcription by mutant derivatives of the latent membrane protein of Epstein-Barr virus. J Virol 69: 2968-76.

Murono S, Inoue H, Tanabe T, Joab I, Yoshizaki T, Furukawa M et al (2001). Induction of cyclooxygenase-2 by Epstein-Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells. Proc Natl Acad Sci U S A 98: 6905-10.

Napione L, Cascone I, Mitola S, Serini G, Bussolino F (2007). Integrins: a flexible platform for endothelial vascular tyrosine kinase receptors. Autoimmun Rev 7: 18-22.

Pattle SB, Farrell PJ (2006). The role of Epstein-Barr virus in cancer. Expert Opin Biol Ther 6: 1193-205.

Regezi JA, Ramos DM, Pytela R, Dekker NP, Jordan RC (2002). Tenascin and beta 6 integrin are overexpressed in floor of mouth in situ carcinomas and invasive squamous cell carcinomas. Oral Oncol 38: 332-6.

Richardson C, Fielding C, Rowe M, Brennan P (2003). Epstein-Barr virus regulates STAT1 through latent membrane protein 1. J Virol 77: 4439-43.

Rickinson A, Kieff E (2001). Epstein-Barr virus. In Field's Virology, Knipe DM, Howley PM. (ed): 2575-2627. Linpincott : Philadephia.

Saito N, Courtois G, Chiba A, Yamamoto N, Nitta T, Hironaka N et al (2003). Two carboxyl-terminal activation regions of Epstein-Barr virus latent membrane protein 1 activate NF-kappaB through distinct signaling pathways in fibroblast cell lines. J Biol Chem 278: 46565-75.

Sample J, Young L, Martin B, Chatman T, Kieff E, Rickinson A (1990). Epstein-Barr virus types 1 and 2 differ in their EBNA-3A, EBNA-3B, and EBNA-3C genes. J Virol 64: 4084-92.

Shair KH, Bendt KM, Edwards RH, Bedford EC, Nielsen JN, Raab-Traub N (2007). EBV latent membrane protein 1 activates Akt, NFkappaB, and Stat3 in B cell lymphomas. PLoS Pathog 3: e166.

Sriuranpong V, Mutirangura A, Gillespie JW, Patel V, Amornphimoltham P, Molinolo AA et al (2004). Global gene expression profile of nasopharyngeal carcinoma by laser capture microdissection and complementary DNA microarrays. Clin Cancer Res 10: 4944-58.

Stevenson D, Charalambous C, Wilson JB (2005). Epstein-Barr virus latent membrane protein 1 (CAO) up-regulates VEGF and TGF alpha concomitant with hyperlasia, with subsequent up-regulation of p16 and MMP9. Cancer Res 65: 8826-35.

Thomas GJ, Nystrom ML, Marshall JF (2006). Alphavbeta6 integrin in wound healing and cancer of the oral cavity. J Oral Pathol Med 35: 1-10.

Thornburg NJ, Kulwichit W, Edwards RH, Shair KH, Bendt KM, Raab-Traub N (2006). LMP1 signaling and activation of NF-kappaB in LMP1 transgenic mice. Oncogene 25: 288-97.

Tsai CN, Tsai CL, Tse KP, Chang HY, Chang YS (2002). The Epstein-Barr virus oncogene product, latent membrane protein 1, induces the downregulation of E-cadherin gene expression via activation of DNA methyltransferases. Proc Natl Acad Sci U S A 99: 10084-9.

Tsai ST, Jin YT, Su IJ (1996). Expression of EBER1 in primary and metastatic nasopharyngeal carcinoma tissues using in situ hybridization. A correlation with WHO histologic subtypes. Cancer 77: 231-6.

Wakisaka N, Murono S, Yoshizaki T, Furukawa M, Pagano JS (2002). Epstein-barr virus latent membrane protein 1 induces and causes release of fibroblast growth factor-2. Cancer Res 62: 6337-44.

Wei WI, Sham JS (2005). Nasopharyngeal carcinoma. Lancet 365: 2041-54.

Wu SJ, Lay JD, Chen CL, Chen JY, Liu MY, Su IJ (1996). Genomic analysis of Epstein-Barr virus in nasal and peripheral T-cell lymphoma: a comparison with nasopharyngeal carcinoma in an endemic area. J Med Virol 50: 314-21.

Zhang L, Hong K, Zhang J, Pagano JS (2004). Multiple signal transducers and activators of transcription are induced by EBV LMP-1. Virology 323: 141-52.

Zheng H, Li LL, Hu DS, Deng XY, Cao Y (2007). Role of Epstein-Barr virus encoded latent membrane protein 1 in the carcinogenesis of nasopharyngeal carcinoma. Cell Mol Immunol 4: 185-96.