臺灣博碩士論文加值系統

癌症長年位居台灣十大死因之首，乳癌更為女性癌症死因第四高。不單是乳癌，在許多癌症中Enhancer of zeste homolog 2（EZH2）都有過量的表現。EZH2是一表觀遺傳調控蛋白質，可以將目標基因組蛋白H3第27號離氨酸進行三甲基化修飾（H3K27me3），抑制目標基因的表現。先前研究發現EZH2的過量表現會促進上皮間質轉化（Epithelial-mesenchymal transition, EMT），進而增強癌細胞轉移（migration）、侵襲（invasion）及抗藥性（drug resistance）能力。我們實驗室之前研究發現EZH2的第487個蘇胺酸（Threonine）可被CDK1（Cyclin-dependent kinase 1）磷酸化，進而抑制EZH2甲基轉移酶活性。若將EZH2 第487個蘇胺酸突變為丙胺酸（Alanine），則會促進癌細胞的轉移及侵襲能力。因此我們進一步探討野生型EZH2及487位點蘇胺酸突變為丙胺酸的突變EZH2是否會影響癌細胞上皮間質轉化及抗藥性。實驗結果顯示，當乳癌細胞EZH2過量表現時，會促進癌細胞的上皮間質轉化、存活率及抗藥性，而表現突變EZH2的乳癌細胞也有同樣結果，並且會促進細胞增殖。利用shRNA抑制EZH2後，確實抑制癌細胞上皮間質轉化、存活率及增殖率，但其調控的機制仍需進一步研究。

Cancer has been the leading cause of death in Taiwan for many years, and breast cancer is the fourth leading cause of female cancer death. In many cancers, including breast cancer, enhancer of zeste homolog 2（EZH2） are overexpressed. EZH2 is an epigenetic regulatory protein that can catalyze trimethylation of histone H3 at lysine 27 （H3K27me3） in targeted gene, leading to silencing of EZH2-targeted gene expression. Previous studies have found that overexpression of EZH2 promotes epithelial-mesenchymal transition（EMT）, enhancing the ability of cancer cells to migration, invasion and drug resistance. Previously our laboratory demonstrated that EZH2 can be phosphorylated at threonine（Thr）487 by cyclin-dependent kinase 1（CDK1）, resulting in inhibiting EZH2 histone methyltransferase activity. If the Thr 487 of EZH2 was mutated to alanine（T487A -EZH2）, the T487A-EZH2 could promote the migratory and invasive abilities of cancer cells. Here, we investigated whether WT-EZH2 and T487A-EZH2 can affect the epithelial-mesenchymal transition and drug resistance of cancer cells. The results show that overexpression of WT-EZH2 and T487A-EZH2 could promote epithelial-mesenchymal transition, cell survival and drug resistance of breast cancer cells. In addition knockdown of WT-EZH2 and T487A-EZH2 using shRNAs inhibited epithelial-mesenchymal transition, cell survival and proliferation of breast cancer cells. However, the underlying regulatory mechanisms heed to be investigated further.

中文摘要 i
Abstract ii
目錄 iii
圖目錄 v
第一章 前言 1
第一節 背景介紹 1
1. 乳癌介紹 1
2. Enhancer of zeste homolog 2 （EZH2） 4
3. Epithelial-mesenchymal transition （EMT） 8
第二節 研究目的與動機 9
第二章 研究材料及方法 11
第一節 藥品 11
第二節 實驗方法 14
1. 人類乳癌細胞（MCF-7）培養方法 14
2. Knockdown of EZH2 gene expression 14
3. 西方墨點法（Western blot） 15
4. 細胞存活率分析（MTT Assay） 17
5. 細胞群落形成分析（Colony formation assay） 17
第三章 結果 18
1. 分析乳癌細胞株中EZH2的蛋白質表現量 18
2. 分析乳癌細胞株中EMT相關蛋白質表現量 18
3. 利用細胞群落實驗分析比較乳癌細胞株的增殖和存活能力 19
4. 利用MTT assay分析EZH2對MCF-7細胞Tamoxifen耐藥性的影響 20

5. 利用shRNAs抑制MCF-7細胞中的EZH2基因，並觀察其對EMT相關蛋白質的影響 21
6. 利用shRNAs抑制MCF-7細胞中的EZH2觀察每孔種入500顆細胞形成細胞群落能力 22
7. 利用shRNAs抑制MCF-7細胞中的EZH2觀察每孔種入300顆細胞形成細胞群落能力 25
第四章 討論和結論 29
第五章 參考資料 32
參考文獻 32
第六章 圖 41

中央研究院分子生物研究所標靶式基因表達核心設施
衛福部國民健康署
財團法人乳癌防治基金會
The American Cancer Society medical and editorial content team
（https://www.cancer.org/cancer/breast-cancer/about/what-is-breast-cancer.html）

Union for International Cancer Control's （UICC）

參考文獻
1.Becker S: A historic and scientific review of breast cancer: The next global healthcare challenge. Int J Gynaecol Obstet 2015, 131 Suppl 1:S36-39.
