口腔癌是頭頸癌的一種，百分之九十以上的口腔癌屬於口腔鱗狀上皮細胞癌。在過去十年間，口腔癌在台灣的死亡率已經上升到第五名，因此口腔癌的研究便顯得非常重要。口腔癌的轉移通常發生在病程的中後期，同時也是它致死的原因。現今，治療口腔癌的方法主要是透過手術切除大部分的癌症，再利用化療以及放射線治療等方式，移除手術後所遺留下來的癌細胞。但是，許多惡性轉移的口腔癌患者在接受治療後，常常會有復發的情形發生。因此，避免口腔癌的轉移便可以作為一種減少患者復發的方法。我們致力於研究口腔癌細胞可能的轉移機制，希望可以降低口腔癌病患復發的風險。在這篇論文中，我們使用了由原位癌分離培養的口腔癌細胞株 (OC3 cells) 以及經篩選後轉移能力較強的細胞株 (IV2 cells)，來探討惡性口腔癌細胞轉移的機制。研究結果顯示，EZH2這個組蛋白甲基轉移酶，在轉移能力較強的IV2 細胞中是下降的。而EZH2的下降會透過甲基化H3K27而增加ADAMTS1、MMP3、ROS1、CXCL1以及GLI1基因的表現，進而增強IV2細胞的轉移能力。同時，我也證實了轉錄因子STAT3為EZH2的上游，它會藉由調控上述的機制而提升IV2細胞的轉移能力。此外，在IV2細胞中IL8訊號以及STAT3間具有反饋迴路 (feedback loop)，互相調控彼此。總結此篇論文研究結果，EZH2在口腔癌中可以藉由調控轉移相關的基因，而扮演一個抑制腫瘤生長的角色。

The cancer mortality rate of oral cancer has risen from the sixth to the fifth place in the past decade in Taiwan. More than 90% of oral cancers are oral squamous cell carcinomas (OSCC). Currently, patients with malignant oral cancer are treated with surgery to remove a large proportion of tumor, followed by radiation therapy or chemotherapy to destroy any remaining cancer cells. However, many patients developed recurrence after treatment, especially for those who suffered from lymphatic metastasis and distant metastasis. Thus, preventing metastasis is a way to reduce recurrence. In this thesis, we investigated the possible mechanism that promotes metastasis of OSCC. To this end, we used an OSCC cell line, OC3, which was derived from a betel nut-chewing oral cancer patient, and a more invasive line, IV2, to study the mechanism of OSCC invasion. We found that the expression of EZH2, a histone methyltransferase responsible for tri-methylation of histone H3 at lysine 27 (H3K27me3), was lower in the more invasive IV2 cells compared to that in OC3 cells. Consistent with this finding, knocking down EZH2 in OC3 cells enhanced migration and invasion. Several target genes of EZH2 have been identified, including ADAMTS1, MMP3, ROS1, CXCL1 and GLI1. To determine what leads to reduced EZH2 in IV2 cells, chromatin immunoprecipitation (ChIP) analysis suggests the involvement of STAT3 in the transcriptional expression of EZH2. In fact, STAT3 level was reduced in IV2 cells compared to OC3 cells. Knocking down STAT3 or inhibiting STAT3 decreased EZH2 expression and increased EZH2 target genes, ADAMTS1, ROS1, CXCL1 and GLI1, to regulate cell migration, invasion and proliferation. We also determined the feedback regulation between STAT3 and IL8 signaling, an upstream of STAT3. Taken together, these findings implicate that EZH2 may serve as a tumor suppressor by inhibiting metastasis-related genes in OSCC cells.

Abstract i
中文摘要 ii
誌謝 iii
Index iv
Abbreviations vi
Introduction 1
Oral cancer 1
Cancer metastasis 2
Histone modifications 2
Polycomb repressive complex 2 (PRC2) 3
Signal transducer and activator of transcription 3 (STAT3) 5
Interleukin 8 (IL8) 6
A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) 6
Matrix metallopeptidases (MMPs) 7
ROS1 8
CXC motif chemokine ligand 1 (CXCL1) 8
GLI family zinc finger 1 (GLI1) 9
Materials and Methods 10
Antibodies and reagents 10
Cell lines and cell culture 10
Knocking down EZH2 and STAT3 by using lentivirus to express shRNA constructs 11
MTT assay 11
Colony formation assay 12
Migration and invasion assay 12
Total RNA extraction, reverse transcription polymerase chain reaction (RT-PCR), polymerase chain reaction (PCR) and semi-quantitative real-time polymerase chain reaction (Q-PCR) 12
Protein extraction and western blotting 13
Chromatin immunoprecipitation (ChIP) analysis 13
Statistical analysis 14
Results 15
EZH2 level is decreased in more invasive IV2 cells 15
Reduced EZH2 enhances cell migration and invasion of OSCC cells 15
Reduced EZH2 increases the expressions of ADAMTS1, MMP3, ROS1, CXCL1 and GLI1 by decreasing H3K27me3 modification in the more invasive IV2 cells 16
Reduced STAT3 contributes to decreased EZH2 in IV2 cells 17
Reduced STAT3 enhances cell proliferation, migration and invasion by increasing EZH2 target genes 18
IL8 signaling induces STAT3 reduction in IV2 cells and STAT3 has a feedback loop to regulate IL8 signaling 19
Discussion 21
Figures 25
Figure 1. The expression of EZH2 is decreased in more invasive IV2 cells 25
Figure 2. Reduced EZH2 induces cell migration and invasion 26
Figure 3. Reduced EZH2 increases the expressions of ADAMTS1, MMP3, ROS1, CXCL1 and GLI1 by H3K27me3 modification 27
Figure 4. STAT3 is an upstream of EZH2 28
Figure 5. Knocking down STAT3 enhances cell migration and invasion by increasing the expressions of EZH2 target genes 29
