鼻咽癌細胞為一種位於鼻咽的惡性上皮細胞癌，其盛行於台灣及東南亞地區。不同於其他頭頸癌，鼻咽癌早期就具有高轉移能力而且晚期之病患，常因其高度侵犯轉移之特性，造成治療之失敗。因此更進一步了解相關分子，將有助於整體治療之成效。先前透過免疫組織化學染色法(IHC)，發現annexin A2蛋白在鼻咽癌病人的腫瘤組織中有較高的表現量，近幾年不同腫瘤研究中也指出annexin A2蛋白質表現與腫瘤惡化相關性，因此進而探討annexin A2在鼻咽癌所扮演的角色。透過基因靜默(gene silence)抑制annexin A2表現，發現抑制annexin A2會降低癌細胞的生長能力，增加細胞對化療藥物 (Cisplatin、5-FU、Vincristine、 Docetaxel)與放射線治療之敏感性。抑制Annexin A2表現會降低細胞移行、黏附和侵犯能力。動物實驗也證實降低annexin A2表現會抑制腫瘤細胞的生長。進一步探討相關調控分子，發現annexin A2會調控上皮-間質細胞轉化（EMT）之相關分子之表現、AKT分子表現與磷酸化現象。在臨床層面分析，發現annexin A2高度表現於晚期鼻咽癌病患且與表現量與N期有相關性。藉由這些研究結果，我們更加了解annexin A2在鼻咽癌細胞的功能，未來annexin A2也許可以當成臨床上治療的一個標的與預後指標。

Nasopharyngeal carcinoma (NPC), originated from the epithelium of the nasopharynx, is a common malignant tumor. NPC mainly occurs in the Southeast Asia including Taiwan. Characteristically, NPC differs from other head and neck carcinomas, especially for its high metastasis character and poor efficiency of clinical treatment. Recently, many reports have indicated that annexin A2 might regulate the metastasis on different kinds of cancer. However, the tumorigenic function of annexin A2 in NPC is not yet understood. Annexin A2 shRNAs were used to evaluate the effects of annexin A2 suppression on NPCs. Annexin A2 silencing reduces the cell proliferation ability and increase chemotherapeutic drugs (Cisplatin, 5-FU, Vincristine and Docetaxel) and irradiation sensitivity. Moreover, annexin A2 not only up-regulates cell adhesion, migration, and invasion abilities on NPCs, but also involves in epithelial-mesenchymal transition (EMT). These cellular results were confirmed using tumor xenografts in mice, as annexin A2 silencing led to suppress tumor growth. Clinicl study reveals that the high level of annexin A2 on late-staged NPC patient tissues and associated with N stage. In summary, annexin A2 not only regulates many malignant phenotypes such as migration, invasion, adhesion and EMT, but also controls therapeutic tolerance in NPCs. Annexin A2 may serve as
prognostic markers and targets in NPC.

中文摘要 v
Abstract vi
Abbreviation vii
Acknowledgement…………………………………………...viii

1. Introduction 1
1.1 Nasopharyneal carcinoma (NPC)……………...…………………1
1.2 Chemo- and radiotherapy for nasopharyngeal carcinoma……..…3
1.3 Tumor-induced immune suppressive response…………………..3
1.3.1 Dendritic cell (DC) and DC-SIGN………………………4
1.4 Identify the DC-SIGN interacted protein: annexin A2…………..5
1.41 Annexin A2………………..………………………………6
1.42 The effect of annexin A2 in nasopharyngeal carcinoma…8
1.5 Epithelial to mesenchymal transition (EMT)……………………8
1.5.1 TGF-β induced EMT……………………………………10
1.5.2 TNF-α induced EMT……………………………………11
1.6 Akt regulating pathways related to cell proliferation, exocytosis
and irradiation resistance………………………………………..12

