頭頸癌是世界上常見的癌症之一，然而頭頸癌發生的頻率在台灣地區有逐年上升的現象，雖然頭頸癌的病徵明顯很容易被診斷，但是頭頸癌患者仍舊對於治療後的復發與轉移承載著高風險，除此之外，在近期的研究中發現到，頭頸癌細胞隨著腫瘤的進程會增加其轉移的能力而進一步的產生近端淋巴轉移，因此，研究探討參與在頭頸癌中的轉移至淋巴的機制是相當重要。Cortactin (CTTN) 不僅僅是一個已知在調控機動蛋白與細胞骨架之間的動態扮演著重要角色，更是一個主要參與癌症侵略性增加的一個因子。在頭頸癌患者中可以看到有30%的病患具有 CTTN 基因大量表現的情況發生。CTTN 過度表達的情況下是會與癌症的侵入能力有正向關係，也表示 CTTN 是可以作為一個檢測癌症侵入程度的生物指標。而文獻中也有探討到 CTTN 會藉由調控機動蛋白的動態分支情況來增加外吐小體的分泌。而其中一個外吐小體-外囊泡就扮演著可以調控細胞間訊息傳遞的工具。而我們的假設就是推測 CTTN 是透過外囊泡的幫助去促進癌症轉移至淋巴。為了探討其中的機制，我們建立了一套實驗去讓 CTTN 大量表現或是減少表現。而實驗結果可以看到當 CTTN 大量表現時確實會增加癌細胞的爬行跟侵襲的能力。除此之外，利用 CTTN 大量表現的細胞所分泌的條件培養基可以提升 CTTN 表現下降的細胞爬行及侵襲的能力。而在我們的實驗中也可以看到 CTTN 可以在不影響淋巴內皮增生能力情況下去增加淋巴內皮管新生的能力。在證明了 CTTN 確實存在於外囊泡中後，我們可以發現當 CTTN 表現量上升時，是可以增加外囊泡分泌但卻不改變其中的內含物。因此我們接下來的實驗也可以看到來自 CTTN 過度表現細胞所分泌的外囊泡是可以提升細胞爬行及侵襲能力。因此在經由以上實驗的證明下，總結來說，我們證明 CTTN 確實在口腔癌中辦演一個致癌基因的角色，而這樣的角色是 CTTN 藉由內分泌提升口腔癌爬行以及侵襲能力再來還有藉由外分泌去增強血管新生的情況去造就 CTTN 可以產生的變化。

Head and neck cancer (HNC) is one of the most common cancers worldwide. Unfortunately, the incidence of people diagnosed with HNC is on the rise in Taiwan. Although the symptoms of the cancer are visible, these patients are still at high risk of death due to the relapse and metastasis. Furthermore, it was recently shown that HNC cells could gain more metastatic capabilities as the tumor progresses to regional lymph nodes. Therefore, it is essential to investigate the mechanisms by which HNC metastasizes to lymph nodes. Cortactin (CTTN) was shown to act not only as a crucial regulator of actin cytoskeletal dynamics, but also as a key player in aggressive cancers. Amplification of the gene that encodes cortactin have been observed in nearly 30% of head and neck cancer. CTTN overexpression has also been linked to invasive cancers, making CTTN being an important biomarker for invasive cancers. CTTN promotes by controlling branched actin dynamics the secretion of exosomes, one type of extracellular vesicles (EVs) that can mediate intercellular communication. We thus hypothesize that CTTN-mediated EVs play a modulatory role in HNC metastasis to lymph nodes. To evaluate the role and action mechanism of CTTN in oral cancer cells, we performed a series of overexpression and depletion experiments. Intratumor CTTN expression promoted oral cancer cell migration and Matrigel invasion. Moreover, CTTN overexpression (CTTN-OE) derived condition medium (CM) enhanced oral cancer cell migration and invasion abilities and the depletion of EVs lost the CTTN-mediated promotion. We also showed a promoting role of CTTN-mediated increase of lymphatic endothelial tube formation but not proliferation. After identifying the presence of CTTN in EVs, its increased expression also enhanced EV secretion without altering the mean particle sizes. CTTN-OE-derived EVs promoted CTTN-depleted cell migration and invasion. Taken together, we provide evidence that CTTN functions as an oncogene in oral cancer through autocrinely promoting oral cancer cell migration and invasion, and paracrinely enhancing angiogenesis.

1. Al Soraj, M., Bargal, S., and Luqmani, Y.A. (2016). Extracellular Vesicles: A Mechanism to Reverse Metastatic Behaviour as a New Approach to Cancer Therapy. In Tumor Metastasis.
2. Argiris, A., Karamouzis, M.V., Raben, D., and Ferris, R.L. (2008). Head and neck cancer. The Lancet 371, 1695-1709.
3. Atay, S., Gercel-Taylor, C., Kesimer, M., and Taylor, D.D. (2011). Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells. Exp Cell Res 317, 1192-1202.
