黑色素癌是由黑色素細胞所產生的癌症，是皮膚癌中最嚴重的一類型，在細胞無限制生長的情況下形成癌症，它會破壞組織的融合、打斷並重新排列正常的組織與改變其功能。正常的黑色素細胞是存在於皮膚的外層並產生棕色的色素，稱為黑色素，而它主要負責皮膚色素的產生。當黑色素細胞發展成癌症，增生且侵入其他組織時，就會形成黑色素癌。POMC是一個帶有241胺基酸的多胜肽荷爾蒙，其中包含了ACTH、α-MSH與β-EP。近年來，我們的研究結果證實在黑色素癌細胞大量表現POMC，不論在體內或體外的實驗上的確會抑制腫瘤生長與轉移的能力。然而，僅管能夠抑制腫瘤的增生與血管新生，B16-F10黑色素癌仍然會在POMC基因治療後存活，其存活機制目前仍不明確。MITF是一個具有螺旋-環狀-螺旋結構的轉錄因子。它不僅與黑色素的生成有關，同時在黑色素細胞的發展與存活上扮演了重要的角色。此外，MITF會結合在HIF-1α的啟動子上並刺激、活化其轉錄作用。在此研究中，我們探討在B16-F10黑色素癌細胞中，POMC基因傳送對MITF/HIF-1α生存路徑的影響。於即時定量PCR與西方墨點分析中顯示了在黑色素癌細胞中，POMC基因傳送會增加MITF mRNA與蛋白質的量；根據報告基因分析來看，發現在黑色素癌細胞中，POMC基因傳送會顯著增加HIF-1α冷光的活性。由轉殖技術與puromycin的篩選，我們得到了具有穩定表現short hairpin RNA抑制MITF特性的MITF-knockdown B16-F10 黑色素癌細胞。而其細胞的生長、侵入與群聚形式相似於vector控制組。經由組織學分析可知以MITF-knockdown細胞殖入處理過的腫瘤會顯著性的減少CD31-positive血管，並且伴隨下降Ki-67-positive增生的細胞數目和增加TUNEL-positive凋亡細胞數目。然而，MITF-KD細胞的殖入會導致黑色素癌腫瘤尺寸顯著減少。ASA是一種被廣泛使用的解熱陣痛劑。而它作用在生物上的影響很廣泛，包括抗熱休克、中風及預防一些癌症的發展；而在我們的研究發現，ASA是可以增加細胞增生的。然而，在侵入試驗中，ASA對於細胞的移動是沒有影響的。此外，經由POMC正向調節的MITF與HIF-1α在MITF-knockdown B16-F10細胞中同樣會明顯的減少。POMC基因傳送會增加HO-1 mRNA與蛋白質的量。HO-1是HIF-1α路徑下游的一個受動器，同時也是一種酵素，它會催化血色素轉換成鐵、一氧化碳與膽綠素。抑制HO-1的活性會增加POMC基因傳送後抑制黑色素癌細胞的增生、移動與細胞生長的能力。而這些研究顯示出MITF/HIF-1α確實會在POMC基因傳送後有助於黑色素癌的生存。

Melanoma is a cancer of the pigment producing cells, melanocytes, and is the most serious type of skin cancer. Cancer is a condition in which one type of cell grows without limit in a disorganized fashion, disrupting and replacing normal tissues and their functions. Normal melanocytes reside in the outer layer of the skin and produce a brown pigment called melanin, which is responsible for skin color. Melanoma occurs when melanocytes become cancerous, grow, and invade other tissues. Pro-opiomelanocortin (POMC) is a precursor polypeptide of 241 amino acids and the prohormone of various neuropeptide, including corticotropin (ACTH),
α-melanocyte-stimulating hormone (α-MSH), and β-endorphin (β-EP). Recently, we demonstrated that systemic POMC overexpression potently suppresses the growth and metastasis of B16-F10 melanoma in vitro and in vivo. However, despite potent inhibition of tumor proliferation and angiogenesis, B16-F10 melanoma still managed to survive after POMC gene therapy. The underlying survival mechanism of B16-F10 melanoma remains unclear. Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix transcription factor that plays a key role not only in melanin synthesis, but also in melanocyte development and survival. Besides, MITF binds to
the hypoxia-inducible factor-1α (HIF-1α) promoter to stimulate its transcriptional activity. In this study, we investigate the influence of POMC gene delivery on the