2. Aim of study 14

3. Materials and Methods
3.1 Cell and Culture………………………………………………..15
3.2 Patient’s tissue sample…………………………………………..15
3.3 Establishment of annexin A2-silenced NPC……………………16
3.4 Western blotting………………………………………….……16
3.5 RT-PCR…………………………………………………………17
3.6 Real-time PCR…………..………………………………………18
3.7 Cell proliferation and cytotoxicity assay………………………18
3.8 Clongenic assays for irradiation …………...............………...…19
3.9 Migration…………..……………………………………………19
3.10 Invasion………………………………………………………..19
3.11 Adhesion………………………………………………………20
3.12 Tube formation assay…………………………………………20
3.13 Immunofluorescence stain……………………………………20
3.14 Xenograft tumor mice model (SCID)…………………………21
3.15 H&E stain……………...………………………………………21
3.16 Immunohistochemistry……...…………………………………22
3.17 Statistic……………...…………………………………………22

4. Results
4.1 Knockdown of annexin A2 inhibits cell proliferation in NPC
cell lines…………..……………………………………………23
4.2 Annexin A2 involves in the resistance of therapeutic reagents
(cisplatin, 5-FU, vincristine and docetaxel)……………….24
4.3 Annexin A2 involves in the resistance of irradiation……...……24
4.4 Annexin A2 promotes cell migration, invasion, vascular
formation, and adhesion of nasopharyngeal carcinoma……...…25
4.5 Expression of annexin A2 induces an epithelial-mesenchymal
transition by activation of TGF-β pathway……………………..26
4.6 Annexin A2 promotes nasopharyngeal carcinoma growth
in vivo………………………………………………………….28
4.7 Expression of annexin A2 in human nasopharyngeal carcinoma
tissues…………………………………………………………..28

