臺灣博碩士論文加值系統

腫瘤細胞的轉移是癌症致死的主要原因，而淋巴轉移是其中最重要的途徑之一。有研究指出，環氧化酶-2的表現常常與癌細胞往淋巴結的轉移有關，而另一方面，細胞趨化素和其相對接受器的交互作用對於癌細胞的轉移也扮演了相當重要的角色。在我們實驗室先前的研究中證明，CCR7是環氧化酶-2的下游目標基因，且環氧化酶-2可透過EP2及EP4 接受器的訊息傳遞路徑誘導CCR7的表現，同時發現了Protein kinase A以及AKT kinase也參與在其中。在本論文中，我們進一步證明了環氧化酶-2是透過活化CCR7啟動子進而刺激CCR7的表現。藉由一系列啟動子片段的活性測試及突變實驗之後，我們發現環氧化酶-2是經由位於轉錄起始點上游-61至-52bp啟動子區域的Sp1結合位來調控CCR7的表現。ChIP assay的結果更確認了環氧化酶-2增強了Sp1結合至CCR7的啟動子。此外，在乳癌細胞株MCF-7中降低Sp1的表現可以抑制PGE2促進CCR7表現的現象，大量表現Sp1則可以提升CCR7的表現量。而in vitro kinase assay的結果顯示，AKT kinase可以直接磷酸化Sp1上S42、T679、S698三個位置。而這樣的磷酸化現象更造成了Sp1蛋白穩定性及與DNA結合力的增加，進而促進Sp1調控基因的表現。最後，免疫組織染色的結果證明，乳癌組織中CCR7與Sp1及磷酸化AKT的表現具有顯著的相關性。綜合來說，環氧化酶-2是透過了EP receptor/PKA/AKT/Sp1的訊息傳遞路徑來調控CCR7的表現。

The metastatic spread of tumor cells is the major lethal aspect of cancer, and lymphatic metastasis is one of the most important routes. Recent studies indicated that cyclooxygenase-2 (COX-2) expression is frequently associated with lymph node metastasis and over-expression of COX-2 can enhance lymphatic invasion of cancer cells. The interaction of chemokines and their cognate receptors also plays a critical role in cancer metastasis. Previous results of our laboratory demonstrated that CCR7 is a downstream target for COX-2 and COX-2 up-regulated CCR7 expression via the EP2 and EP4 receptor. We also found that protein kinase A (PKA) and AKT kinase are involved in COX-2-induced CCR7. In this study, we provided further evidences that COX-2 directly stimulates CCR7 expression via promoter activation. Promoter deletion and mutation assay indicated that COX-2 stimulated CCR7 promoter via the Sp1 binding site located at the -61/-52 bp region upstream of the transcription start site. Increase of Sp1 binding to CCR7 promoter by COX-2 was confirmed by chromatin immunoprecipitation (ChIP) assay. Furthermore, knockdown of Sp1 expression resulted in inhibition of PGE2-induced CCR7, and over-expression of Sp1 potently up-regulated CCR7 in MCF-7 cells. In vitro kinase assay indicated that AKT could directly phosphorylate Sp1 at S42, T679 and S698 sites. And the phosphorylation of Sp1 by AKT led to enhanced protein stability and DNA binding affinity of Sp1. The results of immunohistochemistry indicated that CCR7 expression was significantly associated with Sp1 and phosphor-AKT. Taken together, COX-2 may act via the EP receptor/PKA/AKT/Sp1 signaling pathway to stimulate CCR7 expression in breast cancer cells to promote lymphatic spread.

中文摘要 4
英文摘要 5
縮寫語 6
研究背景 7
研究目的 9
實驗材料 10
實驗方法及步驟 17
實驗結果 35
實驗結果圖表 48
結論圖表 74
討論 75
參考文獻 80
Supplementary Data 85
附錄 88

1.Costa C, Soares R, Reis-Filho JS, Leitao D, Amendoeira I, Schmitt FC. Cyclo-oxygenase 2 expression is associated with angiogenesis and lymph node metastasis in human breast cancer. J Clin Pathol. 2002;55(6):429-34.