pro-survival MITF/HIF-1α pathway in B16-F10 melanoma cells. Quantitative RT-PCR and western blot analysis revealed that POMC gene delivery increased the MITF mRNA and protein level in B16-F10 melanoma cells. Besides, POMC gene delivery significantly enhanced the HIF-1α-driven luciferase activities in melanoma cells. By transfection and puromycin selection, we generated and characterized a MITF-knockdown B16-F10 melanoma cells (MITF KD) stably expressing short hairpin RNA against MITF. The growth, invasion, and colonies formation of MITF-KD were similar to those of vector control. However, implantation of MITF-KD cells led to melanoma with significantly reduced tumor size compared with those in mice implanted with vector control cells. Histological analysis revealed a significant reduction of CD31-positive blood vessels in implantation of MITF-KD cells-treated tumors, which was accompanied with a decrease in Ki-67-positive proliferating cells and an increase in TUNEL-positive apoptotic cells. Moreover, POMC-mediated upregulation of MITF and HIF-1 α was significantly attenuated in MITF KD-B16-F10 cells. Acetylsalicylic acid (aspirin; ASA) is widely used as an
analgesic/antipyretic drug. ASA exhibits a wide range of biological effects, including preventative effects against heart attack, stroke, and the development of some types of cancer. In our study, we found ASA enhanced cell proliferation. However, in invasion test, ASA had no effect on cell migration. POMC gene delivery elevated the mRNA and protein level of hemeoxygenase-1 (HO-1), a downstream effector of HIF-1α pathway and an enzyme catalyzing the converting reaction of heme to carbon monoxide, ion and biliverdine. Inhibition of HO-1 activities augmented the inhibitory effect of POMC gene delivery on proliferation, migration and anchorage-independent
growth of B16-F10 melanoma cells. These studies indicated that activation of MITF/HIF-1α/HO-1 indeed contributes to melanoma survival after POMC gene
delivery.

Abbreviations 3
Abstract in Chinese 4
Abstract in English 6
Introduction 8
Materials and Methods 13
Results 19
Discussion 24
References 27
Figures and Legends 32
Appendix 54

Aix-loop-helix-leucine zipper transcription factor to a subset of E-box elements in vitro and in vivo. Mol Cell Biol. 18:6930-8.
Berberat, P.O., Z. Dambrauskas, A. Gulbinas, T. Giese, N. Giese, B. Kunzli, F. Autschbach, ksan, I., and C.R. Goding. 1998. Targeting the microphthalmia basic helS. Meuer, M.W. Buchler, and H. Friess. 2005. Inhibition of heme oxygenase-1 increases responsiveness of pancreatic cancer cells to anticancer treatment. Clin Cancer Res. 11:3790-8.
Briganti, S., E. Camera, and M. Picardo. 2003. Chemical and instrumental approaches to treat hyperpigmentation. Pigment Cell Res. 16:101-10.
Carreira, S., J. Goodall, I. Aksan, S.A. La Rocca, M.D. Galibert, L. Denat, L. Larue, and C.R. Goding. 2005. Mitf cooperates with Rb1 and activates p21Cip1 expression to regulate cell cycle progression. Nature. 433:764-9.
Catania, A., S. Gatti, G. Colombo, and J.M. Lipton. 2004. Targeting melanocortin receptors as a novel strategy to control inflammation. Pharmacol Rev. 56:1-29.