4.8 Annexin A2 regulates Akt expression in NPC………………….29

5. Conclusion 30

6. Discussion 31

Appendix
A.1 Certificate of TMU-JIRB Approval 35

List of References 36
Figures 41

1.Cho, W.C., Nasopharyngeal carcinoma: molecular biomarker discovery and progress. Mol Cancer, 2007. 6: p. 1.
2.Chen, C.J., et al., Cancer epidemiology and control in Taiwan: a brief review. Jpn J Clin Oncol, 2002. 32 Suppl: p. S66-81.
3.Sun, L.M., et al., Trends in the incidence rates of nasopharyngeal carcinoma among Chinese Americans living in Los Angeles County and the San Francisco metropolitan area, 1992-2002. Am J Epidemiol, 2005. 162(12): p. 1174-8.
4.Niedobitek, G., A. Agathanggelou, and J.M. Nicholls, Epstein-Barr virus infection and the pathogenesis of nasopharyngeal carcinoma: viral gene expression, tumour cell phenotype, and the role of the lymphoid stroma. Semin Cancer Biol, 1996. 7(4): p. 165-74.
5.Spano, J.P., et al., Nasopharyngeal carcinomas: an update. Eur J Cancer, 2003. 39(15): p. 2121-35.
6.Shanmugaratnam, K., Histological typing of nasopharyngeal carcinoma. IARC Sci Publ, 1978(20): p. 3-12.
7.Ma, B.B. and A.T. Chan, Recent perspectives in the role of chemotherapy in the management of advanced nasopharyngeal carcinoma. Cancer, 2005. 103(1): p. 22-31.
8.Kong, L., et al., Neoadjuvant chemotherapy followed by concurrent chemoradiation for locoregionally advanced nasopharyngeal carcinoma: Interim results from 2 prospective phase 2 clinical trials. Cancer, 2013.
9.Dominguez, P.M. and C. Ardavin, Differentiation and function of mouse monocyte-derived dendritic cells in steady state and inflammation. Immunol Rev, 2010. 234(1): p. 90-104.
10.Shortman, K. and S.H. Naik, Steady-state and inflammatory dendritic-cell development. Nat Rev Immunol, 2007. 7(1): p. 19-30.
11.Banchereau, J., et al., Immunobiology of dendritic cells. Annu Rev Immunol, 2000. 18: p. 767-811.
12.Lanzavecchia, A. and F. Sallusto, Regulation of T cell immunity by dendritic cells. Cell, 2001. 106(3): p. 263-6.
13.Sreekumaran, E., et al., Loss of dendritic connectivity in CA1, CA2, and CA3 neurons in hippocampus in rat under aluminum toxicity: antidotal effect of pyridoxine. Brain Res Bull, 2003. 59(6): p. 421-7.
14.Nonaka, M., et al., Glycosylation-dependent interactions of C-type lectin DC-SIGN with colorectal tumor-associated Lewis glycans impair the function and differentiation of monocyte-derived dendritic cells. J Immunol, 2008. 180(5): p. 3347-56.
15.Geijtenbeek, T.B.H., et al., DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell, 2000. 100(5): p. 587-597.
16.Geijtenbeek, T.B.H., et al., Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses. Cell, 2000. 100(5): p. 575-585.
17.den Dunnen, J., S.I. Gringhuis, and T.B. Geijtenbeek, Dusting the sugar fingerprint: C-type lectin signaling in adaptive immunity. Immunol Lett, 2010. 128(1): p. 12-6.
18.Vicari, A.P., C. Caux, and G. Trinchieri, Tumour escape from immune surveillance through dendritic cell inactivation. Seminars in Cancer Biology, 2002. 12(1): p. 33-42.
19.den Dunnen, J., S.I. Gringhuis, and T.B. Geijtenbeek, Innate signaling by the C-type lectin DC-SIGN dictates immune responses. Cancer Immunol Immunother, 2009. 58(7): p. 1149-57.
20.Stambach, N.S. and M.E. Taylor, Characterization of carbohydrate recognition by langerin, a C-type lectin of Langerhans cells. Glycobiology, 2003. 13(5): p. 401-10.
21.Bergman, M.P., et al., Helicobacter pylori modulates the T helper cell 1/T helper cell 2 balance through phase-variable interaction between lipopolysaccharide and DC-SIGN. Journal of Experimental Medicine, 2004. 200(8): p. 979-990.
22.Grutz, G., New insights into the molecular mechanism of interieukin-10-mediated immunosuppression. Journal of Leukocyte Biology, 2005. 77(1): p. 3-15.
23.Waisman, D.M., Annexin II tetramer: structure and function. Mol Cell Biochem, 1995. 149-150: p. 301-22.
24.Menell, J.S., et al., Annexin II and bleeding in acute promyelocytic leukemia. N Engl J Med, 1999. 340(13): p. 994-1004.
