Pro-opiomelanocortin (POMC)是一條241個胺基酸多肽鏈的前軀物，也是一些包括corticotropin (ACTH)、 a-melanocyte-stimulating hormone (a-MSH)、 和b-endorphin (b-EP)的神經性多肽的前軀荷爾蒙。它是一種壓力荷爾蒙，在代謝、能量平衡、和抗發炎反應中都扮演了重要的角色。先前的研究證實，系統性POMC基因傳送透過a-MSH抑制NF-κB/COX2訊息傳遞路徑，對於B16F10黑色素癌細胞不論是活體外(in vitro) 或是活體內(in vivo)的腫瘤生長及轉移能力都有顯著的抑制效果。然而， 系統性POMC基因傳送卻也會引起小鼠的尿量與飲水量的倍增。POMC基因傳送後，使小鼠血漿中的corticosteroids濃度升高及H295R細胞cortisol的產量增加，根據這些證據推測尿量與飲水量倍增可能歸因於固醇生成的增加。Corticosteroids也是強力的抗發炎劑，其所造成的抗發炎反應是否也參與了POMC對腫瘤的抑制，還尚未清楚。為了解開這個謎團，我們製作了包括ACTH (K15A/R17A)在內，一系列帶有不同ACTH突變或是剔除片段的突變型POMC基因的重組腺病毒載體。相較於一般型POMC基因傳送，ACTH (K15A/R17A) 基因傳送在H295R細胞所誘發的cortisol生成量、CYP11B1 mRNA表現量和glucocorticoid responsive element (GRE)-driven luciferase活性皆顯著降低，也不會影響小鼠的尿量和飲水量。而ACTH (K15A/R17A) 基因傳送對於B16F10黑色素癌細胞的腫瘤形成及侵犯能力仍然有顯著的抑制作用。總而言之，在POMC對黑色素癌的抑制作用，固醇生成並非必要存在的因素；此外，ACTH (K15A/R17A) 基因傳送也為黑色素癌的控制提供了更佳的選擇性。

Proopiomelanocortin (POMC) is a 241 amino acids precursor protein, which encodes various neuropeptides including corticotropin (ACTH), a-melanocyte-stimulating hormone (a-MSH), and b-endorphin (b-EP). POMC plays an important role in stress response, metabolism, energy homeostasis and anti-inflammation. Recent studies demonstrated that systemic POMC gene delivery potently suppresses the tumor growth and metastasis of B16-F10 melanoma in vitro and in vivo via inhibition of NF-κB/COX2 pathway. However, systemic POMC expression also led to elevated urine excretion and water intake in mice. This was attributed to enhanced steroidgenesis as evidence by elevated plasma corticosteroids levels in animals and increased cortisol production in adrenal H295R cells after POMC gene delivery. Since corticosteroids are also potent anti-inflammatory agents, it remains unclear whether the ACTH-mediated cortisol synthesis also contributed to the POMC-induced tumor suppression. To address this issue, we generated a series of adenovirus vectors encoding POMC genes with mutation or deletion in ACTH domain including ACTH (K15A/R17A). Unlike the wild type POMC, gene delivery of ACTH (K15A/R17A) resulted in significantly lower cortisol production, CYP11B1 mRNA level, and glucocorticoid responsive element (GRE)-driven luciferase activities in H295R cells. ACTH (K15A/R17A) gene delivery did not affect the urination and water intake in mice. Above all, ACTH (K15A/R17A) gene delivery remained capable of inhibiting the colonies formation and invasiveness of B16-F10 melanoma cells. In summary, steroidgenesis is not essential to POMC-mediated melanoma suppression. In addition, ACTH (K15A/R17A) gene delivery may provide a better alternative for melanoma control.

Abstract in Chinese 4
Abstract in English 5
Abbreviations 6
Introduction 7
Specific Aims 13
Materials and Methods 15
Results 23
Discussion 27
Figures 33
Appendix 50
References 56