Cisowski, J., A. Loboda, A. Jozkowicz, S. Chen, A. Agarwal, and J. Dulak. 2005. Role of heme oxygenase-1 in hydrogen peroxide-induced VEGF synthesis: effect of HO-1 knockout. Biochem Biophys Res Commun. 326:670-6.
Clark, J.E., R. Foresti, C.J. Green, and R. Motterlini. 2000. Dynamics of haem oxygenase-1 expression and bilirubin production in cellular protection against oxidative stress. Biochem J. 348 Pt 3:615-9.
Du, J., H.R. Widlund, M.A. Horstmann, S. Ramaswamy, K. Ross, W.E. Huber, E.K. Nishimura, T.R. Golub, and D.E. Fisher. 2004. Critical role of CDK2 for melanoma growth linked to its melanocyte-specific transcriptional regulation by MITF. Cancer Cell. 6:565-76.
Gaggioli, C., R. Busca, P. Abbe, J.P. Ortonne, and R. Ballotti. 2003. Microphthalmia-associated transcription factor (MITF) is required but is not sufficient to induce the expression of melanogenic genes. Pigment Cell Res. 16:374-82.
Goding, C., and F.L. Meyskens, Jr. 2006. Microphthalmic-associated transcription factor integrates melanocyte biology and melanoma progression. Clin Cancer Res. 12:1069-73.
Hodgkinson CA, M.K., Nakayama A, et al. 1990. Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein. Cell. 74:395-404.
Hodgkinson, C.A., K.J. Moore, A. Nakayama, E. Steingrimsson, N.G. Copeland, N.A. Jenkins, and H. Arnheiter. 1993. Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein. Cell. 74:395-404.
Hughes, M.J., J.B. Lingrel, J.M. Krakowsky, and K.P. Anderson. 1993. A helix-loop-helix transcription factor-like gene is located at the mi locus. J Biol Chem. 268:20687-90.
Javelaud, D., V.I. Alexaki, and A. Mauviel. 2008. Transforming growth factor-beta in cutaneous melanoma. Pigment Cell Melanoma Res. 21:123-32.
Kristensen, D.B., N. Kawada, K. Imamura, Y. Miyamoto, C. Tateno, S. Seki, T. Kuroki, and K. Yoshizato. 2000. Proteome analysis of rat hepatic stellate cells. Hepatology. 32:268-77.
Lerner, A.B., T. Shiohara, R.E. Boissy, K.A. Jacobson, M.L. Lamoreux, and G.E. Moellmann. 1986. A mouse model for vitiligo. J Invest Dermatol. 87:299-304.
Li Volti, G., D. Sacerdoti, B. Sangras, A. Vanella, A. Mezentsev, G. Scapagnini, J.R. Falck, and N.G. Abraham. 2005. Carbon monoxide signaling in promoting angiogenesis in human microvessel endothelial cells. Antioxid Redox Signal. 7:704-10.
Loboda, A., A. Jazwa, B. Wegiel, A. Jozkowicz, and J. Dulak. 2005. Heme oxygenase-1-dependent and -independent regulation of angiogenic genes expression: effect of cobalt protoporphyrin and cobalt chloride on VEGF and IL-8 synthesis in human microvascular endothelial cells. Cell Mol Biol (Noisy-le-grand). 51:347-55.
Loercher, A.E., E.M. Tank, R.B. Delston, and J.W. Harbour. 2005. MITF links differentiation with cell cycle arrest in melanocytes by transcriptional activation of INK4A. J Cell Biol. 168:35-40.
Maines, M.D., and P.E. Gibbs. 2005. 30 some years of heme oxygenase: from a "molecular wrecking ball" to a "mesmerizing" trigger of cellular events. Biochem Biophys Res Commun. 338:568-77.
Malaguarnera, L., S. Quan, M.R. Pilastro, N.G. Abraham, and A. Kappas. 2003. Diminished heme oxygenase potentiates cell death: pyrrolidinedithiocarbamate mediates oxidative stress. Exp Biol Med (Maywood). 228:459-65.