Figure 6. The feedback loop between IL8 signaling and STAT3 30
Figure 7. Working model of reduced EZH2 promoting oral cancer progression 31
Figure 8. The activity of STAT3 may be regulated by SHP1 32
Appendix 33
Figure A1. The expressions of ADAMTS1, MMP3, ROS1, CXCL1 and GLI1 in OSCC-shEZH2 cells 33
Table 34
Table 1. Primer sequences for PCR and Q-PCR 34
Table 2. Primer sequences for ChIP assay 35
References 36

1. Ferlay, J., Soerjomataram, I., Dikshit, R., Eser, S., Mathers, C., Rebelo, M., Parkin, D. M., Forman, D., and Bray, F. (2015) Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. International journal of cancer 136, E359-386
2. Warnakulasuriya, S. (2009) Global epidemiology of oral and oropharyngeal cancer. Oral oncology 45, 309-316
3. Dionne, K. R., Warnakulasuriya, S., Zain, R. B., and Cheong, S. C. (2015) Potentially malignant disorders of the oral cavity: current practice and future directions in the clinic and laboratory. International journal of cancer 136, 503-515
4. Markopoulos, A. K. (2012) Current aspects on oral squamous cell carcinoma. Open Dent J 6, 126-130
5. Petti, S. (2009) Lifestyle risk factors for oral cancer. Oral oncology 45, 340-350
6. Ernani, V., and Saba, N. F. (2015) Oral Cavity Cancer: Risk Factors, Pathology, and Management. Oncology 89, 187-195
7. Steeg, P. S. (2006) Tumor metastasis: mechanistic insights and clinical challenges. Nature medicine 12, 895-904
8. Nguyen, D. X., and Massague, J. (2007) Genetic determinants of cancer metastasis. Nat Rev Genet 8, 341-352
9. Hunter, K. W., Crawford, N. P., and Alsarraj, J. (2008) Mechanisms of metastasis. Breast cancer research : BCR 10 Suppl 1, S2
10. Mimori, K., Kataoka, A., Yoshinaga, K., Ohta, M., Sagara, Y., Yoshikawa, Y., Ohno, S., Barnard, G. F., and Mori, M. (2005) Identification of molecular markers for metastasis-related genes in primary breast cancer cells. Clin Exp Metastasis 22, 59-67
11. Deryugina, E. I., and Quigley, J. P. (2006) Matrix metalloproteinases and tumor metastasis. Cancer metastasis reviews 25, 9-34
12. Yoo, C. B., and Jones, P. A. (2006) Epigenetic therapy of cancer: past, present and future. Nat Rev Drug Discov 5, 37-50
13. Mascolo, M., Siano, M., Ilardi, G., Russo, D., Merolla, F., De Rosa, G., and Staibano, S. (2012) Epigenetic disregulation in oral cancer. International journal of molecular sciences 13, 2331-2353
14. Gasche, J. A., and Goel, A. (2012) Epigenetic mechanisms in oral carcinogenesis. Future Oncol 8, 1407-1425
15. Jithesh, P. V., Risk, J. M., Schache, A. G., Dhanda, J., Lane, B., Liloglou, T., and Shaw, R. J. (2013) The epigenetic landscape of oral squamous cell carcinoma. British journal of cancer 108, 370-379
16. Volkel, P., and Angrand, P. O. (2007) The control of histone lysine methylation in epigenetic regulation. Biochimie 89, 1-20
17. Kouzarides, T. (2007) Chromatin modifications and their function. Cell 128, 693-705
18. Lachner, M., and Jenuwein, T. (2002) The many faces of histone lysine methylation. Curr Opin Cell Biol 14, 286-298
19. Tyan, S. W., Hsu, C. H., Peng, K. L., Chen, C. C., Kuo, W. H., Lee, E. Y., Shew, J. Y., Chang, K. J., Juan, L. J., and Lee, W. H. (2012) Breast cancer cells induce stromal fibroblasts to secrete ADAMTS1 for cancer invasion through an epigenetic change. PloS one 7, e35128
20. Valk-Lingbeek, M. E., Bruggeman, S. W., and van Lohuizen, M. (2004) Stem cells and cancer; the polycomb connection. Cell 118, 409-418
21. Laugesen, A., and Helin, K. (2014) Chromatin repressive complexes in stem cells, development, and cancer. Cell Stem Cell 14, 735-751
22. Han, Z., Xing, X., Hu, M., Zhang, Y., Liu, P., and Chai, J. (2007) Structural basis of EZH2 recognition by EED. Structure 15, 1306-1315
23. Pasini, D., Bracken, A. P., Jensen, M. R., Lazzerini Denchi, E., and Helin, K. (2004) Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity. The EMBO journal 23, 4061-4071
24. Plath, K., Fang, J., Mlynarczyk-Evans, S. K., Cao, R., Worringer, K. A., Wang, H., de la Cruz, C. C., Otte, A. P., Panning, B., and Zhang, Y. (2003) Role of histone H3 lysine 27 methylation in X inactivation. Science 300, 131-135
25. Li, T., Chen, H., Li, W., Cui, J., Wang, G., Hu, X., Hoffman, A. R., and Hu, J. (2014) Promoter histone H3K27 methylation in the control of IGF2 imprinting in human tumor cell lines. Human molecular genetics 23, 117-128