4. Bao, L., You, B., Shi, S., Shan, Y., Zhang, Q., Yue, H., Zhang, J., Zhang, W., Shi, Y., Liu, Y., et al. (2018). Metastasis-associated miR-23a from nasopharyngeal carcinoma-derived exosomes mediates angiogenesis by repressing a novel target gene TSGA10. Oncogene 37, 2873-2889.
5. Becker, A., Thakur, B.K., Weiss, J.M., Kim, H.S., Peinado, H., and Lyden, D. (2016). Extracellular Vesicles in Cancer: Cell-to-Cell Mediators of Metastasis. Cancer Cell 30, 836-848.
6. Ciardiello, C., Cavallini, L., Spinelli, C., Yang, J., Reis-Sobreiro, M., de Candia, P., Minciacchi, V.R., and Di Vizio, D. (2016). Focus on Extracellular Vesicles: New Frontiers of Cell-to-Cell Communication in Cancer. International journal of molecular sciences 17, 175.
7. Costa-Silva, B., Aiello, N.M., Ocean, A.J., Singh, S., Zhang, H., Thakur, B.K., Becker, A., Hoshino, A., Mark, M.T., Molina, H., et al. (2015). Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat Cell Biol 17, 816-826.
8. Fantozzi, I., Grall, D., Cagnol, S., Stanchi, F., Sudaka, A., Brunstein, M.C., Bozec, A., Fischel, J.L., Milano, G., and Van Obberghen-Schilling, E. (2008). Overexpression of cortactin in head and neck squamous cell carcinomas can be uncoupled from augmented EGF receptor expression. Acta oncologica (Stockholm, Sweden) 47, 1502-1512.
9. Folkman, J. (2002). Role of angiogenesis in tumor growth and metastasis. Seminars in oncology 29, 15-18.
10. Gangoda, L., and Mathivanan, S. (2016). Cortactin enhances exosome secretion without altering cargo. J Cell Biol 214, 129-131.
11. Gyorgy, B., Szabo, T.G., Pasztoi, M., Pal, Z., Misjak, P., Aradi, B., Laszlo, V., Pallinger, E., Pap, E., Kittel, A., et al. (2011). Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci 68, 2667-2688.
12. Hida, K., Maishi, N., Annan, D.A., and Hida, Y. (2018). Contribution of Tumor Endothelial Cells in Cancer Progression. International journal of molecular sciences 19.
13. Hoshino, A., Costa-Silva, B., Shen, T.L., Rodrigues, G., Hashimoto, A., Tesic Mark, M., Molina, H., Kohsaka, S., Di Giannatale, A., Ceder, S., et al. (2015). Tumour exosome integrins determine organotropic metastasis. Nature 527, 329-335.
14. Hoshino, D., Branch, K.M., and Weaver, A.M. (2013a). Signaling inputs to invadopodia and podosomes. J Cell Sci 126, 2979-2989.
15. Hoshino, D., Kirkbride, K.C., Costello, K., Clark, E.S., Sinha, S., Grega-Larson, N., Tyska, M.J., and Weaver, A.M. (2013b). Exosome secretion is enhanced by invadopodia and drives invasive behavior. Cell Rep 5, 1159-1168.
16. Jing, X., Wu, H., Ji, X., Wu, H., Shi, M., and Zhao, R. (2016). Cortactin promotes cell migration and invasion through upregulation of the dedicator of cytokinesis 1 expression in human colorectal cancer. Oncol Rep 36, 1946-1952.
17. Karaman, S., and Detmar, M. (2014). Mechanisms of lymphatic metastasis. J Clin Invest 124, 922-928.
18. Kirkbride, K.C., Sung, B.H., Sinha, S., and Weaver, A.M. (2011). Cortactin: a multifunctional regulator of cellular invasiveness. Cell Adh Migr 5, 187-198.
19. Liu, D., Li, C., Trojanowicz, B., Li, X., Shi, D., Zhan, C., Wang, Z., and Chen, L. (2016a). CD97 promotion of gastric carcinoma lymphatic metastasis is exosome dependent. Gastric Cancer 19, 754-766.
20. Liu, Y., Gu, Y., Han, Y., Zhang, Q., Jiang, Z., Zhang, X., Huang, B., Xu, X., Zheng, J., and Cao, X. (2016b). Tumor Exosomal RNAs Promote Lung Pre-metastatic Niche Formation by Activating Alveolar Epithelial TLR3 to Recruit Neutrophils. Cancer Cell 30, 243-256.
21. Lobb, R.J., Lima, L.G., and Moller, A. (2017). Exosomes: Key mediators of metastasis and pre-metastatic niche formation. Semin Cell Dev Biol 67, 3-10.
22. Mao, L., Hong, W.K., and Papadimitrakopoulou, V.A. (2004). Focus on head and neck cancer. Cancer Cell 5, 311-316.
23. Marur, S., and Forastiere, A.A. (2016). Head and Neck Squamous Cell Carcinoma: Update on Epidemiology, Diagnosis, and Treatment. Mayo Clinic proceedings 91, 386-396.