2.Jose Russo, Yun-Fu Hu, Xiaoqi Yang, Russo IH: Chapter 1: Developmental, Cellular, and Molecular Basis of Human Breast Cancer. Journal of the National Cancer Institute Monographs 2000, 2000:17-37.
3.Nounou MI, ElAmrawy F, Ahmed N, Abdelraouf K, Goda S, Syed-Sha-Qhattal H: Breast Cancer: Conventional Diagnosis and Treatment Modalities and Recent Patents and Technologies. Breast Cancer (Auckl) 2015, 9:17-34.
4.Barroso-Sousa R, Metzger-Filho O: Differences between invasive lobular and invasive ductal carcinoma of the breast: results and therapeutic implications. Therapeutic Advances in Medical Oncology 2016, 8:261-266.
5.Chen W, Hoffmann AD, Liu H, Liu X: Organotropism: new insights into molecular mechanisms of breast cancer metastasis. NPJ Precis Oncol 2018, 2:4.
6.Fedele M, Cerchia L, Chiappetta G: The Epithelial-to-Mesenchymal Transition in Breast Cancer: Focus on Basal-Like Carcinomas. Cancers (Basel) 2017, 9.
7.Grimm LJ, Zhang J, Mazurowski MA: Computational approach to radiogenomics of breast cancer: Luminal A and luminal B molecular subtypes are associated with imaging features on routine breast MRI extracted using computer vision algorithms. J Magn Reson Imaging 2015, 42:902-907.
8.Toft DJ, Cryns VL: Minireview: Basal-like breast cancer: from molecular profiles to targeted therapies. Mol Endocrinol 2011, 25:199-211.
9.Ding L, Ellis MJ, Li S, Larson DE, Chen K, Wallis JW, Harris CC, McLellan MD, Fulton RS, Fulton LL, et al: Genome remodelling in a basal-like breast cancer metastasis and xenograft. Nature 2010, 464:999-1005.
10.William J. Gradishar, Benjamin O. Anderson, Ron Balassanian, Sarah L. Blair, Harold J. Burstein, Amy Cyr, Anthony D. Elias, William B. Farrar, Andres Forero, Sharon Hermes Giordano, et al: Breast Cancer, Version 1.2017. Journal of the National Comprehensive Cancer Network 2017, 15:433-451.
11.衛生福利部國民健康署: 107年國人死因統計結果. 2018.
12.衛生福利部國民健康署: 中華民國105年癌症登記報告. 2018.
13.Throckmorton AD, Rhodes DJ, Hughes KS, Degnim AC, Dickson-Witmer D: Dense Breasts: What Do Our Patients Need to Be Told and Why? Annals of Surgical Oncology 2016, 23:3119-3127.
14.Klevos GA, Ezuddin NS, Vinyard A, Ghaddar T, Gort T, Almuna A, Abisch A, Welsh CF: A Breast Cancer Review: Through the Eyes of the Doctor, Nurse, and Patient. Journal of Radiology Nursing 2017, 36:158-165.