26. Pasini, D., Bracken, A. P., and Helin, K. (2004) Polycomb group proteins in cell cycle progression and cancer. Cell cycle 3, 396-400
27. Yoo, K. H., and Hennighausen, L. (2012) EZH2 methyltransferase and H3K27 methylation in breast cancer. International journal of biological sciences 8, 59-65
28. van Leenders, G. J., Dukers, D., Hessels, D., van den Kieboom, S. W., Hulsbergen, C. A., Witjes, J. A., Otte, A. P., Meijer, C. J., and Raaphorst, F. M. (2007) Polycomb-group oncogenes EZH2, BMI1, and RING1 are overexpressed in prostate cancer with adverse pathologic and clinical features. European urology 52, 455-463
29. Margueron, R., and Reinberg, D. (2011) The Polycomb complex PRC2 and its mark in life. Nature 469, 343-349
30. Qian, C., and Zhou, M. M. (2006) SET domain protein lysine methyltransferases: Structure, specificity and catalysis. Cell Mol Life Sci 63, 2755-2763
31. Dillon, S. C., Zhang, X., Trievel, R. C., and Cheng, X. (2005) The SET-domain protein superfamily: protein lysine methyltransferases. Genome Biol 6, 227
32. Zhang, Y., and Reinberg, D. (2001) Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Genes & development 15, 2343-2360
33. Czermin, B., Melfi, R., McCabe, D., Seitz, V., Imhof, A., and Pirrotta, V. (2002) Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites. Cell 111, 185-196
34. Cao, R., and Zhang, Y. (2004) SUZ12 is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex. Molecular cell 15, 57-67
35. Kaneko, S., Li, G., Son, J., Xu, C. F., Margueron, R., Neubert, T. A., and Reinberg, D. (2010) Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA. Genes & development 24, 2615-2620
36. Bracken, A. P., Pasini, D., Capra, M., Prosperini, E., Colli, E., and Helin, K. (2003) EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer. The EMBO journal 22, 5323-5335
37. Lin, Y. W., Ren, L. L., Xiong, H., Du, W., Yu, Y. N., Sun, T. T., Weng, Y. R., Wang, Z. H., Wang, J. L., Wang, Y. C., Cui, Y., Sun, D. F., Han, Z. G., Shen, N., Zou, W., Xu, J., Chen, H. Y., Cao, W., Hong, J., and Fang, J. Y. (2013) Role of STAT3 and vitamin D receptor in EZH2-mediated invasion of human colorectal cancer. The Journal of pathology 230, 277-290
38. Alford, S. H., Toy, K., Merajver, S. D., and Kleer, C. G. (2012) Increased risk for distant metastasis in patients with familial early-stage breast cancer and high EZH2 expression. Breast cancer research and treatment 132, 429-437
39. Chang, C. J., Yang, J. Y., Xia, W., Chen, C. T., Xie, X., Chao, C. H., Woodward, W. A., Hsu, J. M., Hortobagyi, G. N., and Hung, M. C. (2011) EZH2 promotes expansion of breast tumor initiating cells through activation of RAF1-beta-catenin signaling. Cancer cell 19, 86-100
40. Varambally, S., Dhanasekaran, S. M., Zhou, M., Barrette, T. R., Kumar-Sinha, C., Sanda, M. G., Ghosh, D., Pienta, K. J., Sewalt, R. G., Otte, A. P., Rubin, M. A., and Chinnaiyan, A. M. (2002) The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature 419, 624-629
41. Sasaki, M., Ikeda, H., Itatsu, K., Yamaguchi, J., Sawada, S., Minato, H., Ohta, T., and Nakanuma, Y. (2008) The overexpression of polycomb group proteins Bmi1 and EZH2 is associated with the progression and aggressive biological behavior of hepatocellular carcinoma. Laboratory investigation; a journal of technical methods and pathology 88, 873-882
42. Kim, K. H., and Roberts, C. W. (2016) Targeting EZH2 in cancer. Nature medicine 22, 128-134
43. Ntziachristos, P., Tsirigos, A., Van Vlierberghe, P., Nedjic, J., Trimarchi, T., Flaherty, M. S., Ferres-Marco, D., da Ros, V., Tang, Z., Siegle, J., Asp, P., Hadler, M., Rigo, I., De Keersmaecker, K., Patel, J., Huynh, T., Utro, F., Poglio, S., Samon, J. B., Paietta, E., Racevskis, J., Rowe, J. M., Rabadan, R., Levine, R. L., Brown, S., Pflumio, F., Dominguez, M., Ferrando, A., and Aifantis, I. (2012) Genetic inactivation of the polycomb repressive complex 2 in T cell acute lymphoblastic leukemia. Nature medicine 18, 298-301
44. Simon, C., Chagraoui, J., Krosl, J., Gendron, P., Wilhelm, B., Lemieux, S., Boucher, G., Chagnon, P., Drouin, S., Lambert, R., Rondeau, C., Bilodeau, A., Lavallee, S., Sauvageau, M., Hebert, J., and Sauvageau, G. (2012) A key role for EZH2 and associated genes in mouse and human adult T-cell acute leukemia. Genes & development 26, 651-656
45. Darnell, J. E., Jr., Kerr, I. M., and Stark, G. R. (1994) Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264, 1415-1421
