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系統識別號 U0026-2608201603014800
論文名稱(中文) 探討Yes-Associated Protein在口腔癌中所扮演的角色
論文名稱(英文) Studying the Role of Yes-Associated Protein in Oral Cancer
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 104
學期 2
出版年 105
研究生(中文) 謝子勤
研究生(英文) Tzu-Chin Hsieh
學號 T16034056
學位類別 碩士
語文別 英文
論文頁數 42頁
口試委員 指導教授-吳梨華
口試委員-蔣輯武
口試委員-張文粲
中文關鍵字 口腔癌  YAP  TEAD  腫瘤血管新生 
英文關鍵字 oral cancer  YAP  TEAD  tumor angiogenesis 
學科別分類
中文摘要 口腔癌是全球最普遍的癌症之一,其晚期病患的五年存活率少於百分之五十。在台灣,口腔癌是國人第五大癌症死因,並且好發於男性。在先前的研究中利用全基因體定量分析發現在有吸食菸草或是酗酒習慣的口腔癌晚期病患當中,百分之十的病患在染色體區段11q22.1-22.2有增加的現象。同時,此片段的增加也被證明可以作為預後較差的指標。而Yes-Associated Protein (YAP)是位於染色體區段11q22.1-22.2中的基因,其主要功能為調控器官大小並參與細胞凋亡。先前研究指出YAP在不同的癌症中扮演促進癌症或是抑制癌症的角色。先前研究已發現卵黃囊血管新生過程中YAP是必要存在的基因;另外,當在小鼠內皮細胞中過度表現YAP則會促進血管新生。研究皆顯示YAP與血管新生之間有著強烈的連結,但是YAP與腫瘤血管新生之間的關係以及其詳細機制目前尚不清楚。因此,我們想要去探討YAP在口腔癌進程中所扮演的角色以及YAP與腫瘤血管新生之間的關聯性為何。結果顯示當YAP表現被抑制時,口腔癌細胞株的生長、移動以及侵略能力會隨之降低,並且會降低細胞在小鼠中形成腫瘤的能力。反之,當YAP過度表現時則會增強口腔癌細胞株的生長、移動以及侵略能力。這些實驗結果初步證實YAP在口腔癌中扮演的是促進癌症的角色。 此外,我們也發現在口腔癌細胞株中降低YAP的表現量會降低VEGF-A和bFGF的mRNA表現,進而透過旁分泌作用減弱內皮細胞形成管狀能力與生長速率。同時,在口腔癌細胞株中YAP與TEAD所形成的複合體會藉由直接與帶有TEAD結合位的VEGF-A或bFGF啟動子結合並去調控它們的表現。本文發現在口腔癌細胞株中YAP不僅會直接影響口腔癌細胞株轉型的行為,同時會透過旁分泌正向促進腫瘤的血管新生。
英文摘要 Oral cancer is one of the most common cancer types worldwide with a 5-year survival rate less than 50 % for advanced diseases. In Taiwan, oral cancer is the 5th leading cause of cancer death which predominantly occurs among males. Whole-genome copy number profiling revealed that around 10 % of advanced-stage oral cancer patients with tobacco and alcohol habituates had DNA amplifications in chromosome region 11q22.1-22.2. The gain of this region was shown to be one of the strongest predictors for poor clinical outcome for this cancer type. One of the genes in this locus is Yes-associated protein (YAP), previously shown to control organ size and participate in the mediation of apoptosis. A dual function, tumor promoter or suppressor, for YAP has been proposed in different cancer types. Moreover, YAP was also revealed as a vital requirement for yolk sac vasculogenesis, and its overexpression in mouse endothelial cells enhanced angiogenic sprouting. Together, these data provide a strong linkage between YAP and angiogenesis although the detailed mechanism remains elusive. We thus aimed to investigate the role of YAP in oral cancer progression and the relationship between YAP and tumor angiogenesis. Our data demonstrated that YAP knockdown significantly impaired oral cancer cell proliferation, migration and Matrigel invasion. The knockdown also decreased xenograft tumorigenesis in vivo. By contrast, YAP overexpression showed a significant increase in oral cancer cell proliferation, migration and Matrigel invasion. These data revealed an oncogenic role of YAP in oral cancer. A significant reduction of VEGF-A and bFGF mRNA expression in the YAP-depleted oral cancer cells may partly account for the attenuating effect of YAP-depleted conditioned medium (CM) on endothelial proliferation and Matrigel tube formation. Consistent with the notion that YAP mediates angiogenesis, YAP/TEAD complex modulated the transcriptional regulation of VEGF-A and bFGF by directly interacting with their promoters bearing TEAD-binding sites in oral cancer cells. Although more studies are needed, our findings potentially shed mechanistic light on YAP-mediated oncogenic action through both autocrine and paracrine fashions in oral cancer cells.
論文目次 Abstract in Chinese I
Abstract in English II
Acknowledgement IV
Content VI
List of Figures and Tables VIII
Abbreviations IX