1. Miller AJ & Mihm MC, Jr. (2006) N Engl J Med 355, 51-65.
2. Gray-Schopfer V, Wellbrock C, & Marais R (2007) Nature 445, 851-857.
3. Hocker TL, Singh MK, & Tsao H (2008) J Invest Dermatol 128, 2575-2595.
4. de Vries E & Coebergh JW (2005) BMJ 331, 698.
5. Jhappan C, Noonan FP, & Merlino G (2003) Oncogene 22, 3099-3112.
6. Barnhill RL & Mihm MC, Jr. (1993) Semin Diagn Pathol 10, 47-75.
7. Solomon S (1999) Ann N Y Acad Sci 885, 22-40.
8. Catania A, Gatti S, Colombo G, & Lipton JM (2004) Pharmacol Rev 56, 1-29.
9. Benjannet S, Rondeau N, Day R, Chretien M, & Seidah NG (1991) Proc Natl Acad Sci U S A 88, 3564-3568.
10. Zhou A, Bloomquist BT, & Mains RE (1993) J Biol Chem 268, 1763-1769.
11. Zhu X, Zhou A, Dey A, Norrbom C, Carroll R, Zhang C, Laurent V, Lindberg I, Ugleholdt R, Holst JJ, et al. (2002) Proc Natl Acad Sci U S A 99, 10293-10298.
12. Wachira SJ, Hughes-Darden CA, Taylor CV, Ochillo R, & Robinson TJ (2003) Neuropeptides 37, 201-210.
13. Konda Y, Gantz I, DelValle J, Shimoto Y, Miwa H, & Yamada T (1994) J Biol Chem 269, 13162-13166.
14. Schioth HB, Chhajlani V, Muceniece R, Klusa V, & Wikberg JE (1996) Life Sci 59, 797-801.
15. Gantz I, Miwa H, Konda Y, Shimoto Y, Tashiro T, Watson SJ, DelValle J, & Yamada T (1993) J Biol Chem 268, 15174-15179.
16. Puehler W & Stein C (2005) Rheum Dis Clin North Am 31, 103-113, ix.
17. Luger TA, Scholzen TE, Brzoska T, & Bohm M (2003) Ann N Y Acad Sci 994, 133-140.
18. Catania A, Delgado R, Airaghi L, Cutuli M, Garofalo L, Carlin A, Demitri MT, & Lipton JM (1999) Ann N Y Acad Sci 885, 183-187.
19. Vinson GP (2009) Mol Cell Endocrinol 300, 2-6.
20. Connell JM & Davies E (2005) J Endocrinol 186, 1-20.
21. Nimkarn S & New MI (2008) Trends Endocrinol Metab 19, 96-99.
22. Barnes PJ (2006) Br J Pharmacol 148, 245-254.
23. Newton R (2000) Thorax 55, 603-613.
24. Miller WL (2007) Biochim Biophys Acta 1771, 663-676.
25. Kim JK, Summer SN, Wood WM, & Schrier RW (2001) Biochem Biophys Res Commun 289, 1252-1256.
26. Saito T, Kasono K, Otani T, Tamemoto H, Kawakami M, Sasaki S, & Ishikawa S (2009) Acta Physiol (Oxf) 196, 239-247.
27. Wang W, Li C, Summer SN, Falk S, Cadnapaphornchai MA, Chen YC, & Schrier RW (2006) Am J Physiol Renal Physiol 290, F1135-1142.
28. Liu GS, Liu LF, Lin CJ, Tseng JC, Chuang MJ, Lam HC, Lee JK, Yang LC, Chan JH, Howng SL, et al. (2006) Mol Pharmacol 69, 440-451.
29. Costa JL, Bui S, Reed P, Dores RM, Brennan MB, & Hochgeschwender U (2004) Gen Comp Endocrinol 136, 12-16.
30. Rainey WE, Saner K, & Schimmer BP (2004) Mol Cell Endocrinol 228, 23-38.
31. Lin CR, Yang LC, Lee TH, Lee CT, Huang HT, Sun WZ, & Cheng JT (2002) Gene Ther 9, 1247-1253.
32. Tai MH, Cheng H, Wu JP, Liu YL, Lin PR, Kuo JS, Tseng CJ, & Tzeng SF (2003) Exp Neurol 183, 508-515.
33. Kleinman HK & Jacob K (2001) Curr Protoc Cell Biol Chapter 12, Unit 12 12.
34. Soderholm J & Heald R (2005) Chem Biol 12, 263-265.
35. Raffin-Sanson ML, de Keyzer Y, & Bertagna X (2003) Eur J Endocrinol 149, 79-90.
36. Mornet E, Dupont J, Vitek A, & White PC (1989) J Biol Chem 264, 20961-20967.
37. Shackleton CH, Hughes BA, Lavery GG, Walker EA, & Stewart PM (2008) Steroids 73, 1066-1076.
38. Ye P, Kenyon CJ, Mackenzie SM, Nichol K, Seckl JR, Fraser R, Connell JM, & Davies E (2008) J Endocrinol 196, 305-311.
39. Cooke BA (1999) Mol Cell Endocrinol 151, 25-35.
40. Cozza EN, Vila MC, Acevedo-Duncan M, Farese RV, & Gomez-Sanchez CE (1990) Endocrinology 126, 2169-2176.
41. Bicknell AB (2008) J Neuroendocrinol 20, 692-699.
42. Fitzsimons HL, Bland RJ, & During MJ (2002) Methods 28, 227-236.