46. Yu, H., Pardoll, D., and Jove, R. (2009) STATs in cancer inflammation and immunity: a leading role for STAT3. Nat Rev Cancer 9, 798-809
47. Akira, S., Nishio, Y., Inoue, M., Wang, X. J., Wei, S., Matsusaka, T., Yoshida, K., Sudo, T., Naruto, M., and Kishimoto, T. (1994) Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway. Cell 77, 63-71
48. Uddin, N., Kim, R. K., Yoo, K. C., Kim, Y. H., Cui, Y. H., Kim, I. G., Suh, Y., and Lee, S. J. (2015) Persistent activation of STAT3 by PIM2-driven positive feedback loop for epithelial-mesenchymal transition in breast cancer. Cancer science 106, 718-725
49. Lambert, A. W., Wong, C. K., Ozturk, S., Papageorgis, P., Raghunathan, R., Alekseyev, Y., Gower, A. C., Reinhard, B. M., Abdolmaleky, H. M., and Thiagalingam, S. (2016) Tumor Cell-Derived Periostin Regulates Cytokines That Maintain Breast Cancer Stem Cells. Molecular cancer research : MCR 14, 103-113
50. Seo, J. H., Jeong, K. J., Oh, W. J., Sul, H. J., Sohn, J. S., Kim, Y. K., Cho do, Y., Kang, J. K., Park, C. G., and Lee, H. Y. (2010) Lysophosphatidic acid induces STAT3 phosphorylation and ovarian cancer cell motility: their inhibition by curcumin. Cancer letters 288, 50-56
51. Hirano, T., Ishihara, K., and Hibi, M. (2000) Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors. Oncogene 19, 2548-2556
52. Yuan, Z. L., Guan, Y. J., Wang, L., Wei, W., Kane, A. B., and Chin, Y. E. (2004) Central role of the threonine residue within the p+1 loop of receptor tyrosine kinase in STAT3 constitutive phosphorylation in metastatic cancer cells. Molecular and cellular biology 24, 9390-9400
53. Yamanaka, Y., Nakajima, K., Fukada, T., Hibi, M., and Hirano, T. (1996) Differentiation and growth arrest signals are generated through the cytoplasmic region of gp130 that is essential for Stat3 activation. The EMBO journal 15, 1557-1565
54. Lin, W. W., and Karin, M. (2007) A cytokine-mediated link between innate immunity, inflammation, and cancer. The Journal of clinical investigation 117, 1175-1183
55. Selitrennik, M., and Lev, S. (2015) PYK2 integrates growth factor and cytokine receptors signaling and potentiates breast cancer invasion via a positive feedback loop. Oncotarget 6, 22214-22226
56. Xie, T. X., Wei, D., Liu, M., Gao, A. C., Ali-Osman, F., Sawaya, R., and Huang, S. (2004) Stat3 activation regulates the expression of matrix metalloproteinase-2 and tumor invasion and metastasis. Oncogene 23, 3550-3560
57. Garcia, R., Yu, C. L., Hudnall, A., Catlett, R., Nelson, K. L., Smithgall, T., Fujita, D. J., Ethier, S. P., and Jove, R. (1997) Constitutive activation of Stat3 in fibroblasts transformed by diverse oncoproteins and in breast carcinoma cells. Cell Growth Differ 8, 1267-1276
58. Kusaba, T., Nakayama, T., Yamazumi, K., Yakata, Y., Yoshizaki, A., Inoue, K., Nagayasu, T., and Sekine, I. (2006) Activation of STAT3 is a marker of poor prognosis in human colorectal cancer. Oncology reports 15, 1445-1451
59. Tang, Y. J., Sun, Z. L., Wu, W. G., Xing, J., He, Y. F., Xin, D. M., and Han, P. (2015) Inhibitor of signal transducer and activator of transcription 3 (STAT3) suppresses ovarian cancer growth, migration and invasion and enhances the effect of cisplatin in vitro. Genetics and molecular research : GMR 14, 2450-2460
60. Azare, J., Leslie, K., Al-Ahmadie, H., Gerald, W., Weinreb, P. H., Violette, S. M., and Bromberg, J. (2007) Constitutively activated Stat3 induces tumorigenesis and enhances cell motility of prostate epithelial cells through integrin beta 6. Molecular and cellular biology 27, 4444-4453
61. Musteanu, M., Blaas, L., Mair, M., Schlederer, M., Bilban, M., Tauber, S., Esterbauer, H., Mueller, M., Casanova, E., Kenner, L., Poli, V., and Eferl, R. (2010) Stat3 is a negative regulator of intestinal tumor progression in Apc(Min) mice. Gastroenterology 138, 1003-1011 e1001-1005
62. Schneller, D., Machat, G., Sousek, A., Proell, V., van Zijl, F., Zulehner, G., Huber, H., Mair, M., Muellner, M. K., Nijman, S. M., Eferl, R., Moriggl, R., and Mikulits, W. (2011) p19(ARF) /p14(ARF) controls oncogenic functions of signal transducer and activator of transcription 3 in hepatocellular carcinoma. Hepatology 54, 164-172
63. Ettl, T., Stiegler, C., Zeitler, K., Agaimy, A., Zenk, J., Reichert, T. E., Gosau, M., Kuhnel, T., Brockhoff, G., and Schwarz, S. (2012) EGFR, HER2, survivin, and loss of pSTAT3 characterize high-grade malignancy in salivary gland cancer with impact on prognosis. Human pathology 43, 921-931
