惡性腫瘤已經32年蟬連臺灣十大死因之首，根據103年臺灣衛生福利部統計，胰臟癌的死亡率排名第五，與十年前相比上升了兩個名次。癌症轉移是導致胰臟癌病患死亡最主要的原因，超過八成以上的病患發生局部或遠端轉移後才被診斷出胰臟癌；另外胰臟癌初期就會發生癌症轉移，導致胰臟癌病患的五年存活率低於5%。為了更有效治療胰臟癌並提升病患的存活率，了解胰臟癌轉移的機制是目前很重要的課題。本研究中，首先將PANC1細胞經過五次transwell invasion assay篩選建立高侵襲性的PANC1-I5細胞，模擬胰臟癌細胞轉移時的細胞狀態，經由二維差異電泳(2D-DIGE)與基質輔助雷射脫附游離飛行時間質譜儀(MALDI-TOF-MS)分析這兩種細胞株之間蛋白質表現差異，探討胰臟癌轉移的相關的分子機制。本研究結果顯示，PANC1與PANC1-I5細胞之間有88個蛋白質具有顯著的表現量差異，並使用西方墨點法驗證蛋白質表現量。在這些蛋白質中，挑選出具潛力的蛋白Galectin-1，並利用RNA干擾技術抑制Galectin-1蛋白表現量，我們發現可以有效抑制PANC1-I5細胞移動、侵襲與增殖的能力。除此之外，胰臟癌的轉移也會受到Cathepsin D、Annexin A2和Protein S100-A10所調控，未來也有機會可以成為胰臟癌診斷或治療的分子標靶。利用蛋白質體分析技術可以快速鑑定出與胰臟癌轉移機制有關的蛋白質，在未來胰臟癌轉移的診斷與治療上，可以成為很有潛力的標記分子。

In 2014, pancreatic cancer is the 8th leading cause of death in cancer in Taiwan. Cancer metastasis is the major cause of death in pancreatic cancer. Five-year survival rate of pancreatic cancer is lower than 5% due to the early cancer metastasis and delayed diagnosis. In order to reduce the mortality rate of pancreatic cancer, the finding of metastatic biomarkers is essential both in pancreatic cancer prognosis and therapy. In this study, we established a pair of pancreatic ductal adenocarcinoma cells, PANC1 and its highly invasive partner PANC1-I5 as a model system to reveal the metastatic mechanism. We use two-dimensional gel electrophoresis (2D-GIGE) and matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI-TOF-MS) to exam the protein expression changes between PANC1 and PANC1-I5. Proteomic study revealed that the expression of 88 proteins are significantly modulated between PNAC1 cells and PANC1-I5 cells. Potential candidate proteins associated with pancreatic metastasis including Galectin-1 were validated by immunoblotting. Further studies have used RNA interference to evaluate the biological properties of Galectin-1 in regulating cell migration, invasion, and proliferation, in vitro. In addition, our data also demonstrated that the identified proteins such as Cathepsin D、Annexin A2, and Protein S100-A10 were potential cellular targets for modulating the metastatic process of pancreatic cancer. To sum up, proteomics approach provides numerous potential therapeutic candidates and markers for the treatment in pancreatic cancer metastasis.

1 Alexakis, N. et al. Current standards of surgery for pancreatic cancer. Br J Surg 91, 1410-1427, doi:10.1002/bjs.4794 (2004).
2 Ministry of Health and Welfare, T. R. O. C. 2012 statistics of causes of death. (2013).
3 UK, C. R. Worldwide cancer mortality statistics. (2013).
4 UK, C. R. Worldwide cancer incidence statistics. (2013).
5 International, W. C. R. F. Pancreatic cancer statistics. (2012).
6 Fuchs, C. S. et al. A prospective study of cigarette smoking and the risk of pancreatic cancer. Arch Intern Med 156, 2255-2260 (1996).
7 Bosetti, C. et al. Cigarette smoking and pancreatic cancer: an analysis from the International Pancreatic Cancer Case-Control Consortium (Panc4). Ann Oncol 23, 1880-1888, doi:10.1093/annonc/mdr541 (2012).
8 Bracci, P. M. Obesity and pancreatic cancer: overview of epidemiologic evidence and biologic mechanisms. Mol Carcinog 51, 53-63, doi:10.1002/mc.20778 (2012).
9 Morrison, M. Pancreatic cancer and diabetes. Adv Exp Med Biol 771, 229-239 (2012).
10 Cui, Y. & Andersen, D. K. Diabetes and pancreatic cancer. Endocr Relat Cancer 19, F9-F26, doi:10.1530/ERC-12-0105 (2012).