24. Mulcahy, L.A., Pink, R.C., and Carter, D.R. (2014). Routes and mechanisms of extracellular vesicle uptake. Journal of extracellular vesicles 3.
25. Nicolson, G.L. (1993). Paracrine and autocrine growth mechanisms in tumor metastasis to specific sites with particular emphasis on brain and lung metastasis. Cancer metastasis reviews 12, 325-343.
26. Ozawa, P.M.M., Alkhilaiwi, F., Cavalli, I.J., Malheiros, D., de Souza Fonseca Ribeiro, E.M., and Cavalli, L.R. (2018). Extracellular vesicles from triple-negative breast cancer cells promote proliferation and drug resistance in non-tumorigenic breast cells. Breast Cancer Res Treat 172, 713-723.
27. Peinado, H., Zhang, H., Matei, I.R., Costa-Silva, B., Hoshino, A., Rodrigues, G., Psaila, B., Kaplan, R.N., Bromberg, J.F., Kang, Y., et al. (2017). Pre-metastatic niches: organ-specific homes for metastases. Nat Rev Cancer 17, 302-317.
28. Raposo, G., and Stoorvogel, W. (2013). Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200, 373-383.
29. Riches, A., Campbell, E., Borger, E., and Powis, S. (2014). Regulation of exosome release from mammary epithelial and breast cancer cells - a new regulatory pathway. European journal of cancer (Oxford, England : 1990) 50, 1025-1034.
30. Rubin Grandis, J., Melhem, M.F., Gooding, W.E., Day, R., Holst, V.A., Wagener, M.M., Drenning, S.D., and Tweardy, D.J. (1998). Levels of TGF-alpha and EGFR protein in head and neck squamous cell carcinoma and patient survival. Journal of the National Cancer Institute 90, 824-832.
31. Sanchez-Tillo, E., Liu, Y., de Barrios, O., Siles, L., Fanlo, L., Cuatrecasas, M., Darling, D.S., Dean, D.C., Castells, A., and Postigo, A. (2012). EMT-activating transcription factors in cancer: beyond EMT and tumor invasiveness. Cell Mol Life Sci 69, 3429-3456.
32. Sinha, S., Hoshino, D., Hong, N.H., Kirkbride, K.C., Grega-Larson, N.E., Seiki, M., Tyska, M.J., and Weaver, A.M. (2016). Cortactin promotes exosome secretion by controlling branched actin dynamics. J Cell Biol 214, 197-213.
33. Spano, D., and Zollo, M. (2012). Tumor microenvironment: a main actor in the metastasis process. Clin Exp Metastasis 29, 381-395.
34. Spella, M., Marazioti, A., Arendt, K.A.M., and Stathopoulos, G.T. (2017). RAS oncogenes direct metastasis. Molecular & cellular oncology 4, e1345711.
35. Sporn, M.B., and Roberts, A.B. (1985). Autocrine growth factors and cancer. Nature 313, 745-747.
36. Steinbichler, T.B., Dudas, J., Riechelmann, H., and Skvortsova, II (2017). The role of exosomes in cancer metastasis. Semin Cancer Biol 44, 170-181.
37. Thery, C., Amigorena, S., Raposo, G., and Clayton, A. (2006). Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Current protocols in cell biology Chapter 3, Unit 3.22.
38. Timpson, P., Wilson, A.S., Lehrbach, G.M., Sutherland, R.L., Musgrove, E.A., and Daly, R.J. (2007). Aberrant Expression of Cortactin in Head and Neck Squamous Cell Carcinoma Cells Is Associated with Enhanced Cell Proliferation and Resistance to the Epidermal Growth Factor Receptor Inhibitor Gefitinib. 67, 9304-9314.
39. van der Pol, E., Boing, A.N., Harrison, P., Sturk, A., and Nieuwland, R. (2012). Classification, functions, and clinical relevance of extracellular vesicles. Pharmacological reviews 64, 676-705.
40. van Niel, G., D'Angelo, G., and Raposo, G. (2018). Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol 19, 213-228.
41. Vlassov, A.V., Magdaleno, S., Setterquist, R., and Conrad, R. (2012). Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim Biophys Acta 1820, 940-948.
42. Wei, Y., Wang, D., Jin, F., Bian, Z., Li, L., Liang, H., Li, M., Shi, L., Pan, C., Zhu, D., et al. (2017). Pyruvate kinase type M2 promotes tumour cell exosome release via phosphorylating synaptosome-associated protein 23. Nat Commun 8, 14041.
43. Xiao, Z., Luo, G., Liu, C., Wu, C., Liu, L., Liu, Z., Ni, Q., Long, J., and Yu, X. (2014). Molecular mechanism underlying lymphatic metastasis in pancreatic cancer. Biomed Res Int 2014, 925845.
44. Zetter, B.R. (1998). Angiogenesis and tumor metastasis. Annual review of medicine 49, 407-424.