64. Pectasides, E., Egloff, A. M., Sasaki, C., Kountourakis, P., Burtness, B., Fountzilas, G., Dafni, U., Zaramboukas, T., Rampias, T., Rimm, D., Grandis, J., and Psyrri, A. (2010) Nuclear localization of signal transducer and activator of transcription 3 in head and neck squamous cell carcinoma is associated with a better prognosis. Clinical cancer research : an official journal of the American Association for Cancer Research 16, 2427-2434
65. Waugh, D. J., and Wilson, C. (2008) The interleukin-8 pathway in cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 14, 6735-6741
66. Benoy, I. H., Salgado, R., Van Dam, P., Geboers, K., Van Marck, E., Scharpe, S., Vermeulen, P. B., and Dirix, L. Y. (2004) Increased serum interleukin-8 in patients with early and metastatic breast cancer correlates with early dissemination and survival. Clinical cancer research : an official journal of the American Association for Cancer Research 10, 7157-7162
67. Lehrer, S., Diamond, E. J., Mamkine, B., Stone, N. N., and Stock, R. G. (2004) Serum interleukin-8 is elevated in men with prostate cancer and bone metastases. Technology in cancer research & treatment 3, 411
68. Wigmore, S. J., Fearon, K. C., Sangster, K., Maingay, J. P., Garden, O. J., and Ross, J. A. (2002) Cytokine regulation of constitutive production of interleukin-8 and -6 by human pancreatic cancer cell lines and serum cytokine concentrations in patients with pancreatic cancer. International journal of oncology 21, 881-886
69. Brew, R., Erikson, J. S., West, D. C., Kinsella, A. R., Slavin, J., and Christmas, S. E. (2000) Interleukin-8 as an autocrine growth factor for human colon carcinoma cells in vitro. Cytokine 12, 78-85
70. Itoh, Y., Joh, T., Tanida, S., Sasaki, M., Kataoka, H., Itoh, K., Oshima, T., Ogasawara, N., Togawa, S., Wada, T., Kubota, H., Mori, Y., Ohara, H., Nomura, T., Higashiyama, S., and Itoh, M. (2005) IL-8 promotes cell proliferation and migration through metalloproteinase-cleavage proHB-EGF in human colon carcinoma cells. Cytokine 29, 275-282
71. Hu, M., and Polyak, K. (2008) Microenvironmental regulation of cancer development. Current opinion in genetics & development 18, 27-34
72. Tan Ide, A., Ricciardelli, C., and Russell, D. L. (2013) The metalloproteinase ADAMTS1: a comprehensive review of its role in tumorigenic and metastatic pathways. International journal of cancer 133, 2263-2276
73. Porter, S., Clark, I. M., Kevorkian, L., and Edwards, D. R. (2005) The ADAMTS metalloproteinases. The Biochemical journal 386, 15-27
74. Shindo, T., Kurihara, H., Kuno, K., Yokoyama, H., Wada, T., Kurihara, Y., Imai, T., Wang, Y., Ogata, M., Nishimatsu, H., Moriyama, N., Oh-hashi, Y., Morita, H., Ishikawa, T., Nagai, R., Yazaki, Y., and Matsushima, K. (2000) ADAMTS-1: a metalloproteinase-disintegrin essential for normal growth, fertility, and organ morphology and function. The Journal of clinical investigation 105, 1345-1352
75. Kuno, K., Kanada, N., Nakashima, E., Fujiki, F., Ichimura, F., and Matsushima, K. (1997) Molecular cloning of a gene encoding a new type of metalloproteinase-disintegrin family protein with thrombospondin motifs as an inflammation associated gene. The Journal of biological chemistry 272, 556-562
76. Brown, H. M., Dunning, K. R., Robker, R. L., Pritchard, M., and Russell, D. L. (2006) Requirement for ADAMTS-1 in extracellular matrix remodeling during ovarian folliculogenesis and lymphangiogenesis. Developmental biology 300, 699-709
77. Iruela-Arispe, M. L., Carpizo, D., and Luque, A. (2003) ADAMTS1: a matrix metalloprotease with angioinhibitory properties. Annals of the New York Academy of Sciences 995, 183-190
78. Kuno, K., Okada, Y., Kawashima, H., Nakamura, H., Miyasaka, M., Ohno, H., and Matsushima, K. (2000) ADAMTS-1 cleaves a cartilage proteoglycan, aggrecan. FEBS letters 478, 241-245
79. Lind, T., Birch, M. A., and McKie, N. (2006) Purification of an insect derived recombinant human ADAMTS-1 reveals novel gelatin (type I collagen) degrading activities. Molecular and cellular biochemistry 281, 95-102
80. Rodriguez-Manzaneque, J. C., Carpizo, D., Plaza-Calonge Mdel, C., Torres-Collado, A. X., Thai, S. N., Simons, M., Horowitz, A., and Iruela-Arispe, M. L. (2009) Cleavage of syndecan-4 by ADAMTS1 provokes defects in adhesion. The international journal of biochemistry & cell biology 41, 800-810
81. Egeblad, M., and Werb, Z. (2002) New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2, 161-174
