泌尿道上皮細胞癌是台灣最常見的癌症之一，大多數的泌尿道上皮細胞癌，尤其是膀胱癌即使在根除性手術之後，常因伴隨高復發率及遠端轉移而具有相當侵襲性。目前對泌尿道上皮癌進展之機轉仍未完全釐清；換言之，針對該腫瘤的基因及分子變異與其發生、侵犯、以及病患預後的關連性仍有待探究。臨床上該腫瘤的特性為復發率高且在之後會發生轉移，並缺乏可靠的生物標記與治療方式，目前也無法充分解釋該腫瘤侵犯過程，意味著有其它可能調控機轉的存在。
P21 activated protein kinase -1(PAK1)為Small Rho GTPase，Rac1和 Cdc42 之下游的加速子，主要調控了有關細胞的形態的生成，細胞的移動性，有絲分裂及血管生成。目前已知PAK1的表現在乳癌及大腸癌其PAK1 都有過度表現的現象，而且與癌症的轉移有關，而其它相關研究也發現高度磷酸化的PAK1與癌細胞的移行有關。在本實驗中，我們想要探討在泌尿道上皮細胞癌的細胞株PAK1和磷酸化的PAK是否有過度表現，同時也會探討在泌尿道上皮癌的細胞株中PAK1的表現的多寡是否與PAK磷酸化程度有關。此外，在本研究中我們將會利用PAK抑制劑：PF-3758309與所選定的泌尿道上皮細胞癌的細胞株RT9 、BFTC909和TSGH8301來看其對細胞株的毒殺作用的成效，並釐清PAK1藥理抑制對細胞的毒殺效果所代表的生物學和相關治療意涵。
從實驗結果得知，PAK1的表現愈高，PAK1磷酸化的機會就愈高，由此可推測磷酸化的PAK1可能會引發下游細胞訊號的進行，而造成細胞的增殖、移行和抗細胞凋亡的進行；相對的，PAK1抑制劑：PF-3758309，會造成細胞淍亡，主要是透過抑制PAK1下游訊號的進行，抑制了PAK1 的磷酸化進而促使細胞走向凋亡。

Urothelial carcinoma of urinary bladder (UBUC) and upper tract (UTUC) are common cancer in Taiwan. Most of urothelial carcinoma, especially those of urinary bladder, are superficially invading and often cured by curettage. Some urinary bladder cancer developed unexpected local recurrence that followed by distal spreading. However, the molecular aberrations responsible for its tumor development and progression remain largely unclear. Therefore, it is beneficial to identify novel prognostic biomarkers and therapeutic targets for this malignancy.
P21-activated protein kinase1(PAK1) is a downstream effector of small Rho GTPase, Rac and Cdc42, plays an important role in the regulation of cell morphogenesis, motility, mitosis and angiogenesis. Several studies report that PAK1 overexpressed in breast cancer and colon cancer, and it is correlate with metastasis. Other studies show that hyperphosphorylation of PAK1 promote cell migration . The aim of this study was to investigate the expression of PAK1 and phosphorylated PAK in cell lines of human urotheliel carcinoma and clarify whether PAK1 expression associated with phosphoryled PAK1. We also used PF-3758309, which is a ATP competitive inhibitor of PAK to assess the therapeutic efficacy of PF-3758309 in RT9,BFTC909 and TSGH8301 cell lines and identify the biological and therapeutic relevance of PAK1 in urothelial carcinoma.
According our results, there is a positive correlation between PAK1 expression and phosphorylated PAK. Phosphorylated PAK may induce a cascade of cell signal resulting cell proliferation, migration and anti-apoptosis; In opposition, PAK inhibitor, PF-3758309 could cause cell apoptosis by inhibiting phosphorylaton of PAK1.