15.Ganesh N. Sharma, Rahul Dave, Jyotsana Sanadya, Piush Sharma, Sharma aKK: VARIOUS TYPES AND MANAGEMENT OF BREAST CANCER: AN OVERVIEW. Journal of Advanced Pharmaceutical Technology & Research 2010, 1:109-126.
16.Irina Sachelarie, Michael L. Grossbard, Manjeet Chadha, Sheldon Feldman, Munir Ghesani, Blum RH: Primary Systemic Therapy of Breast Cancer. The Oncologist 2006, 11:574-589.
17.Amoroso V, Generali D, Buchholz T, Cristofanilli M, Pedersini R, Curigliano G, Daidone MG, Di Cosimo S, Dowsett M, Fox S, et al: International Expert Consensus on Primary Systemic Therapy in the Management of Early Breast Cancer: Highlights of the Fifth Symposium on Primary Systemic Therapy in the Management of Operable Breast Cancer, Cremona, Italy (2013). J Natl Cancer Inst Monogr 2015, 2015:90-96.
18.Clarke R, Tyson JJ, Dixon JM: Endocrine resistance in breast cancer--An overview and update. Mol Cell Endocrinol 2015, 418 Pt 3:220-234.
19.Johnston SRD: Targeted Combinations forHormone Receptor–Positive Advanced Breast Cancer: Who Benefits? JOURNAL OF CLINICAL ONCOLOGY 2016, 34:393-395.
20.Xiao T, Li W, Wang X, Xu H, Yang J, Wu Q, Huang Y, Geradts J, Jiang P, Fei T, et al: Estrogen-regulated feedback loop limits the efficacy of estrogen receptor-targeted breast cancer therapy. Proceedings of the National Academy of Sciences of the United States of America 2018, 115:7869-7878.
21.Shi Y, Wang XX, Zhuang YW, Jiang Y, Melcher K, Xu HE: Structure of the PRC2 complex and application to drug discovery. Acta Pharmacol Sinica 2017, 38:963-976.
22.Eskeland R, Leeb M, Grimes GR, Kress C, Boyle S, Sproul D, Gilbert N, Fan Y, Skoultchi AI, Wutz A, Bickmore WA: Ring1B compacts chromatin structure and represses gene expression independent of histone ubiquitination. Mol Cell 2010, 38:452-464.
23.Lawrence CL, Baldwin AS: Non-Canonical EZH2 Transcriptionally Activates RelB in Triple Negative Breast Cancer. PLoS One 2016, 11:e0165005.
24.Lee ST, Li Z, Wu Z, Aau M, Guan P, Karuturi RK, Liou YC, Yu Q: Context-specific regulation of NF-kappaB target gene expression by EZH2 in breast cancers. Mol Cell 2011, 43:798-810.
25.Lian R, Ma H, Wu Z, Zhang G, Jiao L, Miao W, Jin Q, Li R, Chen P, Shi H, Yu W: EZH2 promotes cell proliferation by regulating the expression of RUNX3 in laryngeal carcinoma. Molecular and Cellular Biochemistry 2018, 439:35-43.
26.Cheng T, Xu Y: Effects of Enhancer of Zeste Homolog 2 (EZH2) Expression on Brain Glioma Cell Proliferation and Tumorigenesis. Medical Science Monitor 2018, 24:7249-7255.
27.Li L-Y: EZH2 novel therapeutic target for human cancer. BioMedicine 2014, 4:1-6.
28.Stacchiotti S, Schoffski P, Jones R, Agulnik M, Villalobos VM, Jahan TM, Chen TW-W, Italiano A, Demetri GD, Cote GM, et al: Safety and efficacy of tazemetostat, a first-in-class EZH2 inhibitor, in patients (pts) with epithelioid sarcoma (ES) (NCT02601950). Journal of Clinical Oncology 2019, 37:11003-11003.
29.Italiano A, Soria J-C, Toulmonde M, Michot J-M, Lucchesi C, Varga A, Coindre J-M, Blakemore SJ, Clawson A, Suttle B, et al: Tazemetostat, an EZH2 inhibitor, in relapsed or refractory B-cell non-Hodgkin lymphoma and advanced solid tumours: a first-in-human, open-label, phase 1 study. The Lancet Oncology 2018, 19:649-659.