82. Van Lint, P., and Libert, C. (2007) Chemokine and cytokine processing by matrix metalloproteinases and its effect on leukocyte migration and inflammation. J Leukoc Biol 82, 1375-1381
83. Ye, S., Eriksson, P., Hamsten, A., Kurkinen, M., Humphries, S. E., and Henney, A. M. (1996) Progression of coronary atherosclerosis is associated with a common genetic variant of the human stromelysin-1 promoter which results in reduced gene expression. The Journal of biological chemistry 271, 13055-13060
84. Eguchi, T., Kubota, S., Kawata, K., Mukudai, Y., Uehara, J., Ohgawara, T., Ibaragi, S., Sasaki, A., Kuboki, T., and Takigawa, M. (2008) Novel transcription-factor-like function of human matrix metalloproteinase 3 regulating the CTGF/CCN2 gene. Molecular and cellular biology 28, 2391-2413
85. Sun, N., Zhang, Q., Xu, C., Zhao, Q., Ma, Y., Lu, X., Wang, L., and Li, W. (2014) Molecular regulation of ovarian cancer cell invasion. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 35, 11359-11366
86. Li, X., and Tai, H. H. (2012) Increased expression of matrix metalloproteinases mediates thromboxane A2-induced invasion in lung cancer cells. Curr Cancer Drug Targets 12, 703-715
87. Kmiecik, A. M., Pula, B., Suchanski, J., Olbromski, M., Gomulkiewicz, A., Owczarek, T., Kruczak, A., Ambicka, A., Rys, J., Ugorski, M., Podhorska-Okolow, M., and Dziegiel, P. (2015) Metallothionein-3 Increases Triple-Negative Breast Cancer Cell Invasiveness via Induction of Metalloproteinase Expression. PloS one 10, e0124865
88. Li, N., Dhar, S. S., Chen, T. Y., Kan, P. Y., Wei, Y., Kim, J. H., Chan, C. H., Lin, H. K., Hung, M. C., and Lee, M. G. (2016) JARID1D Is a Suppressor and Prognostic Marker of Prostate Cancer Invasion and Metastasis. Cancer research 76, 831-843
89. Zhang, M., Dai, C., Zhu, H., Chen, S., Wu, Y., Li, Q., Zeng, X., Wang, W., Zuo, J., Zhou, M., Xia, Z., Ji, G., Saiyin, H., Qin, L., and Yu, L. (2011) Cyclophilin A promotes human hepatocellular carcinoma cell metastasis via regulation of MMP3 and MMP9. Molecular and cellular biochemistry 357, 387-395
90. Ramos, D. M., But, M., Regezi, J., Schmidt, B. L., Atakilit, A., Dang, D., Ellis, D., Jordan, R., and Li, X. (2002) Expression of integrin beta 6 enhances invasive behavior in oral squamous cell carcinoma. Matrix Biol 21, 297-307
91. Kurahara, S., Shinohara, M., Ikebe, T., Nakamura, S., Beppu, M., Hiraki, A., Takeuchi, H., and Shirasuna, K. (1999) Expression of MMPS, MT-MMP, and TIMPs in squamous cell carcinoma of the oral cavity: correlations with tumor invasion and metastasis. Head Neck 21, 627-638
92. Birchmeier, C., Sharma, S., and Wigler, M. (1987) Expression and rearrangement of the ROS1 gene in human glioblastoma cells. Proceedings of the National Academy of Sciences of the United States of America 84, 9270-9274
93. Bergethon, K., Shaw, A. T., Ou, S. H., Katayama, R., Lovly, C. M., McDonald, N. T., Massion, P. P., Siwak-Tapp, C., Gonzalez, A., Fang, R., Mark, E. J., Batten, J. M., Chen, H., Wilner, K. D., Kwak, E. L., Clark, J. W., Carbone, D. P., Ji, H., Engelman, J. A., Mino-Kenudson, M., Pao, W., and Iafrate, A. J. (2012) ROS1 rearrangements define a unique molecular class of lung cancers. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 30, 863-870
94. Rikova, K., Guo, A., Zeng, Q., Possemato, A., Yu, J., Haack, H., Nardone, J., Lee, K., Reeves, C., Li, Y., Hu, Y., Tan, Z., Stokes, M., Sullivan, L., Mitchell, J., Wetzel, R., Macneill, J., Ren, J. M., Yuan, J., Bakalarski, C. E., Villen, J., Kornhauser, J. M., Smith, B., Li, D., Zhou, X., Gygi, S. P., Gu, T. L., Polakiewicz, R. D., Rush, J., and Comb, M. J. (2007) Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 131, 1190-1203
95. Birch, A. H., Arcand, S. L., Oros, K. K., Rahimi, K., Watters, A. K., Provencher, D., Greenwood, C. M., Mes-Masson, A. M., and Tonin, P. N. (2011) Chromosome 3 anomalies investigated by genome wide SNP analysis of benign, low malignant potential and low grade ovarian serous tumours. PloS one 6, e28250
96. Lee, J., Lee, S. E., Kang, S. Y., Do, I. G., Lee, S., Ha, S. Y., Cho, J., Kang, W. K., Jang, J., Ou, S. H., and Kim, K. M. (2013) Identification of ROS1 rearrangement in gastric adenocarcinoma. Cancer 119, 1627-1635
97. Jun, H. J., Johnson, H., Bronson, R. T., de Feraudy, S., White, F., and Charest, A. (2012) The oncogenic lung cancer fusion kinase CD74-ROS activates a novel invasiveness pathway through E-Syt1 phosphorylation. Cancer research 72, 3764-3774
