近年來已發現，組蛋白去甲基酶4（KDM4A-KDM4D）會選擇性地去除組蛋白H3上的甲基化，並且在癌症進展的表觀遺傳調控上是一個重要的調控者。KDM4A和KDM4B過度表現於許多惡性腫瘤，其中，包括前列腺癌。KDM4家族中的成員都會與AR（Androgen receptor）共同結合，同時調控AR的功能，藉此增加前列腺癌細胞的增生。在實驗室之前的研究中，已解出KDM4B的結構，並且篩選出具有抑制KDM4A和KDM4B的新穎抑制劑來對抗激素敏感性前列腺癌（androgen-dependent PCa）LNCaP。然而，KDM4B在抗去勢型攝護腺癌（castration resistant prostate cancer/ CRPC）的治療潛力和癌細胞增生機制上所扮演的角色仍有待釐清。在此研究中，我們首先針對KDM4B是否為一個具有潛力治療CRPC的標靶進行探討，發現敲減（knockdown）KDM4B除了可以抑制LNCaP的增長外，也同時抑制了AR-independent 的細胞株C4-2B的增長。活體實驗上，異種移殖動物模型驗證KDM4B之敲減可以減少前列腺癌C4-2B細胞株腫瘤生長的能力。進行荷爾蒙療法時，會造成前列腺癌細胞產生缺氧的現象。而實驗上發現，當LNCaP和C4-2B細胞株培養於缺氧環境下，會使得KDM4B被過度表現。不管在有氧與無氧環境下，KDM4B在癌細胞的能量代謝與適應上的調控皆扮演著重要的角色，增進前列腺癌代謝上的適應，並調控著前列腺癌的增長與生存。

Histone lysine demethylase 4 (KDM4A-KDM4D) that selectively removes the methyl marks of histone 3 has been recently recognized as an important class of epigenetic regulators in cancer progression. KDM4A and KDM4B are over-expressed in many cancers including prostate cancer. All KDM4 members interact with androgen receptor (AR), modulating AR to stimulate the cell proliferation in prostate cancer. Dr. Wang’s prior work has determined structure of KDM4B and identified a novel inhibitor of KDM4A and KDM4B against the AR-dependent LNCaP cells. However, the therapeutic potential of KDM4B against castration-resistant prostate cancer (CRPC) and its underlying mechanism that leads to cancer progression remains largely unclear. In this study, we aimed to characterize whether KDM4B was a promising therapeutic target against CRPC. Knocking down the expression of KDM4B inhibited cell proliferation of not only LNCaP but also AR-independent C4-2B cells. In vivo study showed that down regulation of KDM4B significantly impaired tumor growth of C4-2B in xenografts of nude mice. KDM4B was upregulated under hypoxia mimicking a hormone-therapy environment in LNCaP and C4-2B cells. KDM4B knockdown significantly reduced the expression of genes in glycolytic flux under normoxia and hypoxia. Our results suggest that KDM4B contributes to metabolic adaptation in prostate tumor progression and survival.

中文摘要 i
Abstract II
第一章. 序言 1
1.1前列腺癌之簡介 1
1.2前列腺癌的診斷與治療 1
1.3雄激素受體在前列腺中的角色 2
1.4前列腺癌治療的困難 2
1.5抗去勢型攝護腺癌 2
1.6 表關遺傳與癌症 3
1.7組蛋白修飾與組蛋白去甲基酶 3
1.8 KDM4家族和前列腺癌 4
1.9前列腺癌細胞微環境 5
1.10 缺氧環境下的重要因子HIF-1α 6
1.11 HIF-1α與c-Myc在代謝途徑上的角色 6
1.12 缺氧環境下HIF-1α與c-Myc的關係 7
1.13 研究目的 7
第二章. 實驗方法 9
2.1實驗細胞株與細胞培養 9
2.2細胞毒性測試 9
2.3細胞增長分析 10
2.4西方墨點法 10
2.5 RNA分析 10
2.6環境壓力測試 11
2.7 shRNA lentivirus的製造 12
2.8建立敲減KDM4s前列腺癌細胞株 12
2.9免疫共沉澱法 12
2.10螢光素酶檢測法 13
2.11海馬生物能量測定 13
2.12異體移植動物模型 13
2.13統計分析 14
第三章 實驗結果 15
3.1 KDM4A及KDM4B在前列腺癌是被高度表現的 15
3.2 KDM4B廣泛調控致癌代謝途徑 15
3.3敲減KDM4B會影響具有AR的前列腺癌細胞癌細胞的增生與形成腫瘤的能力 16
3.4 KDM4B在缺氧的環境中是會被激活且大量表現的 17
3.5探討C4-2B前列腺癌細胞在缺氧環境下代謝途徑上的調控 18
3.6敲減KDM4B會使得C4-2B細胞在缺氧環境，明顯降低代謝途徑基因之表現 19
3.7敲減KDM4B降低lipid synthesis和glutaminolysis途徑上的基因表現 19
3.8使用資料庫UCSC的資料分析，發現KDM4B調控的代謝基因的啟動子上同時具有c-Myc的辨認位 20
3.9 KDM4B在有氧或缺氧環境下均會調控HIF-1α和c-Myc的transactivation activity 20
3.10 KDM4B分別與HIF-1α和c-Myc結合 21
3.11敲減KDM4B會降低HIF-1α和c-Myc所共同調控的基因HK2，PDK1和VEGFA之表現 20
第四章. 結果與討論 23
4.1 KDM4B為一個治療具有雄激素受體的抗去勢前列腺癌的潛力標靶 23
4.2 KDM4B在AR-positive CRPC前列腺癌與HIF-1α和c-Myc共同參與代謝途徑之調控 23
參考文獻 26

1. Feldman, B.J. and D. Feldman, The development of androgen-independent prostate cancer. Nat Rev Cancer, 2001. 1(1): p. 34-45.