30.Anwar T, Arellano-Garcia C, Ropa J, Chen YC, Kim HS, Yoon E, Grigsby S, Basrur V, Nesvizhskii AI, Muntean A, et al: p38-mediated phosphorylation at T367 induces EZH2 cytoplasmic localization to promote breast cancer metastasis. Nat Commun 2018, 9:2801.
31.Celina G. Kleer, Qi Cao, Sooryanarayana Varambally, Ronglai Shen, Ichiro Ota, A. S, Tomlins, Debashis Ghosh, Richard G. A. B. Sewalt, Arie P. Otte, et al: EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells. proceedings of the national academy of sciences of the united states of america 2003, 100:11606-11611.
32.Zhang Q, Dong P, Liu X, Sakuragi N, Guo SW: Enhancer of Zeste homolog 2 (EZH2) induces epithelial-mesenchymal transition in endometriosis. Scientific Reports 2017, 7:6804.
33.Ting Han, Feng Jiao, Hai Hu, Cuncun Yuan, Lei Wang, Zi-Liang Jin, Wei-feng Song, Wang L-W: EZH2 promotes cell migration and invasion but not alters cell proliferation by suppressing E-cadherin, partly through association with MALAT-1 in pancreatic cancer. Oncotarget 2016, 7:11194-11207.
34.Tianfu Yu, Yingyi Wang, Qi Hu, WeiNing Wu, Youzhi Wu, Wenjin Wei, Dongfeng Han, Yongping You, Liu NLaN: The EZH2 inhibitor GSK343 suppresses cancer stem-like phenotypes and reverses mesenchymal transition in glioma cells. Oncotarget 2017, 8:98348-98359.
35.Zhu P, Wang Y, Huang G, Ye B, Liu B, Wu J, Du Y, He L, Fan Z: lnc-beta-Catm elicits EZH2-dependent beta-catenin stabilization and sustains liver CSC self-renewal. Nature Structural & Molecular Biology 2016, 23:631-639.
36.Kim KH, Roberts CW: Targeting EZH2 in cancer. Nat Med 2016, 22:128-134.
37.Gan L, Yang Y, Li Q, Feng Y, Liu T, Guo W: Epigenetic regulation of cancer progression by EZH2: from biological insights to therapeutic potential. Biomarker Research 2018, 6:10.
38.Cai L, Wang Z, Liu D: Interference with endogenous EZH2 reverses the chemotherapy drug resistance in cervical cancer cells partly by up-regulating Dicer expression. Tumour Biol 2016, 37:6359-6369.
39.Wu Y, Zhang Z, Cenciarini ME, Proietti CJ, Amasino M, Hong T, Yang M, Liao Y, Chiang HC, Kaklamani VG, et al: Tamoxifen Resistance in Breast Cancer Is Regulated by the EZH2-ERalpha-GREB1 Transcriptional Axis. Cancer Research 2018, 78:671-684.
40.Chen S, Yao F, Xiao Q, Liu Q, Yang Y, Li X, Jiang G, Kuno T, Fang Y: EZH2 inhibition sensitizes tamoxifenresistant breast cancer cells through cell cycle regulation. Molecular Medicine Reports 2018, 17:2642-2650.
41.Frasor J, El-Shennawy L, Stender JD, Kastrati I: NFkappaB affects estrogen receptor expression and activity in breast cancer through multiple mechanisms. Mol Cell Endocrinol 2015, 418 Pt 3:235-239.
42.Reijm EA, Timmermans AM, Look MP, Meijer-van Gelder ME, Stobbe CK, van Deurzen CH, Martens JW, Sleijfer S, Foekens JA, Berns PM, Jansen MP: High protein expression of EZH2 is related to unfavorable outcome to tamoxifen in metastatic breast cancer. Ann Oncol 2014, 25:2185-2190.
43.Greenburg G: Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells. The Journal of Cell Biology 1982, 95:333-339.