McGill, G.G., M. Horstmann, H.R. Widlund, J. Du, G. Motyckova, E.K. Nishimura, Y.L. Lin, S. Ramaswamy, W. Avery, H.F. Ding, S.A. Jordan, I.J. Jackson, S.J. Korsmeyer, T.R. Golub, and D.E. Fisher. 2002. Bcl2 regulation by the melanocyte master regulator Mitf modulates lineage survival and melanoma cell viability. Cell. 109:707-18.
Newell-Price, J., P. King, and A.J. Clark. 2001. The CpG island promoter of the human proopiomelanocortin gene is methylated in nonexpressing normal tissue and tumors and represses expression. Mol Endocrinol. 15:338-48.
Otterbein, L.E., and A.M. Choi. 2000. Heme oxygenase: colors of defense against cellular stress. Am J Physiol Lung Cell Mol Physiol. 279:L1029-37.
Otterbein, L.E., B.S. Zuckerbraun, M. Haga, F. Liu, R. Song, A. Usheva, C. Stachulak, N. Bodyak, R.N. Smith, E. Csizmadia, S. Tyagi, Y. Akamatsu, R.J. Flavell, T.R. Billiar, E. Tzeng, F.H. Bach, A.M. Choi, and M.P. Soares. 2003. Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury. Nat Med. 9:183-90.
Raffin-Sanson, M.L., Y. de Keyzer, and X. Bertagna. 2003. Proopiomelanocortin, a polypeptide precursor with multiple functions: from physiology to pathological conditions. Eur J Endocrinol. 149:79-90.
Regehly, M., K. Greish, F. Rancan, H. Maeda, F. Bohm, and B. Roder. 2007. Water-soluble polymer conjugates of ZnPP for photodynamic tumor therapy. Bioconjug Chem. 18:494-9.
Sato, S., K. Roberts, G. Gambino, A. Cook, T. Kouzarides, and C.R. Goding. 1997. CBP/p300 as a co-factor for the Microphthalmia transcription factor. Oncogene. 14:3083-92.
Selzer, E., V. Wacheck, T. Lucas, E. Heere-Ress, M. Wu, K.N. Weilbaecher, W. Schlegel, P. Valent, F. Wrba, H. Pehamberger, D. Fisher, and B. Jansen. 2002. The melanocyte-specific isoform of the microphthalmia transcription factor affects the phenotype of human melanoma. Cancer Res. 62:2098-103.
Semenza, G.L. 2002. Involvement of hypoxia-inducible factor 1 in human cancer. Intern Med. 41:79-83.
Solano, F., S. Briganti, M. Picardo, and G. Ghanem. 2006. Hypopigmenting agents: an updated review on biological, chemical and clinical aspects. Pigment Cell Res. 19:550-71.
Solomon, S. 1999. POMC-derived peptides and their biological action. Ann N Y Acad Sci. 885:22-40.
Steingrimsson, E., N.G. Copeland, and N.A. Jenkins. 2004. Melanocytes and the microphthalmia transcription factor network. Annu Rev Genet. 38:365-411.
Steingrimsson E, M.K., Lamoreux. 1994. Molecular basis of mouse microphthalmia (mi) mutations helps explain their developmental and phenotypic consequences. Nat Genet. 8:256-63.
Tachibana, M. 2000. MITF: a stream flowing for pigment cells. Pigment Cell Res. 13:230-40.
Ugurel, S., R. Houben, D. Schrama, H. Voigt, M. Zapatka, D. Schadendorf, E.B. Brocker, and J.C. Becker. 2007. Microphthalmia-associated transcription factor gene amplification in metastatic melanoma is a prognostic marker for patient survival, but not a predictive marker for chemosensitivity and chemotherapy response. Clin Cancer Res. 13:6344-50.
Weissmann, G. 1991. Aspirin. Sci Am. 264:84-90.