11 Vincent, A., Herman, J., Schulick, R., Hruban, R. H. & Goggins, M. Pancreatic cancer. Lancet 378, 607-620, doi:10.1016/S0140-6736(10)62307-0 (2011).
12 Society, A. C. Signs and symptoms of pancreatic cancer. (2015).
13 Surveillance, E., and End Results Program. SEER Stat Fact Sheets: Pancreas Cancer. (2015).
14 Hruban, R. H., Goggins, M., Parsons, J. & Kern, S. E. Progression model for pancreatic cancer. Clin Cancer Res 6, 2969-2972 (2000).
15 Rhim, A. D. et al. EMT and dissemination precede pancreatic tumor formation. Cell 148, 349-361, doi:10.1016/j.cell.2011.11.025 (2012).
16 Wolfgang, C. L. et al. Recent progress in pancreatic cancer. CA Cancer J Clin 63, 318-348, doi:10.3322/caac.21190 (2013).
17 Kleeff, J. et al. Surgery for recurrent pancreatic ductal adenocarcinoma. Ann Surg 245, 566-572, doi:10.1097/01.sla.0000245845.06772.7d (2007).
18 Croce, C. M. Oncogenes and cancer. N Engl J Med 358, 502-511, doi:10.1056/NEJMra072367 (2008).
19 Wirtz, D., Konstantopoulos, K. & Searson, P. C. The physics of cancer: the role of physical interactions and mechanical forces in metastasis. Nat Rev Cancer 11, 512-522, doi:10.1038/nrc3080 (2011).
20 Zetter, B. R. Angiogenesis and tumor metastasis. Annu Rev Med 49, 407-424, doi:10.1146/annurev.med.49.1.407 (1998).
21 Valastyan, S. & Weinberg, R. A. Tumor metastasis: molecular insights and evolving paradigms. Cell 147, 275-292, doi:10.1016/j.cell.2011.09.024 (2011).
22 Royer, C. & Lu, X. Epithelial cell polarity: a major gatekeeper against cancer? Cell Death Differ 18, 1470-1477, doi:10.1038/cdd.2011.60 (2011).
23 Thiery, J. P. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2, 442-454, doi:10.1038/nrc822 (2002).
24 Lamouille, S., Xu, J. & Derynck, R. Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol 15, 178-196, doi:10.1038/nrm3758 (2014).
25 Wasinger, V. C. et al. Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium. Electrophoresis 16, 1090-1094 (1995).
26 Wilkins, M. R. et al. From proteins to proteomes: large scale protein identification by two-dimensional electrophoresis and amino acid analysis. Biotechnology (N Y) 14, 61-65 (1996).
27 O'Farrell, P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250, 4007-4021 (1975).
28 Macko, V. & Stegemann, H. Free electrofocusing in a coil of polyethylene tubing. Anal Biochem 37, 186-190 (1970).
29 Kondo, T. & Hirohashi, S. Application of 2D-DIGE in cancer proteomics toward personalized medicine. Methods Mol Biol 577, 135-154, doi:10.1007/978-1-60761-232-2_11 (2009).
30 Unlu, M., Morgan, M. E. & Minden, J. S. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18, 2071-2077, doi:10.1002/elps.1150181133 (1997).
31 Timms, J. F. & Cramer, R. Difference gel electrophoresis. Proteomics 8, 4886-4897, doi:10.1002/pmic.200800298 (2008).
32 Westermeier, R. & Scheibe, B. Difference gel electrophoresis based on lys/cys tagging. Methods Mol Biol 424, 73-85, doi:10.1007/978-1-60327-064-9_7 (2008).
33 Lieber, M., Mazzetta, J., Nelson-Rees, W., Kaplan, M. & Todaro, G. Establishment of a continuous tumor-cell line (panc-1) from a human carcinoma of the exocrine pancreas. Int J Cancer 15, 741-747 (1975).
34 Liu, F. T. & Rabinovich, G. A. Galectins as modulators of tumour progression. Nat Rev Cancer 5, 29-41, doi:10.1038/nrc1527 (2005).
35 Camby, I., Le Mercier, M., Lefranc, F. & Kiss, R. Galectin-1: a small protein with major functions. Glycobiology 16, 137R-157R, doi:10.1093/glycob/cwl025 (2006).
36 Clausse, N., van den Brule, F., Waltregny, D., Garnier, F. & Castronovo, V. Galectin-1 expression in prostate tumor-associated capillary endothelial cells is increased by prostate carcinoma cells and modulates heterotypic cell-cell adhesion. Angiogenesis 3, 317-325 (1999).