I. Introduction 1
1-1 Oral cancer 1
1-2 Hippo pathway and its key effector, Yes-associated protein (YAP), in tissue homeostasis and cancer 1
1-3 The regulation of YAP activity by phosphorylation 3
1-4 TEAD/TEF family 3
1-5 Angiogenesis 4
1-6 The role of YAP in tumor angiogenesis 4
III. Specific aims 6
IV. Materials and methods 8
4-1 Cell culture 8
4-2 Lentivirus transduction 8
4-3 Western blotting 8
4-4 Doubling time 9
4-5 Wound-healing assay 9
4-6 Invasion assay 9
4-7 Xenograft tumorigenesis 10
4-8 Conditioned medium (CM) preparation 10
4-9 Endothelial tube formation assay 10
4-10 MTS assay 11
4-11 Quantitative RT-PCR (qRT-PCR) 11
4-12 Promoter-driven luciferase assay 11
4-13 Chromatin immunoprecipitation-qPCR (ChIP-qPCR) 11
V. Result 13
5-1 YAP knockdown reduced oral cancer cell proliferation 13
5-2 YAP knockdown decreased the migration and invasion ability of oral cancer cells…. 13
5-3 YAP overexpression increased oral cancer cell proliferation, migration and invasion 13
5-4 YAP knockdown decreased xenograft tumorigenesis in vivo. 14
5-5 YAP-depletion reduced the ability of HSC-3-derived CM to modulate angiogenesis 14
5-6 YAP modulated the expression of angiogenic VEGF-A and bFGF as a transcriptional co-activator 15
5-7 YAP directly interacted with the proximal TEAD-binding site in either VEGF-A or bFGF promoter region by forming a complex with TEAD 16
VI. Discussion 17
VII. References 20
VIII. Appendix 42

參考文獻 Ambatipudi, S., Gerstung, M., Gowda, R., Pai, P., Borges, A.M., Schaffer, A.A., Beerenwinkel, N., and Mahimkar, M.B. (2011). Genomic profiling of advanced-stage oral cancers reveals chromosome 11q alterations as markers of poor clinical outcome. PLoS One 6, e17250.

Barry, E.R., Morikawa, T., Butler, B.L., Shrestha, K., de la Rosa, R., Yan, K.S., Fuchs, C.S., Magness, S.T., Smits, R., Ogino, S., et al. (2013). Restriction of intestinal stem cell expansion and the regenerative response by YAP. Nature 493, 106-110.

Choi, H.J., Zhang, H., Park, H., Choi, K.S., Lee, H.W., Agrawal, V., Kim, Y.M., and Kwon, Y.G. (2015). Yes-associated protein regulates endothelial cell contact-mediated expression of angiopoietin-2. Nature communications 6, 6943.

Chung, A.S., Lee, J., and Ferrara, N. (2010). Targeting the tumour vasculature: insights from physiological angiogenesis. Nat Rev Cancer 10, 505-514.

Consortium, I.P.T.o.t.I.C.G. (2013). Mutational landscape of gingivo-buccal oral squamous cell carcinoma reveals new recurrently-mutated genes and molecular subgroups. Nature communications 4, 2873.

Cross, M.J., and Claesson-Welsh, L. (2001). FGF and VEGF function in angiogenesis: signalling pathways, biological responses and therapeutic inhibition. Trends Pharmacol Sci 22, 201-207.

Farnsworth, R.H., Lackmann, M., Achen, M.G., and Stacker, S.A. (2014). Vascular remodeling in cancer. Oncogene 33, 3496-3505.

Ferlay, J., Shin, H.R., Bray, F., Forman, D., Mathers, C., and Parkin, D.M. (2010). Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 127, 2893-2917.
Guo, X., and Zhao, B. (2013). Integration of mechanical and chemical signals by YAP and TAZ transcription coactivators. Cell & Bioscience 3, 33-33.

Halder, G., and Johnson, R.L. (2011). Hippo signaling: growth control and beyond. Development (Cambridge, England) 138, 9-22.

Hanahan, D., and Weinberg, R.A. (2011). Hallmarks of cancer: the next generation. Cell 144, 646-674.

