現今針對前期攝護腺癌患者的治療，通常以根除性攝護腺切除術以及賀爾蒙治療法為主，其中95%的患者可痊癒。然而，其餘患者有機率復發，並且對賀爾蒙治療產生抗性而形成去勢性攝護腺癌 (CRPC)，三年內死亡率高達77%。去勢性攝護腺癌的分子進程源自於遺傳或表觀遺傳的改變。近年來，人們開始高度關注人類組蛋白離胺酸特異性去甲基化酶 (KDM4B)在癌症中扮演的角色，其主要針對H3K9以及H3K36兩個位置的雙甲基或三甲基去甲基化。在本研究中，KDM4B的降減會造成糖解能力下降但是粒線體氧化能力的提升。代謝基因組學的分析中也可以看到，KDM4B的降減會使麩醯胺酸和氧化型麩胱甘肽的總產量上升，麩胺酸和谷胱甘肽的產量下降。這些結果可以表達，當攝護腺癌細胞中的KDM4B被降減之後，葡萄糖的代謝途徑可能從無氧糖解轉為氧化磷酸化途徑。而作為編碼轉錄因子的調節基因，c-Myc在能量的代謝途徑中扮演很重要的角色。由過去的文獻指出，它會直接調控乳酸脫氫酶 (LDHA)的基因表現，同時當KDM4B被降減時，LDHA的mRNA表現量也下降了。因此我們推斷KDM4B和c-Myc有關連，從免疫沉澱法的實驗結果可以證明兩者會結合在一起。接著，利用報導基因冷光活性試驗(promoter reporter assay)，也證實KDM4B會加強c-Myc對LDHA啟動子的活性表現。由這些實驗結果，我們推斷KDM4B擔任c-Myc的共激活因子，共同調控CRPC的代謝適應。

The standard treatment for initial-stage androgen-dependent prostate cancer (PCa) patients is androgen deprivation therapy (ADT). However, castration-resistant prostate cancer (CRPC) inevitably occurs within 2-3 years after ADT and causes about 77% deaths within 3 years (1). The molecular evolution of CRPC arises from complicated genetic/epigenetic alterations. Recently, the role of histone lysine-specific demethylase KDM4B/JMJD2B which targets H3K9me2/me3, H3K36me2/me3 in CRPC has drawn much attention. Here, we show that the depletion of KDM4B resulted in downregulation of glucose metabolism whereas upregulation of mitochondria oxidation. Metabolomic analysis of KDM4B-knockdown cells revealed a reduced level of glutamate and glutathione but increased level of glutamine and oxidative glutathione as compared with that of the control groups. These results suggest that KDM4B-knockdown cells shifted from aerobic glycolysis to oxidative mitochondrial metabolism. c-Myc, an important regulator of energy metabolism that drives the expression of lactate dehydrogenase A (LDHA) was also downregulated in KDM4B-knockdown cells (2). Accordingly, there was a significantly reduced level of LDHA expression after the depletion of KDM4B. Immunoprecipitation experiments revealed that KDM4B physically interacted with c-Myc. Additionally, KDM4B and c-Myc synergistically enhanced the LDHA promoter transcriptional activity. Taken together, our results suggest that KDM4B functions as a c-Myc coactivator to regulate CRPC metabolic adaptation.

人類組蛋白去甲基酵素KDM4B在抗去勢前列腺癌之代謝適應上扮演關鍵的角色........i
中文摘要..........................................................ii
Abstract.........................................................iii
Abbreviation.....................................................iv
1 Introduction..................................................5
1.1 About prostate cancer........................................5
1.2 Symptoms, diagnose and therapy of prostate cancer............5
1.3 Androgen receptor............................................6
1.4 The progression of castration-resistance prostate cancer.....6
1.5 The mechanism and therapy of CRPC............................7
1.6 Epigenetic regulation........................................8
1.7 JMJD2/KDM4 subfamily and KDM4B...............................9
1.8 The role of c-Myc in metabolic adaptation....................9
1.9 Purpose of this research.....................................10
1.10 Warburg effect and cancer proliferation.....................11
2 Material and methods..........................................12
2.1 Cell culture.................................................12
2.2 Lentivirus production and knockdown cell lines establishment .................................................................12
2.3 Cell viability test..........................................12
2.4 RNA extraction...............................................13
2.5 qRT-PCR......................................................14
2.6 Immunoprecipitation and Immunoblotting.......................14
2.7 OCR and ECAR.................................................15
2.8 Lactate production and PDH activity..........................16
2.9 Luciferase assay.............................................17
2.10 ChIP assay..................................................17
2.11 Fluorescence image assay....................................18
2.12 Statistical analysis........................................18
3 Results.......................................................20
3.1 A reduced level of the glycolytic flux after the depletion of KMD4B............................................................20
3.2 Knockdown of KDM4B induces mitochondrial oxidative phosphorylation .................................................................20
3.3 KDM4B modulates the energetic metabolism.....................21
3.4 KDM4B associates with c-Myc..................................22
3.5 KDM4B is crucial for c-Myc-regulated LDHA transactivation activity.........................................................23
3.6 Both KDM4B and c-Myc are recruited to the LDHA loci..........24
3.7 M2 acts as a KDM4B inhibitor and suppresses CRPC.............24
3.8 M2 and enzalutamide or abiraterone acetate synergistically inhibit the survival of CRPC cells.......................................25
4. Conclusion and discussion....................................27
4.1 KDM4B and c-Myc co-regulate metabolic pathway in CRPC........27
4.2 M2 inhibits KDM4B and enhances treatment of enzalutamide and abiraterone acetate..............................................29
4.3 The role of KDM4B in other CRPC and drug resistant cell lines needs to be confirmed............................................30
5. Reference....................................................31

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