37 van den Brule, F. A. et al. Galectin-1 modulates human melanoma cell adhesion to laminin. Biochem Biophys Res Commun 209, 760-767 (1995).
38 Astorgues-Xerri, L. et al. Unraveling galectin-1 as a novel therapeutic target for cancer. Cancer Treat Rev 40, 307-319, doi:10.1016/j.ctrv.2013.07.007 (2014).
39 Sakaguchi, M. et al. A carbohydrate-binding protein, Galectin-1, promotes proliferation of adult neural stem cells. Proc Natl Acad Sci U S A 103, 7112-7117, doi:10.1073/pnas.0508793103 (2006).
40 Lutomski, D. et al. Externalization and binding of galectin-1 on cell surface of K562 cells upon erythroid differentiation. Glycobiology 7, 1193-1199 (1997).
41 Gu, M., Wang, W., Song, W. K., Cooper, D. N. & Kaufman, S. J. Selective modulation of the interaction of alpha 7 beta 1 integrin with fibronectin and laminin by L-14 lectin during skeletal muscle differentiation. J Cell Sci 107 ( Pt 1), 175-181 (1994).
42 Jung, T. Y. et al. Role of galectin-1 in migration and invasion of human glioblastoma multiforme cell lines. J Neurosurg 109, 273-284, doi:10.3171/JNS/2008/109/8/0273 (2008).
43 Kim, H. J. et al. High galectin-1 expression correlates with poor prognosis and is involved in epithelial ovarian cancer proliferation and invasion. Eur J Cancer 48, 1914-1921, doi:10.1016/j.ejca.2012.02.005 (2012).
44 Spano, D. et al. Galectin-1 and its involvement in hepatocellular carcinoma aggressiveness. Mol Med 16, 102-115, doi:10.2119/molmed.2009.00119 (2010).
45 Ito, K. et al. Thiodigalactoside inhibits murine cancers by concurrently blocking effects of galectin-1 on immune dysregulation, angiogenesis and protection against oxidative stress. Angiogenesis 14, 293-307, doi:10.1007/s10456-011-9213-5 (2011).
46 Dalotto-Moreno, T. et al. Targeting galectin-1 overcomes breast cancer-associated immunosuppression and prevents metastatic disease. Cancer Res 73, 1107-1117, doi:10.1158/0008-5472.CAN-12-2418 (2013).
47 Kim, H. J. et al. Galectin 1 expression is associated with tumor invasion and metastasis in stage IB to IIA cervical cancer. Hum Pathol 44, 62-68 (2013).
48 Chen, R. et al. Pancreatic cancer proteome: The proteins that underlie invasion, metastasis, and immunologic escape. Gastroenterology 129, 1187-1197 (2005).
49 Mendez, M. G., Kojima, S. & Goldman, R. D. Vimentin induces changes in cell shape, motility, and adhesion during the epithelial to mesenchymal transition. FASEB J 24, 1838-1851, doi:10.1096/fj.09-151639 (2010).
50 Zhang, W. et al. Annexin A2 promotes the migration and invasion of human hepatocellular carcinoma cells in vitro by regulating the shedding of CD147-harboring microvesicles from tumor cells. Plos One 8, e67268, doi:10.1371/journal.pone.0067268 (2013).
51 Berberat, P. O. et al. Comparative analysis of galectins in primary tumors and tumor metastasis in human pancreatic cancer. J Histochem Cytochem 49, 539-549 (2001).
52 Barondes, S. H. et al. Galectins: a family of animal beta-galactoside-binding lectins. Cell 76, 597-598 (1994).
53 Barrow, H., Rhodes, J. M. & Yu, L. G. The role of galectins in colorectal cancer progression. Int J Cancer 129, 1-8, doi:10.1002/ijc.25945 (2011).
54 Szoke, T. et al. Prognostic significance of endogenous adhesion/growth-regulatory lectins in lung cancer. Oncology 69, 167-174, doi:10.1159/000087841 (2005).
55 Chow, S. N. et al. Analysis of protein profiles in human epithelial ovarian cancer tissues by proteomic technology. Eur J Gynaecol Oncol 31, 55-62 (2010).
56 Hsu, Y. L. et al. Galectin-1 promotes lung cancer tumor metastasis by potentiating integrin alpha6beta4 and Notch1/Jagged2 signaling pathway. Carcinogenesis 34, 1370-1381, doi:10.1093/carcin/bgt040 (2013).
