臺灣博碩士論文加值系統

人類粒線體NAD(P)+-依賴型蘋果酸酶 (ME2)為一個可將蘋果酸以脫羧反應轉變成丙酮酸之代謝酵素，目前已有研究指出在肺癌、白血病、黑色素細胞瘤等癌症中ME2對於腫瘤的增生扮演關鍵性的角色。然而，腫瘤微環境(例如氧氣或養份之供應)的改變是否會影響ME2表現及其調控腫瘤增生能力之詳細機制目前仍沒有被詳盡的研究。
我們利用88種黑色素細胞(包含良性與惡性)的基因表達陣列數據發現ME2與缺氧誘導因子(HIF-1α)及其下游基因，如血管內皮生長因子-A(VEGF-A)、胰島素樣生長因子結合蛋白3(IGFBP3)等基因表現皆呈現負相關性。因此將黑色素瘤細胞置於缺氧環境或給予模擬缺氧藥物氯化鈷 (CoCl2)，ME2蛋白質表達量會受到抑制。
然而我們在缺氧下利用siRNA抑制HIF-1α發現缺氧誘導之ME2蛋白質下降可能並非透過HIF-1α調控路徑而是透過DEC1或DEC2這兩個轉錄因子影響其轉錄活性。總結我們的實驗結果顯示，在黑色素細胞在缺氧環境下會抑制ME2表現，而ME2的過度表達能提供惡性黑色素細胞瘤增生的代謝優勢，因此發展抑制ME2的小分子藥物可做為未來治療惡性黑色素細胞瘤的一個可行性的治療策略。

Human mitochondrial NAD(P)+-dependent malic enzyme (ME2) is an metabolic enzyme which catabolizes the conversion of malate into pyruvate via a decarboxylation reaction. Several lines of evidence suggest that it coordinates cell metabolism and tumor growth, such as lung cancer, leukemia and melanoma. Nevertheless, the detailed mechanisms by which how ME2 is upregulated during tumorigenesis and its functional role in response to tumor microenvironment remain elusive. Intriguingly, we found the ME2 levels inversely correlated with the expression level of the hypoxia markers HIF1α, and its downstream targets, VEGF-A and IGFBP3, in an expression array data set which consists of primary melanoma specimens and cell lines. In addition, we observed ME2 was suppressed under hypoxia and the treatment of CoCl2, a hypoxia mimetic, in melanoma cells. Surprisingly, we found that hypoxia-induced ME2 downregulation via a HIF1α-independent pathway by siRNA targeting HIF1α under hypoxia, and the repression of ME2 under hypoxia may result from transcription repression by DEC1 and DEC2. These results illustrated a physiologic hypoxia response in pigment cells resulting in ME2 repression, while ME2 overexpression was regarded as a metabolic advantage for malignant melanoma proliferation and thus provided an attractive therapeutic strategy for suppression of the ME2 in melanoma treatment.

目錄 I
圖文目錄 III
縮寫表 IV
中文摘要 V
英文摘要 VI
第一章 緒論 1
第一節 皮膚 1
第二節 黑色素細胞瘤 1
第三節 黑色素細胞瘤的分期 2
第四節 黑色素細胞瘤的治療 4
第五節 蘋果酸酶 5
第六節 缺氧誘導因子 6
第七節 研究動機 7
第二章 實驗材料與方法 8
第一節 實驗材料 8
第二節 實驗方法 13
第三章 結果 25
第一節 分析在黑色素細胞瘤細胞中HIF-1α及其下游基因與ME2的關係 25
第二節 探討在缺氧的條件下ME2在人類黑色素細胞瘤的表現 25
第三節 探討在缺氧的條件下HIF-1α對於ME2之間的影響 27
第四節 探討缺氧條件下DEC1/2的表達與抑制ME2之間的影響 28
第五節 探討缺氧條件下對於ME2啟動子活性的影響 30
第四章 討論 32
第一節 人類黑色素細胞瘤與ME2之相關性 32
第二節 缺氧對於ME2的影響 33
第三節 缺氧與轉錄調控造成ME2蛋白質下降之關係 35
第四節 DEC1/2對於ME2的影響 36
第五節 HIF-1α、DEC1/2對於ME2啟動子報告載體的相關性 37
第五章 結論 39
第六章 參考文獻 49

1.Nickoloff, B.J. and Y. Naidu, Perturbation of epidermal barrier function correlates with initiation of cytokine cascade in human skin. Journal of the American Academy of Dermatology, 1994. 30(4): p. 535-546.