25.Bharadwaj, A., et al., Annexin A2 heterotetramer: structure and function. Int J Mol Sci, 2013. 14(3): p. 6259-305.
26.Burger, A., et al., The crystal structure and ion channel activity of human annexin II, a peripheral membrane protein. J Mol Biol, 1996. 257(4): p. 839-47.
27.Chiang, Y., et al., Specific down-regulation of annexin II expression in human cells interferes with cell proliferation. Mol Cell Biochem, 1999. 199(1-2): p. 139-47.
28.Ling, Q. and K.A. Hajjar, Inhibition of endothelial cell thromboresistance by homocysteine. J Nutr, 2000. 130(2S Suppl): p. 373S-376S.
29.Liu, L., J.Q. Tao, and U.J. Zimmerman, Annexin II binds to the membrane of A549 cells in a calcium-dependent and calcium-independent manner. Cell Signal, 1997. 9(3-4): p. 299-304.
30.Protzel, C., et al., The role of annexins I, II and IV in tumor development, progression and metastasis of human penile squamous cell carcinomas. World J Urol, 2011. 29(3): p. 393-8.
31.Lokman, N.A., et al., Annexin A2 is regulated by ovarian cancer-peritoneal cell interactions and promotes metastasis. Oncotarget, 2013. 4(8): p. 1199-1211.
32.Zhang, H.J., et al., Annexin A2 silencing inhibits invasion, migration, and tumorigenic potential of hepatoma cells. World J Gastroenterol, 2013. 19(24): p. 3792-801.
33.Chan, C.M., et al., Proteomic comparison of nasopharyngeal cancer cell lines C666-1 and NP69 identifies down-regulation of annexin II and beta2-tubulin for nasopharyngeal carcinoma. Arch Pathol Lab Med, 2008. 132(4): p. 675-83.
34.Luo, W., et al., Epstein-Barr virus latent membrane protein 1 mediates serine 25 phosphorylation and nuclear entry of annexin A2 via PI-PLC-PKCalpha/PKCbeta pathway. Mol Carcinog, 2008. 47(12): p. 934-46.
35.Savagner, P., et al., Modulations of the epithelial phenotype during embryogenesis and cancer progression. Cancer Treat Res, 1994. 71: p. 229-49.
36.Thiery, J.P., Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer, 2002. 2(6): p. 442-54.
37.Thompson, E.W., D.F. Newgreen, and D. Tarin, Carcinoma invasion and metastasis: a role for epithelial-mesenchymal transition? Cancer Res, 2005. 65(14): p. 5991-5; discussion 5995.
38.Folkman, J., Tumor angiogenesis: therapeutic implications. N Engl J Med, 1971. 285(21): p. 1182-6.
39.Thiery, J.P., et al., Epithelial-mesenchymal transitions in development and disease. Cell, 2009. 139(5): p. 871-90.
40.Holian, J., et al., Role of Kruppel-like factor 6 in transforming growth factor-beta1-induced epithelial-mesenchymal transition of proximal tubule cells. Am J Physiol Renal Physiol, 2008. 295(5): p. F1388-96.
41.Lv, Z.D., et al., Induction of gastric cancer cell adhesion through transforming growth factor-beta1-mediated peritoneal fibrosis. Journal of Experimental & Clinical Cancer Research, 2010. 29.
42.Miyazono, K., Transforming growth factor-beta signaling in epithelial-mesenchymal transition and progression of cancer. Proc Jpn Acad Ser B Phys Biol Sci, 2009. 85(8): p. 314-23.
43.Peinado, H., D. Olmeda, and A. Cano, Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer, 2007. 7(6): p. 415-28.
44.Massague, J. and D. Wotton, Transcriptional control by the TGF-beta/Smad signaling system. Embo Journal, 2000. 19(8): p. 1745-1754.
45.Oft, M., K.H. Heider, and H. Beug, TGF beta signaling is necessary for carcinoma cell invasiveness and metastasis. Current Biology, 1998. 8(23): p. 1243-1252.
46.Roberts, A.B. and L.M. Wakefield, The two faces of transforming growth factor beta in carcinogenesis. Proc Natl Acad Sci U S A, 2003. 100(15): p. 8621-3.
47.Oft, M., et al., TGF-beta1 and Ha-Ras collaborate in modulating the phenotypic plasticity and invasiveness of epithelial tumor cells. Genes Dev, 1996. 10(19): p. 2462-77.
48.Xu, J., S. Lamouille, and R. Derynck, TGF-beta-induced epithelial to mesenchymal transition. Cell Res, 2009. 19(2): p. 156-72.
49.Locksley, R.M., N. Killeen, and M.J. Lenardo, The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell, 2001. 104(4): p. 487-501.