Hao, Y., Chun, A., Cheung, K., Rashidi, B., and Yang, X. (2008). Tumor suppressor LATS1 is a negative regulator of oncogene YAP. The Journal of biological chemistry 283, 5496-5509.

Harvey, K.F., Zhang, X., and Thomas, D.M. (2013). The Hippo pathway and human cancer. Nat Rev Cancer 13, 246-257.

Hiemer, S.E., Zhang, L., Kartha, V.K., Packer, T.S., Almershed, M., Noonan, V., Kukuruzinska, M., Bais, M.V., Monti, S., and Varelas, X. (2015). A YAP/TAZ-Regulated Molecular Signature Is Associated with Oral Squamous Cell Carcinoma. Mol Cancer Res 13, 957-968.
Holmes, D.I.R., and Zachary, I. (2005). The vascular endothelial growth factor (VEGF) family: angiogenic factors in health and disease. Genome Biology 6, 209-209.

Jacquemin, P., Sapin, V., Alsat, E., Evain-Brion, D., Dolle, P., and Davidson, I. (1998). Differential expression of the TEF family of transcription factors in the murine placenta and during differentiation of primary human trophoblasts in vitro. Developmental dynamics : an official publication of the American Association of Anatomists 212, 423-436.

Jiang, S.W., Desai, D., Khan, S., and Eberhardt, N.L. (2000). Cooperative binding of TEF-1 to repeated GGAATG-related consensus elements with restricted spatial separation and orientation. DNA Cell Biol 19, 507-514.

Jornet, P.L., Garcia, F.J., Berdugo, M.L., Perez, F.P., and Lopez, A.P. (2015). Mouth self-examination in a population at risk of oral cancer. Aust Dent J 60, 59-64.

Kaneko, K.J., Cullinan, E.B., Latham, K.E., and DePamphilis, M.L. (1997). Transcription factor mTEAD-2 is selectively expressed at the beginning of zygotic gene expression in the mouse. Development (Cambridge, England) 124, 1963-1973.

Lamar, J.M., Stern, P., Liu, H., Schindler, J.W., Jiang, Z.G., and Hynes, R.O. (2012). The Hippo pathway target, YAP, promotes metastasis through its TEAD-interaction domain. Proc Natl Acad Sci U S A 109, E2441-2450.

Lin, Y.T., Chien, C.Y., Lu, C.T., Lou, S.D., Lu, H., Huang, C.C., Fang, F.M., Li, S.H., Huang, T.L., and Chuang, H.C. (2015). Triple-positive pathologic findings in oral cavity cancer are related to a dismal prognosis. Laryngoscope 125, E300-305.

Lorenzetto, E., Brenca, M., Boeri, M., Verri, C., Piccinin, E., Gasparini, P., Facchinetti, F., Rossi, S., Salvatore, G., Massimino, M., et al. (2014). YAP1 acts as oncogenic target of 11q22 amplification in multiple cancer subtypes. Oncotarget 5, 2608-2621.

Marti, P., Stein, C., Blumer, T., Abraham, Y., Dill, M.T., Pikiolek, M., Orsini, V., Jurisic, G., Megel, P., Makowska, Z., et al. (2015). YAP promotes proliferation, chemoresistance, and angiogenesis in human cholangiocarcinoma through TEAD transcription factors. Hepatology 62, 1497-1510.

Meng, Z., Moroishi, T., and Guan, K.L. (2016). Mechanisms of Hippo pathway regulation. Genes Dev 30, 1-17.

Messadi, D.V., Wilder-Smith, P., and Wolinsky, L. (2009). Improving oral cancer survival: the role of dental providers. J Calif Dent Assoc 37, 789-798.

Mo, J.-S., Meng, Z., Kim, Y.C., Park, H.W., Hansen, C.G., Kim, S., Lim, D.-S., and Guan, K.-L. (2015). Cellular energy stress induces AMPK-mediated regulation of YAP and the Hippo pathway. Nat Cell Biol 17, 500-510.

Mo, J.S., Park, H.W., and Guan, K.L. (2014). The Hippo signaling pathway in stem cell biology and cancer. EMBO Rep 15, 642-656.

Morin-Kensicki, E.M., Boone, B.N., Howell, M., Stonebraker, J.R., Teed, J., Alb, J.G., Magnuson, T.R., O'Neal, W., and Milgram, S.L. (2006). Defects in yolk sac vasculogenesis, chorioallantoic fusion, and embryonic axis elongation in mice with targeted disruption of Yap65. Molecular and cellular biology 26, 77-87.