2.Cichorek, M., M. Wachulska, A. Stasiewicz, and A. Tyminska, Skin melanocytes: biology and development. Postepy Dermatol Alergol, 2013. 30(1): p. 30-41.
3.Tsatmali, M., J. Ancans, and A.J. Thody, Melanocyte Function and Its Control by Melanocortin Peptides. Journal of Histochemistry & Cytochemistry, 2002. 50(2): p. 125-133.
4.Chen, J., R. Shao, X.D. Zhang, and C. Chen, Applications of nanotechnology for melanoma treatment, diagnosis, and theranostics. Applications of nanotechnology for melanoma treatment, diagnosis, and theranostics, 2013. 34(6): p. 1481-1489.
5.Word Health Organization[Homepage on the Internet] Skin Cancer. Available from:http://www.who.int/uv/faq/skincancer/en/index1.html. Accessed May 4, 2016.
6.Helmbach, H., E. Rossmann, M.A. Kern, and D. Schadendorf, Drug resistance in human melanoma. International Journal of Cancer, 2001. 93(5): p. 617-622.
7.Skin Cancer Foundation [Homepage on the Internet] Types of melanoma. Available from:http://www.skincancer.org/skin-cancer-information/melanoma/types-of-melanoma. Accessed May 4,2016.
8.Bellew, S., D.J.Q. Rosso, and G.K. Kim, Skin cancer in asians: part 2: melanoma. The Journal of clinical and aesthetic dermatology, 2009. 2(10): p. 34-36.
9.Autier, P., D. Epimel, and J. Group, Influence of sun exposures during childhood and during adulthood on melanoma risk. International Journal of Cancer, 1998. 77(4): p. 533-537.
10.Abbasi, N.R., H.M. Shaw, D.S. Rigel, and R.J. Friedman, Early diagnosis of cutaneous melanoma: revisiting the ABCD criteria. Jama, 2004. 292(22): p. 2771-2776.
11.American Joint Committee on Cancer [Homepage on the Internet] Melanoma of the Skin Staging.Available from: https://cancerstaging.org/references-tools/quickreferences/documents/melanomasmall.pdf .Accessed May 4.
12.Cancer Research UK [Homepage on the Internet] Skin cancer incidence by stage at diagnosis. Available from: http://www.cancerresearchuk.org/health-professional/cancer-statistics/statistics-by-cancer-type/skin-cancer/incidence#heading-Three. Accessed May 4, 2016
13.Bhatia, S., S.S. Tykodi, and J.A. Thompson, Treatment of metastatic melanoma: an overview. Oncology, 2009. 23(6): p. 488-496.
14.Villanueva, J., A. Vultur, J.T. Lee, and R. Somasundaram, Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer cell, 2010. 18(6): p. 683-695.
15.Zheng, B. and D.E. Fisher, Metabolic vulnerability in melanoma: a ME2 (me too) story. The Journal of investigative dermatology, 2015. 135(3): p. 657-659.
16.Chang, Y.-L., H.-W. Gao, C.-P. Chiang, W.-M.M. Wang, S.-M. Huang, C.-F. Ku, G.-Y. Liu, and H.-C. Hung, Human mitochondrial NAD(P)(+)-dependent malic enzyme participates in cutaneous melanoma progression and invasion. The Journal of investigative dermatology, 2015. 135(3): p. 807-815.
17.Ren, J.-G.G., P. Seth, C.B. Clish, P.K. Lorkiewicz, R.M. Higashi, A.N. Lane, T.W. Fan, and V.P. Sukhatme, Knockdown of malic enzyme 2 suppresses lung tumor growth, induces differentiation and impacts PI3K/AKT signaling. Scientific reports, 2014. 4(5414): p. 1-11.