57 Laderach, D. J. et al. A unique galectin signature in human prostate cancer progression suggests galectin-1 as a key target for treatment of advanced disease. Cancer Res 73, 86-96, doi:10.1158/0008-5472.CAN-12-1260 (2013).
58 Tang, D. et al. Pancreatic satellite cells derived galectin-1 increase the progression and less survival of pancreatic ductal adenocarcinoma. Plos One 9, e90476, doi:10.1371/journal.pone.0090476 (2014).
59 Scott, K. & Weinberg, C. Galectin-1: a bifunctional regulator of cellular proliferation. Glycoconj J 19, 467-477, doi:10.1023/B:GLYC.0000014076.43288.89 (2004).
60 Fischer, C. et al. Galectin-1 interacts with the {alpha}5{beta}1 fibronectin receptor to restrict carcinoma cell growth via induction of p21 and p27. J Biol Chem 280, 37266-37277, doi:10.1074/jbc.M411580200 (2005).
61 Wells, V., Davies, D. & Mallucci, L. Cell cycle arrest and induction of apoptosis by beta galactoside binding protein (beta GBP) in human mammary cancer cells. A potential new approach to cancer control. Eur J Cancer 35, 978-983 (1999).
62 Banh, A. et al. Tumor galectin-1 mediates tumor growth and metastasis through regulation of T-cell apoptosis. Cancer Res 71, 4423-4431, doi:10.1158/0008-5472.CAN-10-4157 (2011).
63 Li, H., Wang, Y. & Zhou, F. Effect of ex vivo-expanded gammadelta-T cells combined with galectin-1 antibody on the growth of human cervical cancer xenografts in SCID mice. Clin Invest Med 33, E280-289 (2010).
64 Fernandez-Medarde, A. & Santos, E. Ras in cancer and developmental diseases. Genes Cancer 2, 344-358, doi:10.1177/1947601911411084 (2011).
65 Paz, A., Haklai, R., Elad-Sfadia, G., Ballan, E. & Kloog, Y. Galectin-1 binds oncogenic H-Ras to mediate Ras membrane anchorage and cell transformation. Oncogene 20, 7486-7493, doi:10.1038/sj.onc.1204950 (2001).
66 Lee, M. Y., Lee, S. H., Park, J. H. & Han, H. J. Interaction of galectin-1 with caveolae induces mouse embryonic stem cell proliferation through the Src, ERas, Akt and mTOR signaling pathways. Cell Mol Life Sci 66, 1467-1478, doi:10.1007/s00018-009-8691-8 (2009).
67 Vousden, K. H. & Ryan, K. M. p53 and metabolism. Nat Rev Cancer 9, 691-700, doi:10.1038/nrc2715 (2009).
68 Montcourrier, P. et al. Cathepsin-D in Breast-Cancer Cells Can Digest Extracellular-Matrix in Large Acidic Vesicles. Cancer Research 50, 6045-6054 (1990).
69 Tan, G. J., Peng, Z. K., Lu, J. P. & Tang, F. Q. Cathepsins mediate tumor metastasis. World J Biol Chem 4, 91-101, doi:10.4331/wjbc.v4.i4.91 (2013).
70 Wang, C. Y. & Lin, C. F. Annexin A2: its molecular regulation and cellular expression in cancer development. Dis Markers 2014, 308976, doi:10.1155/2014/308976 (2014).
71 Rao, J. S. Molecular mechanisms of glioma invasiveness: the role of proteases. Nat Rev Cancer 3, 489-501, doi:10.1038/nrc1121 (2003).
72 Etienne-Manneville, S. Cdc42--the centre of polarity. J Cell Sci 117, 1291-1300, doi:10.1242/jcs.01115 (2004).
73 Matas, O. B., Martinez-Menarguez, J. A. & Egea, G. Association of Cdc42/N-WASP/Arp2/3 signaling pathway with Golgi membranes. Traffic 5, 838-846, doi:10.1111/j.1600-0854.2004.00225.x (2004).
74 Liu, Z. et al. PDZ and LIM domain protein 1(PDLIM1)/CLP36 promotes breast cancer cell migration, invasion and metastasis through interaction with alpha-actinin. Oncogene 34, 1300-1311, doi:10.1038/onc.2014.64 (2015).
75 Naik, M. U. & Naik, U. P. Junctional adhesion molecule-A-induced endothelial
cell migration on vitronectin is integrin alpha v beta 3 specific. J Cell Sci 119,
490-499, doi:10.1242/jcs.02771 (2006).