50.Rhyu, D.Y., et al., Role of reactive oxygen species in TGF-beta1-induced mitogen-activated protein kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells. J Am Soc Nephrol, 2005. 16(3): p. 667-75.
51.Liu, X.X., et al., NADPH oxidase-dependent formation of reactive oxygen species contributes to transforming growth factor beta1-induced epithelial-mesenchymal transition in rat peritoneal mesothelial cells, and the role of astragalus intervention. Chin J Integr Med, 2012.
52.Wang, H., et al., Epithelial-mesenchymal transition (EMT) induced by TNF-alpha requires AKT/GSK-3beta-mediated stabilization of snail in colorectal cancer. PLoS One, 2013. 8(2): p. e56664.
53.Li, C.W., et al., Epithelial-mesenchymal transition induced by TNF-alpha requires NF-kappaB-mediated transcriptional upregulation of Twist1. Cancer Res, 2012. 72(5): p. 1290-300.
54.Castellino, R.C. and D.L. Durden, Mechanisms of disease: the PI3K-Akt-PTEN signaling node--an intercept point for the control of angiogenesis in brain tumors. Nat Clin Pract Neurol, 2007. 3(12): p. 682-93.
55.Theys, J., et al., E-Cadherin loss associated with EMT promotes radioresistance in human tumor cells. Radiother Oncol, 2011. 99(3): p. 392-7.
56.Liu, Y., et al., Activation of AKT is associated with metastasis of nasopharyngeal carcinoma. Tumour Biol, 2012. 33(1): p. 241-5.
57.Chen, W., et al., Effect of AKT inhibition on epithelial-mesenchymal transition and ZEB1-potentiated radiotherapy in nasopharyngeal carcinoma. Oncol Lett, 2013. 6(5): p. 1234-1240.
58.Larue, L. and A. Bellacosa, Epithelial-mesenchymal transition in development and cancer: role of phosphatidylinositol 3'' kinase/AKT pathways. Oncogene, 2005. 24(50): p. 7443-54.
59.Rodrigo, J.P., et al., Clinical significance of annexin A2 downregulation in oral squamous cell carcinoma. Head Neck, 2011. 33(12): p. 1708-14.
60.Zhong, L.P., et al., Increased expression of Annexin A2 in oral squamous cell carcinoma. Arch Oral Biol, 2009. 54(1): p. 17-25.
61.Liao, S.K., et al., Chromosomal abnormalities of a new nasopharyngeal carcinoma cell line (NPC-BM1) derived from a bone marrow metastatic lesion. Cancer Genet Cytogenet, 1998. 103(1): p. 52-8.
62.Folberg, R., M.J. Hendrix, and A.J. Maniotis, Vasculogenic mimicry and tumor angiogenesis. Am J Pathol, 2000. 156(2): p. 361-81.
63.Lorusso, A., et al., Annexin2 coating the surface of enlargeosomes is needed for their regulated exocytosis. EMBO J, 2006. 25(23): p. 5443-56.
64.Cocucci, E., et al., Enlargeosome traffic: exocytosis triggered by various signals is followed by endocytosis, membrane shedding or both. Traffic, 2007. 8(6): p. 742-57.
65.Prada, I., et al., The Ca2+-dependent exocytosis of enlargeosomes is greatly reinforced by genistein via a non-tyrosine kinase-dependent mechanism. FEBS Lett, 2007. 581(25): p. 4932-6.
66.Madureira, P.A., et al., Genotoxic agents promote the nuclear accumulation of annexin A2: role of annexin A2 in mitigating DNA damage. PLoS One, 2012. 7(11): p. e50591.
67.Chow, B.H., et al., Increased expression of annexin I is associated with drug-resistance in nasopharyngeal carcinoma and other solid tumors. Proteomics Clin Appl, 2009. 3(6): p. 654-62.
68.Zeng, G.Q., et al., Annexin A1: a new biomarker for predicting nasopharyngeal carcinoma response to radiotherapy. Med Hypotheses, 2013. 81(1): p. 68-70.
69.Hucumenoglu, S., et al., Annexin A2, A7, and A11 expression in head and neck squamous cell carcinomas. Turkish Journal of Medical Sciences, 2009. 39(4): p. 547-555.
70.Cheng, J.C., et al., Radiation-enhanced hepatocellular carcinoma cell invasion with MMP-9 expression through PI3K/Akt/NF-kappaB signal transduction pathway. Oncogene, 2006. 25(53): p. 7009-18.
71.Sakamoto, K. and G.D. Holman, Emerging role for AS160/TBC1D4 and TBC1D1 in the regulation of GLUT4 traffic. Am J Physiol Endocrinol Metab, 2008. 295(1): p. E29-37.
72.Rajendran, L., H.J. Knolker, and K. Simons, Subcellular targeting strategies for drug design and delivery. Nat Rev Drug Discov, 2010. 9(1): p. 29-42.