18.Ren, J.-G., P. Seth, P. Everett, C.B. Clish, and V.P. Sukhatme, Induction of Erythroid Differentiation in Human Erythroleukemia Cells by Depletion of Malic Enzyme 2. PLoS ONE, 2010. 5(9): p. 1-12.
19.Jiang, P., W. Du, A. Mancuso, K.E. Wellen, and X. Yang, Reciprocal regulation of p53 and malic enzymes modulates metabolism and senescence. Nature, 2013. 493(7434): p. 689-693.
20.Feige, E., S. Yokoyama, C. Levy, M. Khaled, V. Igras, R.J. Lin, S. Lee, H.R. Widlund, S.R. Granter, A.L. Kung, and D.E. Fisher, Hypoxia-induced transcriptional repression of the melanoma-associated oncogene MITF. Proceedings of the National Academy of Sciences of the United States of America, 2011. 108(43): p. 33.
21.Giordano, F.J. and R.S. Johnson, Angiogenesis: the role of the microenvironment in flipping the switch. Current opinion in genetics & development, 2001. 11(1): p. 35-40.
22.Covello, K.L. and M.C. Simon, HIFs, hypoxia, and vascular development. Current topics in developmental biology, 2004. 62: p. 37-54.
23.Kaelin Jr, W.G., Proline hydroxylation and gene expression. Annu. Rev. Biochem., 2005. 74: p. 115-128.
24.Carroll, V.A. and M. Ashcroft, HIF-1a regulation by proline hydroxylation. Expert Reviews in Molecular Medicine, 2005. 7(6): p. 1.
25.Maxwell, P.H., M.S. Wiesener, G.W. Chang, and S.C. Clifford, The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature, 1999. 399: p. 271-275.
26.Sadri, N. and P.J. Zhang, Hypoxia-inducible factors: mediators of cancer progression; prognostic and therapeutic targets in soft tissue sarcomas. Cancers, 2013. 5(2): p. 320-333.
27.Semenza, G.L., Hydroxylation of HIF-1: oxygen sensing at the molecular level. Physiology, 2004. 19(4): p. 176-182.
28.Feige, E., S. Yokoyama, C. Levy, M. Khaled, V. Igras, R.J. Lin, S. Lee, H.R. Widlund, S.R. Granter, and A.L. Kung, Hypoxia-induced transcriptional repression of the melanoma-associated oncogene MITF. Proceedings of the National Academy of Sciences, 2011. 108(43): p. 924-933.
29.Talks, K.L., H. Turley, K.C. Gatter, and P.H. Maxwell, The expression and distribution of the hypoxia-inducible factors HIF-1α and HIF-2α in normal human tissues, cancers, and tumor-associated macrophages. American Journal of Pathology, 2000. 157(2): p. 411-421.
30.Lin, W.M., A.C. Baker, R. Beroukhim, W. Winckler, and W. Feng, Modeling genomic diversity and tumor dependency in malignant melanoma. Cancer research, 2008. 68(3): p. 664-673.
31.Semenza, G.L., Hypoxia-inducible factor 1: control of oxygen homeostasis in health and disease. Pediatric research, 2001. 49(5): p. 614-617.
32.Hoeflich, K.P., S. Herter, J. Tien, L. Wong, L. Berry, J. Chan, C. O'Brien, Z. Modrusan, S. Seshagiri, and M. Lackner, Antitumor efficacy of the novel RAF inhibitor GDC-0879 is predicted by BRAFV600E mutational status and sustained extracellular signal-regulated kinase/mitogen-activated protein kinase pathway suppression. Cancer research, 2009. 69(7): p. 3042-3051.
33.Xing, F., Y. Persaud, C.A. Pratilas, B.S. Taylor, M. Janakiraman, Q.B. She, H. Gallardo, C. Liu, T. Merghoub, and B. Hefter, Concurrent loss of the PTEN and RB1 tumor suppressors attenuates RAF dependence in melanomas harboring V600EBRAF. Oncogene, 2012. 31(4): p. 446-457.
34.Wu, H., V. Goel, and F.G. Haluska, PTEN signaling pathways in melanoma. Oncogene, 2003. 22(20): p. 3113-3122.
35.Jiang, D. and L.D. Attardi, Engaging the p53 metabolic brake drives senescence. Cell Res, 2013. 23(6): p. 739-40.