98. Haghnegahdar, H., Du, J., Wang, D., Strieter, R. M., Burdick, M. D., Nanney, L. B., Cardwell, N., Luan, J., Shattuck-Brandt, R., and Richmond, A. (2000) The tumorigenic and angiogenic effects of MGSA/GRO proteins in melanoma. J Leukoc Biol 67, 53-62
99. Owen, J. D., Strieter, R., Burdick, M., Haghnegahdar, H., Nanney, L., Shattuck-Brandt, R., and Richmond, A. (1997) Enhanced tumor-forming capacity for immortalized melanocytes expressing melanoma growth stimulatory activity/growth-regulated cytokine beta and gamma proteins. International journal of cancer 73, 94-103
100. Kuo, P. L., Shen, K. H., Hung, S. H., and Hsu, Y. L. (2012) CXCL1/GROalpha increases cell migration and invasion of prostate cancer by decreasing fibulin-1 expression through NF-kappaB/HDAC1 epigenetic regulation. Carcinogenesis 33, 2477-2487
101. Kawanishi, H., Matsui, Y., Ito, M., Watanabe, J., Takahashi, T., Nishizawa, K., Nishiyama, H., Kamoto, T., Mikami, Y., Tanaka, Y., Jung, G., Akiyama, H., Nobumasa, H., Guilford, P., Reeve, A., Okuno, Y., Tsujimoto, G., Nakamura, E., and Ogawa, O. (2008) Secreted CXCL1 is a potential mediator and marker of the tumor invasion of bladder cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 14, 2579-2587
102. Cheng, W. L., Wang, C. S., Huang, Y. H., Tsai, M. M., Liang, Y., and Lin, K. H. (2011) Overexpression of CXCL1 and its receptor CXCR2 promote tumor invasion in gastric cancer. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO 22, 2267-2276
103. Zhang, J., Tu, K., Yang, W., Li, C., Yao, Y., Zheng, X., and Liu, Q. (2014) Evaluation of Jagged2 and Gli1 expression and their correlation with prognosis in human hepatocellular carcinoma. Molecular medicine reports 10, 749-754
104. Rossi, M., Magnoni, L., Miracco, C., Mori, E., Tosi, P., Pirtoli, L., Tini, P., Oliveri, G., Cosci, E., and Bakker, A. (2011) beta-catenin and Gli1 are prognostic markers in glioblastoma. Cancer biology & therapy 11, 753-761
105. Kubo, M., Nakamura, M., Tasaki, A., Yamanaka, N., Nakashima, H., Nomura, M., Kuroki, S., and Katano, M. (2004) Hedgehog signaling pathway is a new therapeutic target for patients with breast cancer. Cancer research 64, 6071-6074
106. Chen, J. S., Huang, X. H., Wang, Q., Huang, J. Q., Zhang, L. J., Chen, X. L., Lei, J., and Cheng, Z. X. (2013) Sonic hedgehog signaling pathway induces cell migration and invasion through focal adhesion kinase/AKT signaling-mediated activation of matrix metalloproteinase (MMP)-2 and MMP-9 in liver cancer. Carcinogenesis 34, 10-19
107. Leovic, D., Sabol, M., Ozretic, P., Musani, V., Car, D., Marjanovic, K., Zubcic, V., Sabol, I., Sikora, M., Grce, M., Glavas-Obrovac, L., and Levanat, S. (2012) Hh-Gli signaling pathway activity in oral and oropharyngeal squamous cell carcinoma. Head Neck 34, 104-112
108. Wang, Y. F., Chang, C. J., Lin, C. P., Chang, S. Y., Chu, P. Y., Tai, S. K., Li, W. Y., Chao, K. S., and Chen, Y. J. (2012) Expression of hedgehog signaling molecules as a prognostic indicator of oral squamous cell carcinoma. Head Neck 34, 1556-1561
109. Lin, S. C., Liu, C. J., Chiu, C. P., Chang, S. M., Lu, S. Y., and Chen, Y. J. (2004) Establishment of OC3 oral carcinoma cell line and identification of NF-kappa B activation responses to areca nut extract. J Oral Pathol Med 33, 79-86
110. Lee, T. I., Johnstone, S. E., and Young, R. A. (2006) Chromatin immunoprecipitation and microarray-based analysis of protein location. Nat Protoc 1, 729-748
111. O'Donnell, R. K., Kupferman, M., Wei, S. J., Singhal, S., Weber, R., O'Malley, B., Cheng, Y., Putt, M., Feldman, M., Ziober, B., and Muschel, R. J. (2005) Gene expression signature predicts lymphatic metastasis in squamous cell carcinoma of the oral cavity. Oncogene 24, 1244-1251
112. Song, H., Wang, R., Wang, S., and Lin, J. (2005) A low-molecular-weight compound discovered through virtual database screening inhibits Stat3 function in breast cancer cells. Proceedings of the National Academy of Sciences of the United States of America 102, 4700-4705
113. Li, Z., Wang, Y., Qiu, J., Li, Q., Yuan, C., Zhang, W., Wang, D., Ye, J., Jiang, H., Yang, J., and Cheng, J. (2013) The polycomb group protein EZH2 is a novel therapeutic target in tongue cancer. Oncotarget 4, 2532-2549
114. Leslie, K., Lang, C., Devgan, G., Azare, J., Berishaj, M., Gerald, W., Kim, Y. B., Paz, K., Darnell, J. E., Albanese, C., Sakamaki, T., Pestell, R., and Bromberg, J. (2006) Cyclin D1 is transcriptionally regulated by and required for transformation by activated signal transducer and activator of transcription 3. Cancer research 66, 2544-2552