2. Egger, G., et al., Epigenetics in human disease and prospects for epigenetic therapy. Nature, 2004. 429(6990): p. 457-63.
3. Sharma, S., T.K. Kelly, and P.A. Jones, Epigenetics in cancer. Carcinogenesis, 2010. 31(1): p. 27-36.
4. Wagner, E.J. and P.B. Carpenter, Understanding the language of Lys36 methylation at histone H3. Nat Rev Mol Cell Biol, 2012. 13(2): p. 115-26.
5. Bannister, A.J. and T. Kouzarides, Regulation of chromatin by histone modifications. Cell Res, 2011. 21(3): p. 381-95.
6. Varier, R.A. and H.T. Timmers, Histone lysine methylation and demethylation pathways in cancer. Biochim Biophys Acta, 2011. 1815(1): p. 75-89.
7. Bannister, A.J., R. Schneider, and T. Kouzarides, Histone methylation: dynamic or static? Cell, 2002. 109(7): p. 801-6.
8. Berry, W.L. and R. Janknecht, KDM4/JMJD2 histone demethylases: epigenetic regulators in cancer cells. Cancer Res, 2013. 73(10): p. 2936-42.
9. Coffey, K., et al., The lysine demethylase, KDM4B, is a key molecule in androgen receptor signalling and turnover. Nucleic Acids Res, 2013. 41(8): p. 4433-46.
10. Shin, S. and R. Janknecht, Activation of androgen receptor by histone demethylases JMJD2A and JMJD2D. Biochem Biophys Res Commun, 2007. 359(3): p. 742-6.
11. Wissmann, M., et al., Cooperative demethylation by JMJD2C and LSD1 promotes androgen receptor-dependent gene expression. Nat Cell Biol, 2007. 9(3): p. 347-53.
12. Chu, C.H., et al., KDM4B as a target for prostate cancer: structural analysis and selective inhibition by a novel inhibitor. J Med Chem, 2014. 57(14): p. 5975-85.
13. Al-Ubaidi, F.L., et al., Castration therapy of prostate cancer results in downregulation of HIF-1alpha levels. Int J Radiat Oncol Biol Phys, 2012. 82(3): p. 1243-8.
14. Milosevic, M., et al., Tumor hypoxia predicts biochemical failure following radiotherapy for clinically localized prostate cancer. Clin Cancer Res, 2012. 18(7): p. 2108-14.
15. Yip, K. and R. Alonzi, Carbogen gas and radiotherapy outcomes in prostate cancer. Ther Adv Urol, 2013. 5(1): p. 25-34.
16. Fernandez, E.V., et al., Dual targeting of the androgen receptor and hypoxia-inducible factor 1alpha pathways synergistically inhibits castration-resistant prostate cancer cells. Mol Pharmacol, 2015. 87(6): p. 1006-12.
17. Wang, L., et al., Enrichment of prostate cancer stem-like cells from human prostate cancer cell lines by culture in serum-free medium and chemoradiotherapy. Int J Biol Sci, 2013. 9(5): p. 472-9.
18. Hanahan, D. and R.A. Weinberg, The hallmarks of cancer. Cell, 2000. 100(1): p. 57-70.
19. Hanahan, D. and R.A. Weinberg, Hallmarks of cancer: the next generation. Cell, 2011. 144(5): p. 646-74.
20. Vander Heiden, M.G., L.C. Cantley, and C.B. Thompson, Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science, 2009. 324(5930): p. 1029-33.