2.Sheng H, Shao J, Washington MK, DuBois RN. Prostaglandin E2 increases growth and motility of colorectal carcinoma cells. J Biol Chem. 2001;276(21):18075-81.
3.Pai R, Soreghan B, Szabo IL, Pavelka M, Baatar D, Tarnawski AS. Prostaglandin E2 transactivates EGF receptor: a novel mechanism for promoting colon cancer growth and gastrointestinal hypertrophy. Nat Med. 2002;8(3):289-93.
4.Barnes NL, Warnberg F, Farnie G, White D, Jiang W, Anderson E, Bundred NJ. Cyclooxygenase-2 inhibition: effects on tumour growth, cell cycling and lymphangiogenesis in a xenograft model of breast cancer. Br J Cancer. 2007;96(4):575-82.
5.Timoshenko AV, Chakraborty C, Wagner GF, Lala PK. COX-2-mediated stimulation of the lymphangiogenic factor VEGF-C in human breast cancer. Br J Cancer. 2006;94(8):1154-63.
6.Su JL, Shih JY, Yen ML, Jeng YM, Chang CC, Hsieh CY, Wei LH, Yang PC, Kuo ML. Cyclooxygenase-2 induces EP1- and HER-2/Neu-dependent vascular endothelial growth factor-C up-regulation: a novel mechanism of lymphangiogenesis in lung adenocarcinoma. Cancer Res. 2004;64(2):554-64.
7.Raman D, Baugher PJ, Thu YM, Richmond A. Role of chemokines in tumor growth. Cancer Lett. 2007;256(2):137-165.
8.Muller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, McClanahan T, Murphy E, Yuan W, Wagner SN, Barrera JL, Mohar A, Verástegui E, Zlotnik A. Involvement of chemokine receptors in breast cancer metastasis. Nature. 2001;410(6824):50-6.
9.Koizumi K, Hojo S, Akashi T, Yasumoto K, Saiki I. Chemokine receptors in cancer metastasis and cancer cell-derived chemokines in host immune response. Cancer Sci. 2007;98(11):1652-8.
10. Zlotnik A. Chemokines and cancer. Int J Cancer. 2006;119(9):2026-9.
11. Kakinuma T, Hwang ST. Chemokines, chemokine receptors, and cancer metastasis. J Leukoc Biol. 2006;79(4):639-51.
12. Pan MR, Hou MF, Chang HC, Hung WC. Cyclooxygenase-2 up-regulates CCR7 via EP2/EP4 receptor signaling pathways to enhance lymphatic invasion of breast cancer cells. J Biol Chem. 2008;283(17):11155-63.
13. Mantovani A, Allavena P, Sica A, Balkwill F. Cancer-related inflammation. Nature. 2008;454(7203):436-44. Review.
14. Colotta F, Allavena P, Sica A, Garlanda C, Mantovani A. Cancer-related inflammation, the seventh hallmark of cancer: links to genetic instability. Carcinogenesis. 2009;30(7):1073-81.
15. Bernard MP, Bancos S, Sime PJ, Phipps RP. Targeting cyclooxygenase-2 in hematological malignancies: rationale and promise. Curr Pharm Des. 2008;14(21):2051-60. Review.
16. Ghosh N, Chaki R, Mandal V, Mandal SC. COX-2 as a target for cancer chemotherapy. Pharmacol Rep. 2010;62(2):233-44.
17. NSAIDs and cancer prevention: targets downstream of COX-2. Cha YI, DuBois RN. Annu Rev Med. 2007;58:239-52. Review.
18. Denkert C, Winzer KJ, Muller BM, Weichert W, Pest S, Kobel M, Kristiansen G, Reles A, Siegert A, Guski H, Hauptmann S. Elevated expression of cyclooxygenase-2 is a negative prognostic factor for disease free survival and overall survival in patients with breast carcinoma. Cancer. 2003;97(12):2978-87.
19. Zheng Y, Ritzenthaler JD, Sun X, Roman J, Han S. Prostaglandin E2 stimulates human lung carcinoma cell growth through induction of integrin-linked kinase: the involvement of EP4 and Sp1. Cancer Res. 2009;69(3):896-904.