Narayanan, S.P., Singh, S., Gupta, A., Yadav, S., Singh, S.R., and Shukla, S. (2015). Integrated genomic analyses identify KDM1A's role in cell proliferation via modulating E2F signaling activity and associate with poor clinical outcome in oral cancer. Cancer Lett 367, 162-172.

Oka, T., Mazack, V., and Sudol, M. (2008). Mst2 and Lats kinases regulate apoptotic function of Yes kinase-associated protein (YAP). The Journal of biological chemistry 283, 27534-27546.

Sankaranarayanan, R., Ramadas, K., Thomas, G., Muwonge, R., Thara, S., Mathew, B., and Rajan, B. (2005). Effect of screening on oral cancer mortality in Kerala, India: a cluster-randomised controlled trial. Lancet 365, 1927-1933.

Sciubba, J.J. (2001). Oral cancer. The importance of early diagnosis and treatment. Am J Clin Dermatol 2, 239-251.

Shah, F.D., Begum, R., Vajaria, B.N., Patel, K.R., Patel, J.B., Shukla, S.N., and Patel, P.S. (2011). A review on salivary genomics and proteomics biomarkers in oral cancer. Indian J Clin Biochem 26, 326-334.

Shing, Y., Folkman, J., Sullivan, R., Butterfield, C., Murray, J., and Klagsbrun, M. (1984). Heparin affinity: purification of a tumor-derived capillary endothelial cell growth factor. Science (New York, NY) 223, 1296-1299.

Su, C.-W., Huang, Y.-W., Chen, M.-K., Su, S.-C., Yang, S.-F., and Lin, C.-W. (2015). Polymorphisms and Plasma Levels of Tissue Inhibitor of Metalloproteinase-3: Impact on Genetic Susceptibility and Clinical Outcome of Oral Cancer. Medicine 94, e2092.

Tsai, S.T., Jin, Y.T., Tsai, W.C., Wang, S.T., Lin, Y.C., Chang, M.T., and Wu, L.W. (2005). S100A2, a potential marker for early recurrence in early-stage oral cancer. Oral oncology 41, 349-357.

Vassilev, A., Kaneko, K.J., Shu, H., Zhao, Y., and DePamphilis, M.L. (2001). TEAD/TEF transcription factors utilize the activation domain of YAP65, a Src/Yes-associated protein localized in the cytoplasm. Genes & Development 15, 1229-1241.

Walsh, T., Liu, J.L., Brocklehurst, P., Glenny, A.M., Lingen, M., Kerr, A.R., Ogden, G., Warnakulasuriya, S., and Scully, C. (2013). Clinical assessment to screen for the detection of oral cavity cancer and potentially malignant disorders in apparently healthy adults. Cochrane Database Syst Rev, Cd010173.

Weis, S.M., and Cheresh, D.A. (2011). Tumor angiogenesis: molecular pathways and therapeutic targets. Nat Med 17, 1359-1370.

Yoshikawa, K., Noguchi, K., Nakano, Y., Yamamura, M., Takaoka, K., Hashimoto-Tamaoki, T., and Kishimoto, H. (2015). The Hippo pathway transcriptional co-activator, YAP, confers resistance to cisplatin in human oral squamous cell carcinoma. Int J Oncol 46, 2364-2370.

Zender, L., Spector, M.S., Xue, W., Flemming, P., Cordon-Cardo, C., Silke, J., Fan, S.-T., Luk, J.M., Wigler, M., Hannon, G.J., et al. (2006). Identification and Validation of Oncogenes in Liver Cancer Using an Integrative Oncogenomic Approach. Cell 125, 1253-1267.

Zeng, Q., and Hong, W. (2008). The emerging role of the hippo pathway in cell contact inhibition, organ size control, and cancer development in mammals. Cancer cell 13, 188-192.
Zhang, S.K., Zheng, R., Chen, Q., Zhang, S., Sun, X., and Chen, W. (2015). Oral cancer incidence and mortality in China, 2011. Chin J Cancer Res 27, 44-51.

Zhao, B., Li, L., Tumaneng, K., Wang, C.-Y., and Guan, K.-L. (2010). A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(β-TRCP). Genes & Development 24, 72-85.

Zhao, B., Wei, X., Li, W., Udan, R.S., Yang, Q., Kim, J., Xie, J., Ikenoue, T., Yu, J., Li, L., et al. (2007). Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes & Development 21, 2747-2761.

Zhao, B., Ye, X., Yu, J., Li, L., Li, W., Li, S., Yu, J., Lin, J.D., Wang, C.-Y., Chinnaiyan, A.M., et al. (2008). TEAD mediates YAP-dependent gene induction and growth control. Genes & Development 22, 1962-1971.
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