論文審定書…………………………………………………………...………………….i
誌謝 ………………………………………………………………………….…………ii
中文摘要………………………………...……………………………………....…...iii
Abstract …………………………………………………………………………….…..iv
圖表目錄…………………………………………….……………...…………………viii
壹、 前言…………………………………………………………...…………..………..1
1 泌尿道上皮細胞癌……………………………………………...………………..…..1
1.1 泌尿道上皮癌的症狀與診斷…………………………………………….....……...1
1.2 泌尿上皮癌的治療……………………………………………………...……...…..2
1.3 致癌危險因子…………………………………………….………………….....…. 2
2 P21-activated kinase(PAKs) ………………...……….…………………...…………2
2.2 PAK 在癌症上的調節………………….………………….……………………..…4
2.3 促進細胞生長…………………………………….……………..…...……………. 4
2.4 促進細胞生存…………………………………….………………...…………..…. 5
2.5 活化侵犯與轉移的路徑………………………………….………...…………..…. 5
2.6 血管新生作用…………………………………….………………………...…..…. 6
3.PF-3758309……………………………………….…………………………...…..…. 7
4.研究動機…………………………………………………………...………........…….8
貳、材料與方法…………………………….……………………...…………….…..…. 9
1. 實驗儀器…………………………….……………………...………………………. 9
2. 細胞繼代培養…………………………….…………………...……………….….. 10
3. XTT細胞存活率分析(XTT cell viability assay) ………….……………...……….. 12
4.蛋白質萃取.………….…………..………...…………………….………….......….. 13
5.蛋白質定量分析……………………………………………………………………..14
6.西方墨點法…………………….…………………..…....……………....…………. 14
7. RNA 萃取…………………….…………………..…....……………...……...……. 18
8.反轉錄聚合酶連鎖反應(Reverse Transcriptase Polymerase Chain Reaction) .….... 19
9.流式細胞儀分析(flow cytometry analysis)………………………………………….20
9.1 細胞週期分析(cell cycle analysis).……………………………...…………....…. 20
9.2 細胞凋亡分析(Apoptosis analysis) ………………………………………………21
參、實驗結果…..…....…………………...……………………………….……..…….23
肆、討論…..…....…………………...…………………………………….……..……. 26
伍、參考文獻…..…....…………………...……………………………….……..……..30
陸、圖/表(Figures/Tables)說明…..…....…………………...……..……………….34

1. Hall MC, Womack S, Sagalowsky AI, Carmody T. Prognostic factors, recurrence, and
survival in transitional cell carcinoma of the upper urinary tract: a 30-year experience
in 252 patients. Urology. 1998 Oct;52(4):594-601
2. Kuroda K, Asakuma J, Asano T, Horiguchi A, Isono M. Clinical significance of p21-activated kinase 1 expression level in patients with upper urinary tract urothelial carcinoma. Jpn J Clin Oncol. 2015 Jan;45(1):103-10.
3. Lin J, Spitz MR, Dinney CP, Etzel CJ, Grossman HB, Wu X. Bladder cancer risk as modified by family history and smoking.Cancer. 2006;107(4):705.
4. Chen CJ, Chuang YC, You SL. A retrospective study on malignant neoplasms of bladder, lung and liver in blackfoot disease endemic area in Taiwan. Br J Cancer. 1986;53(3):399
5. Tsai SM, Wang TN, Ko YC. Cancer mortality trends in a blackfoot disease endemic community of Taiwan following water source replacement. J Toxicol Environ Health A. 1998;55(6):389.
6. Molli PR, Li DQ, Murray BW, Rayala SK, Kumar R. PAK signaling in oncogenesis Oncogene (2009) 28, 2545–2555
7. Kumar R, Gururaj AE, Barnes CJ. p21-activated kinases in cancer. Nat Rev Cancer. 2006 Jun;6(6):459-71. Review.
8. Murray BW, Guo C, Piraino J, Westwick JK, Zhang C. Small-molecule p21-activated kinase inhibitor PF-3758309 is a potent inhibitor of oncogenic signaling and tumor growth. Proc Natl Acad Sci U S A. 2010 May 18;107(20):9446-51
9. Radu M, Semenova G, Kosoff R, Chernoff J. PAK signalling during the development and progression of cancer. Nat Rev Cancer. 2014 Jan;14(1):13-25. Review.
10. Shrestha Y, Schafer EJ, Boehm JS, Thomas SR. PAK1 is a breast cancer oncogene that coordinately activates MAPK and MET signaling. Oncogene. 2012 Jul19;31 (29):3397-408
11. Arias-Romero, L. E., Villamar-Cruz, O., Huang, M. Pak1 Kinase Links ErbB2 to β-Catenin in Transformation of Breast Epithelial Cells. Cancer Res. 2013 Jun 15;73 (12):3671-82
12. Jin S, Zhuo Y, Guo W, Field J. p21-activated Kinase 1 (Pak1)-dependent phosphorylation of Raf-1 regulates its mitochondrial localization, phosphorylation of BAD, and Bcl-2 association. J Biol Chem. 2005 Jul 1;280(26):24698-705
13. Schürmann A, Mooney AF, Sanders LC, Sells MA, Wang HG, Reed JC, Bokoch GM. p21-activated kinase 1 phosphorylates the death agonist bad and protects cells from apoptosis. Mol Cell Biol. 2000 Jan;20(2):453-61
14. Ye DZ, Jin S, Zhuo Y, Field J. p21-Activated kinase 1 (Pak1) phosphorylates BAD directly at serine 111 in vitro and indirectly through Raf-1 at serine 112. PLoS One. 2011;6(11):e27637. doi: 10.1371/journal.pone.0027637
15. Friedland JC, Lakins JN, Kazanietz MG, Chernoff J. alpha6beta4 integrin activates Rac-dependent p21-activated kinase 1 to drive NF-kappaB-dependent resistance to apoptosis in 3D mammary acini. J Cell Sci. 2007 Oct 15;120(Pt 20):3700-12.