36.Hofer, T., R. Wenger, and M. Gassmann, Oxygen sensing, HIF-1a stabilization and potential therapeutic strategies. Pflügers Archiv European Journal of Physiology, 2002. 443(4): p. 503-507.
37.Han, Y.H., H.J. Moon, and B.R. You, The effect of MG132, a proteasome inhibitor on HeLa cells in relation to cell growth, reactive oxygen species and GSH. Oncology reports, 2009. 22(1): p. 215-221.
38.Sato, F., U.K. Bhawal, T. Yoshimura, and Y. Muragaki, DEC1 and DEC2 crosstalk between circadian rhythm and tumor progression. Journal of Cancer, 2016. 7(2): p. 153-159.
39.Wen, Y., L. Xu, F.-l.L. Chen, J. Gao, J.-y.Y. Li, L.-h.H. Hu, and J. Li, Discovery of a novel inhibitor of NAD(P)(+)-dependent malic enzyme (ME2) by high-throughput screening. Acta pharmacologica Sinica, 2014. 35(5): p. 674-684.
40.Hsieh, J.-Y., S.-Y. Li, W.-C. Tsai, J.-H. Liu, C.-L. Lin, G.-Y. Liu, and H.-C. Hung, A small-molecule inhibitor suppresses the tumor-associated mitochondrial NAD (P)+-dependent malic enzyme (ME2) and induces cellular senescence. Oncotarget, 2015. 6(24): p. 20084.
41.Mo, J., B. Sun, X. Zhao, Q. Gu, X. Dong, Z. Liu, and Y. Ma, Hypoxia-induced senescence contributes to the regulation of microenvironment in melanomas. Pathology–Research and Practice, 2013. 209(10): p. 640-647.
42.Kaplon, J., L. Zheng, K. Meissl, B. Chaneton, V.A. Selivanov, G. Mackay, S.H. van der Burg, E.M. Verdegaal, M. Cascante, T. Shlomi, E. Gottlieb, and D.S. Peeper, A key role for mitochondrial gatekeeper pyruvate dehydrogenase in oncogene-induced senescence. Nature, 2013. 498(7452): p. 109-112.
43.Harris, A.L., Hypoxia--a key regulatory factor in tumour growth. Nature reviews. Cancer, 2002. 2(1): p. 38-47.
44.Minchenko, A., T. Bauer, S. Salceda, and J. Caro, Hypoxic stimulation of vascular endothelial growth factor expression in vitro and in vivo. Laboratory investigation; a journal of technical methods and pathology, 1994. 71(3): p. 374-379.
45.Stone, J., A. Itin, T. Alon, J. Pe'Er, H. Gnessin, T. Chan-Ling, and E. Keshet, Development of retinal vasculature is mediated by hypoxia-induced vascular endothelial growth factor (VEGF) expression by neuroglia. The Journal of neuroscience, 1995. 15(7): p. 4738-4747.
46.Greijer, A.E., The role of hypoxia inducible factor 1 (HIF-1) in hypoxia induced apoptosis. Journal of Clinical Pathology, 2004. 57(10): p. 1009-1014.
47.Papandreou, I., A.L. Lim, and K. Laderoute, Hypoxia signals autophagy in tumor cells via AMPK activity, independent of HIF-1, BNIP3, and BNIP3L. Cell Death and Differentiation, 2008. 15(10): p. 1572-1581.
48.Wu, Y., H. Sato, T. Suzuki, T. Yoshizawa, S. Morohashi, H. Seino, T. Kawamoto, K. Fujimoto, Y. Kato, and H. Kijima, Involvement of c-Myc in the proliferation of MCF-7 human breast cancer cells induced by bHLH transcription factor DEC2. International Journal of Molecular Medicine, 2014. 35(3): p. 815-820.
49.Fujimoto, K., H. Hamaguchi, T. Hashiba, T. Nakamura, T. Kawamoto, F. Sato, M. Noshiro, U.K. Bhawal, K. Suardita, and Y. Kato, Transcriptional repression by the basic helix-loop-helix protein Dec2: multiple mechanisms through E-box elements. International journal of molecular medicine, 2007. 19(6): p. 925-932.