44.Roche J: The Epithelial-to-Mesenchymal Transition in Cancer. Cancers (Basel) 2018, 10.
45.Nieto MA, Huang Ruby Y-J, Jackson Rebecca A, Thiery Jean P: Emt: 2016. Cell 2016, 166:21-45.
46.Marcucci F, Stassi G, De Maria R: Epithelial-mesenchymal transition: a new target in anticancer drug discovery. Nat Rev Drug Discov 2016, 15:311-325.
47.Lamouille S, Xu J, Derynck R: Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol 2014, 15:178-196.
48.Dongre A, Weinberg RA: New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer. Nat Rev Mol Cell Biol 2019, 20:69-84.
49.Chao YL, Shepard CR, Wells A: Breast carcinoma cells re-express E-cadherin during mesenchymal to epithelial reverting transition. Mol Cancer 2010, 9:179.
50.Nieman MT, Prudoff RS, Johnson KR, Wheelock MJ: N-Cadherin Promotes Motility in Human Breast Cancer Cells Regardless of Their E-Cadherin Expression. The Journal of Cell Biology 1999, 147:631-644.
51.De Craene B, Berx G: Regulatory networks defining EMT during cancer initiation and progression. Nat Rev Cancer 2013, 13:97-110.
52.Eduard Batlle, Elena Sancho, Clara Francí, David Domínguez, Mercè Monfar, Baulida J, Herreros AGd: The transcription factor Snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nature Cell Biology 2000, 2:84-89.
53.Aristidis Moustakas, Herreros AGd: Epithelial–mesenchymal transition in cancer. molecular oncology 2017, 11:715-717.
54.Tran HD, Luitel K, Kim M, Zhang K, Longmore GD, Tran DD: Transient SNAIL1 Expression Is Necessary for Metastatic Competence in Breast Cancer. Cancer Research 2014, 74:6330-6340.
55.Zheng X, Carstens JL, Kim J, Scheible M, Kaye J, Sugimoto H, Wu C-C, LeBleu VS, Kalluri R: Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature 2015, 527:525-530.
56.Krebs AM, Mitschke J, Lasierra Losada M, Schmalhofer O, Boerries M, Busch H, Boettcher M, Mougiakakos D, Reichardt W, Bronsert P, et al: The EMT-activator Zeb1 is a key factor for cell plasticity and promotes metastasis in pancreatic cancer. Nature Cell Biology 2017, 19:518-529.
57.Wei Y, Chen YH, Li LY, Lang J, Yeh SP, Shi B, Yang CC, Yang JY, Lin CY, Lai CC, Hung MC: CDK1-dependent phosphorylation of EZH2 suppresses methylation of H3K27 and promotes osteogenic differentiation of human mesenchymal stem cells. Nat Cell Biol 2011, 13:87-94.
58.Chen. C, Wan. H: 慢病毒载体及其研究进展. Chinese Journal of Lung Cancer 2014, 17:870-876.
59.Arya SK, Lee KC, Dah'alan DuB, Daniel, Rahman ARA: Breast tumor cell detection at single cell resolution using an electrochemical impedance technique. Lab on a Chip 2012, 12:2362.
60.Valastyan S, Weinberg RA: Tumor metastasis: molecular insights and evolving paradigms. Cell 2011, 147:275-292.
61.Hu C, Liu Y, Teng M, Jiao K, Zhen J, Wu M, Li Z: Resveratrol inhibits the proliferation of estrogen receptor-positive breast cancer cells by suppressing EZH2 through the modulation of ERK1/2 signaling. Cell Biology and Toxicology 2019.
62.Dominic B. Bernkopf, Gabriele Daum, Martina Brückner, Behrens aJ: Sulforaphane inhibits growth and blocks Wnt β-catenin signaling. Oncotarget 2018, 9:33982-33994.
63.Yu J, Liu D, Sun X, Yang K, Yao J, Cheng C, Wang C, Zheng J: CDX2 inhibits the proliferation and tumor formation of colon cancer cells by suppressing Wnt/beta-catenin signaling via transactivation of GSK-3beta and Axin2 expression. Cell Death Dis 2019, 10:26.