16. Ching YP, Leong VY, Lee MF, Xu HT, Jin DY, Ng IO. P21-activated protein kinase is overexpressed in hepatocellular carcinoma and enhances cancer metastasis involving c-Jun NH2-terminal kinase activation and paxillin phosphorylation. Cancer Res. 2007 Apr 15;67(8):3601-8
17. Edwards DC, Sanders LC, Bokoch GM, Gill GN. Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics. Nat Cell Biol. 1999 Sep;1(5):253-9
18. Zhou L, Yan C, Gieling RG, Kida Y, Garner W. Tumor necrosis factor-alpha induced expression of matrix metalloproteinase-9 through p21-activated kinase-1. BMC Immunol. 2009 Mar 19;10:15. doi: 10.1186/1471-2172-10-15
19. Hu GD, Chen YH, Zhang L, Tong WC. The generation of the endothelial specific cdc42-deficient mice and the effect of cdc42 deletion on the angiogenesis and embryonic development. Chin Med J (Engl). 2011 Dec;124(24):4155-9
20. Bagheri-Yarmand R, Vadlamudi RK, Wang RA, Mendelsohn J, Kumar R. Vascular endothelial growth factor up-regulation via p21-activated kinase-1 signaling regulates heregulin-beta1-mediated angiogenesis. J Biol Chem. 2000 Dec15;275(50)
:39451-7
21. Zhao ZS, Manser E. Do PAKs make good drug targets? F1000 Biol Rep. 2010 Sep 23;2:70. doi: 10.3410/B2-70.
22. Murray BW, Guo C, Piraino J, Westwick JK, Zhang C, Lamerdin J. Small-molecule p21-activated kinase inhibitor PF-3758309 is a potent inhibitor of oncogenic signaling and tumor growth. Proc Natl Acad Sci U S A. 2010 May18;107(20): 9446-51.
23. Maruta H. Effective neurofibromatosis therapeutics blocking the oncogenic kinase PAK1. Drug Discov Ther. 2011 Dec;5(6):266-78
24. Li CF, Shen KH, Huang LC, et al. Annexin-I overexpression is associated with tumour progression and independently predicts inferior disease-specific and metastasis-free survival in urinary bladder urothelial carcinoma. Pathology. 2010; 42, 43-49.
25. Persico A, Cervigni RI, Barretta ML, Corda D. Golgi partitioning controls mitotic entry through Aurora-A kinase. Mol Biol Cell. 2010 Nov 1;21(21):3708-21.
26. Kuželová K, Grebeňová D, Holoubek A, Röselová P, Obr A. Group I PAK inhibitor IPA-3 induces cell death and affects cell adhesivity to fibronectin in human hematopoietic cells. PLoS One. 2014 Mar 24;9(3)
27. Ong CC, Jubb AM, Haverty PM, et.al. Targeting p21-activated kinase 1 (PAK1) to induce apoptosis of tumor cells. Proc Natl Acad Sci U S A. 2011 Apr 26;108(17): 7177-82.
28. Ito M1, Nishiyama H, Kawanishi H, Matsui S. P21-activated kinase 1: a new molecular marker for intravesical recurrence after transurethral resection of bladder cancer. J Urol. 2007 Sep;178(3 Pt 1):1073-9
29. Choi YJ, Lee SH, Lee JL, Ahn JH, Lee KH, You D, Hong B. Phase II study of pemetrexed in combination with cisplatin in patients with advanced urothelial cancer: the PECULIAR study (KCSG 10-17). Br J Cancer. 2015 Jan 20; 112(2): 260-5.
30. Wang T, Chuan Pan C, Rui Yu J, Long Y. Association between TYMS expression and efficacy of pemetrexed-based chemotherapy in advanced non-small cell lung cancer: a meta-analysis. PLoS One. 2013 Sep 10;8(9):e74284.
31. https://en.wikipedia.org/wiki/Transitional_cell_carcinoma