21. Sun, R.C. and N.C. Denko, Hypoxic regulation of glutamine metabolism through HIF1 and SIAH2 supports lipid synthesis that is necessary for tumor growth. Cell Metab, 2014. 19(2): p. 285-92.
22. Kishton, R.J. and J.C. Rathmell, Novel therapeutic targets of tumor metabolism. Cancer J, 2015. 21(2): p. 62-9.
23. Folkman, J., The role of angiogenesis in tumor growth. Semin Cancer Biol, 1992. 3(2): p. 65-71.
24. Folkman, J., Role of angiogenesis in tumor growth and metastasis. Semin Oncol, 2002. 29(6 Suppl 16): p. 15-8.
25. Semenza, G.L., HIF-1 and mechanisms of hypoxia sensing. Curr Opin Cell Biol, 2001. 13(2): p. 167-71.
26. Kenneth, N.S. and S. Rocha, Regulation of gene expression by hypoxia. Biochem J, 2008. 414(1): p. 19-29.
27. Semenza, G.L., L.A. Shimoda, and N.R. Prabhakar, Regulation of gene expression by HIF-1. Novartis Found Symp, 2006. 272: p. 2-8; discussion 8-14, 33-6.
28. Mahon, P.C., K. Hirota, and G.L. Semenza, FIH-1: a novel protein that interacts with HIF-1alpha and VHL to mediate repression of HIF-1 transcriptional activity. Genes Dev, 2001. 15(20): p. 2675-86.
29. Semenza, G.L., HIF-1, O(2), and the 3 PHDs: how animal cells signal hypoxia to the nucleus. Cell, 2001. 107(1): p. 1-3.
30. DeBerardinis, R.J., et al., The biology of cancer: metabolic reprogramming fuels cell growth and proliferation. Cell Metab, 2008. 7(1): p. 11-20.
31. Eigenbrodt, E., et al., Double role for pyruvate kinase type M2 in the expansion of phosphometabolite pools found in tumor cells. Crit Rev Oncog, 1992. 3(1-2): p. 91-115.
32. Yeung, S.J., J. Pan, and M.H. Lee, Roles of p53, MYC and HIF-1 in regulating glycolysis - the seventh hallmark of cancer. Cell Mol Life Sci, 2008. 65(24): p. 3981-99.
33. Chen, L. and H. Cui, Targeting Glutamine Induces Apoptosis: A Cancer Therapy Approach. Int J Mol Sci, 2015. 16(9): p. 22830-55.
34. Edmunds, L.R., et al., c-Myc programs fatty acid metabolism and dictates acetyl-CoA abundance and fate. J Biol Chem, 2015. 290(33): p. 20100.
35. Huang, L.E., Carrot and stick: HIF-alpha engages c-Myc in hypoxic adaptation. Cell Death Differ, 2008. 15(4): p. 672-7.
36. Kim, J.W., et al., Hypoxia-inducible factor 1 and dysregulated c-Myc cooperatively induce vascular endothelial growth factor and metabolic switches hexokinase 2 and pyruvate dehydrogenase kinase 1. Mol Cell Biol, 2007. 27(21): p. 7381-93.
37. Chen, J.Q. and J. Russo, Dysregulation of glucose transport, glycolysis, TCA cycle and glutaminolysis by oncogenes and tumor suppressors in cancer cells. Biochim Biophys Acta, 2012. 1826(2): p. 370-84.
38. Galluzzi, L., et al., Metabolic targets for cancer therapy. Nat Rev Drug Discov, 2013. 12(11): p. 829-46.
39. Pertega-Gomes, N., et al., A glycolytic phenotype is associated with prostate cancer progression and aggressiveness: a role for monocarboxylate transporters as metabolic targets for therapy. J Pathol, 2015. 236(4): p. 517-30.
40. Zhao, Y., et al., Emerging metabolic targets in cancer therapy. Front Biosci (Landmark Ed), 2011. 16: p. 1844-60.
41. Degenhardt, K., et al., Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell, 2006. 10(1): p. 51-64.
42. Johansson, A., et al., Targeting castration-induced tumour hypoxia enhances the acute effects of castration therapy in a rat prostate cancer model. BJU Int, 2011. 107(11): p. 1818-24.
43. Salminen, A., K. Kaarniranta, and A. Kauppinen, Hypoxia-Inducible Histone Lysine Demethylases: Impact on the Aging Process and Age-Related Diseases. Aging Dis, 2016. 7(2): p. 180-200.
44. Wong, W.J., et al., MYC degradation under low O2 tension promotes survival by evading hypoxia-induced cell death. Mol Cell Biol, 2013. 33(17): p. 3494-504.
45. Mongiardi, M.P., et al., c-MYC inhibition impairs hypoxia response in glioblastoma multiforme. Oncotarget, 2016.