20. Bradbury D, Clarke D, Seedhouse C, Corbett L, Stocks J, Knox A. Vascular endothelial growth factor induction by prostaglandin E2 in human airway smooth muscle cells is mediated by E prostanoid EP2/EP4 receptors and SP-1 transcription factor binding sites. J Biol Chem. 2005;280(34):29993-30000.
21. Salcedo R, Zhang X, Young HA, Michael N, Wasserman K, Ma WH, Martins-Green M, Murphy WJ, Oppenheim JJ. Angiogenic effects of prostaglandin E2 are mediated by up-regulation of CXCR4 on human microvascular endothelial cells. Blood. 2003;102(6):1966-77.
22. Pore N, Liu S, Shu HK, Li B, Haas-Kogan D, Stokoe D, Milanini-Mongiat J, Pages G, O''Rourke DM, Bernhard E, Maity A. Sp1 is involved in Akt-mediated induction of VEGF expression through an HIF-1-independent mechanism. Mol Biol Cell. 2004;15(11):4841-53.
23.Engelman JA. Targeting PI3K signalling in cancer: opportunities, challenges and limitations. Nat Rev Cancer. 2009;9(8):550-62. Review.
24.Wong KK, Engelman JA, Cantley LC. Targeting the PI3K signaling pathway in cancer. Curr Opin Genet Dev. 2010;20(1):87-90. Review.
25. Wohlschlaeger J, Schmitz KJ, Palatty J, Takeda A, Takeda N, Vahlhaus C, Levkau B, Stypmann J, Schmid C, Schmid KW, Baba HA. Roles of cyclooxygenase-2 and phosphorylated Akt (Thr308) in cardiac hypertrophy regression mediated by left-ventricular unloading. J Thorac Cardiovasc Surg. 2007;133(1):37-43.
26. Wu T, Leng J, Han C, Demetris AJ. The cyclooxygenase-2 inhibitor celecoxib blocks phosphorylation of Akt and induces apoptosis in human cholangiocarcinoma cells. Mol Cancer Ther. 2004;3(3):299-307.
27. Fayard E, Tintignac LA, Baudry A, Hemmings BA. Protein kinase B/Akt at a glance. J Cell Sci. 2005;118(Pt 24):5675-8. Review.
28. Liang J, Zubovitz J, Petrocelli T, Kotchetkov R, Connor MK, Han K, Lee JH, Ciarallo S, Catzavelos C, Beniston R, Franssen E, Slingerland JM. PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest. Nat Med. 2002;8(10):1153-60.
29. Dan HC, Sun M, Kaneko S, Feldman RI, Nicosia SV, Wang HG, Tsang BK, Cheng JQ. Akt phosphorylation and stabilization of X-linked inhibitor of apoptosis protein (XIAP). J Biol Chem. 2004;279(7):5405-12.
30. Suske G. The Sp-family of transcription factors. Gene. 1999;238(2):291-300. Review
31. Bouwman P, Philipsen S. Regulation of the activity of Sp1-related transcription factors. Mol Cell Endocrinol. 2002;195(1-2):27-38.
32. Chu S, Ferro TJ. Sp1: regulation of gene expression by phosphorylation. Gene. 2005;348:1-11. Review.
33. Tan NY, Khachigian LM. Sp1 phosphorylation and its regulation of gene transcription. Mol Cell Biol. 2009;29(10):2483-8.
34. Philipsen S, Suske G. A tale of three fingers: the family of mammalian Sp/XKLF transcription factors. Nucleic Acids Res. 1999 27(15):2991-3000.
35. Wang L, Wei D, Huang S, Peng Z, Le X, Wu TT, Yao J, Ajani J, Xie K. Transcription factor Sp1 expression is a significant predictor of survival in human gastric cancer. Clin Cancer Res. 2003;9(17):6371-80.
36.Yao JC, Wang L, Wei D, Gong W, Hassan M, Wu TT, Mansfield P, Ajani J, Xie K. Association between expression of transcription factor Sp1 and increased vascular endothelial growth factor expression, advanced stage, and poor survival in patients with resected gastric cancer. Clin Cancer Res. 2004;10(12 Pt 1):4109-17.