115. Wang, Z., Zhu, S., Shen, M., Liu, J., Wang, M., Li, C., Wang, Y., Deng, A., and Mei, Q. (2013) STAT3 is involved in esophageal carcinogenesis through regulation of Oct-1. Carcinogenesis 34, 678-688
116. Niu, G., Wright, K. L., Ma, Y., Wright, G. M., Huang, M., Irby, R., Briggs, J., Karras, J., Cress, W. D., Pardoll, D., Jove, R., Chen, J., and Yu, H. (2005) Role of Stat3 in regulating p53 expression and function. Molecular and cellular biology 25, 7432-7440
117. Cao, Q., Yu, J., Dhanasekaran, S. M., Kim, J. H., Mani, R. S., Tomlins, S. A., Mehra, R., Laxman, B., Cao, X., Yu, J., Kleer, C. G., Varambally, S., and Chinnaiyan, A. M. (2008) Repression of E-cadherin by the polycomb group protein EZH2 in cancer. Oncogene 27, 7274-7284
118. Guo, J., Cai, J., Yu, L., Tang, H., Chen, C., and Wang, Z. (2011) EZH2 regulates expression of p57 and contributes to progression of ovarian cancer in vitro and in vivo. Cancer science 102, 530-539
119. Yu, J., Cao, Q., Yu, J., Wu, L., Dallol, A., Li, J., Chen, G., Grasso, C., Cao, X., Lonigro, R. J., Varambally, S., Mehra, R., Palanisamy, N., Wu, J. Y., Latif, F., and Chinnaiyan, A. M. (2010) The neuronal repellent SLIT2 is a target for repression by EZH2 in prostate cancer. Oncogene 29, 5370-5380
120. Shuai, K., and Liu, B. (2003) Regulation of JAK-STAT signalling in the immune system. Nat Rev Immunol 3, 900-911
121. Carow, B., and Rottenberg, M. E. (2014) SOCS3, a Major Regulator of Infection and Inflammation. Front Immunol 5, 58
122. Matsumura, Y., Kobayashi, T., Ichiyama, K., Yoshida, R., Hashimoto, M., Takimoto, T., Tanaka, K., Chinen, T., Shichita, T., Wyss-Coray, T., Sato, K., and Yoshimura, A. (2007) Selective expansion of foxp3-positive regulatory T cells and immunosuppression by suppressors of cytokine signaling 3-deficient dendritic cells. Journal of immunology 179, 2170-2179
123. Shen, X., Hong, F., Nguyen, V. A., and Gao, B. (2000) IL-10 attenuates IFN-alpha-activated STAT1 in the liver: involvement of SOCS2 and SOCS3. FEBS letters 480, 132-136
124. Schmitz, J., Weissenbach, M., Haan, S., Heinrich, P. C., and Schaper, F. (2000) SOCS3 exerts its inhibitory function on interleukin-6 signal transduction through the SHP2 recruitment site of gp130. The Journal of biological chemistry 275, 12848-12856
125. Sasaki, A., Yasukawa, H., Suzuki, A., Kamizono, S., Syoda, T., Kinjyo, I., Sasaki, M., Johnston, J. A., and Yoshimura, A. (1999) Cytokine-inducible SH2 protein-3 (CIS3/SOCS3) inhibits Janus tyrosine kinase by binding through the N-terminal kinase inhibitory region as well as SH2 domain. Genes Cells 4, 339-351
126. Yasukawa, H., Misawa, H., Sakamoto, H., Masuhara, M., Sasaki, A., Wakioka, T., Ohtsuka, S., Imaizumi, T., Matsuda, T., Ihle, J. N., and Yoshimura, A. (1999) The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop. The EMBO journal 18, 1309-1320
127. Liu, B., Liao, J., Rao, X., Kushner, S. A., Chung, C. D., Chang, D. D., and Shuai, K. (1998) Inhibition of Stat1-mediated gene activation by PIAS1. Proceedings of the National Academy of Sciences of the United States of America 95, 10626-10631
128. Shuai, K., and Liu, B. (2005) Regulation of gene-activation pathways by PIAS proteins in the immune system. Nat Rev Immunol 5, 593-605
129. Zhang, J., Somani, A. K., and Siminovitch, K. A. (2000) Roles of the SHP-1 tyrosine phosphatase in the negative regulation of cell signalling. Semin Immunol 12, 361-378
130. Wu, C., Sun, M., Liu, L., and Zhou, G. W. (2003) The function of the protein tyrosine phosphatase SHP-1 in cancer. Gene 306, 1-12
131. Chim, C. S., Fung, T. K., Cheung, W. C., Liang, R., and Kwong, Y. L. (2004) SOCS1 and SHP1 hypermethylation in multiple myeloma: implications for epigenetic activation of the Jak/STAT pathway. Blood 103, 4630-4635
132. Yuan, A., Chen, J. J., Yao, P. L., and Yang, P. C. (2005) The role of interleukin-8 in cancer cells and microenvironment interaction. Front Biosci 10, 853-865
133. Mao, Y., Keller, E. T., Garfield, D. H., Shen, K., and Wang, J. (2013) Stromal cells in tumor microenvironment and breast cancer. Cancer metastasis reviews 32, 303-315
134. Palena, C., Hamilton, D. H., and Fernando, R. I. (2012) Influence of IL-8 on the epithelial-mesenchymal transition and the tumor microenvironment. Future Oncol 8, 713-722