根據美國癌症協會2013年的估計，前列腺癌(prostate cancer)是男性最常罹患的癌症，每7人就會有一位被診斷出患有前列腺癌；且前列腺癌在美國男性癌症死亡排名第二。亞洲國家男性罹患前列腺癌的比率比西方國家低很多，但在台灣也是男性十大癌症之一（7. 3 %；台灣登記中心）。由此可知，前列腺癌治療的相關研究是有其重要且必要性的。
前列腺癌的治療方式是由芝加哥大學的查理哈更斯(Charles Huggins)教授在1941年提出。哈更斯發現前列腺癌細胞的生長需要倚賴男性荷爾蒙(androgen)，稱為男性荷爾蒙依賴型前列腺癌 (androgen- dependent prostate cancer)。他證明以手術去勢抑制病患體內男性荷爾蒙，可以讓前列腺腫瘤萎縮而治癒；目前臨床上醫生使用促性腺釋放激素(luteinizing hormone-releasing hormone，LHRH) 類似物(analogue) 抑制病患體內的男性荷爾蒙。然而，80-90%的前列腺癌病患經過男性荷爾蒙抑制治療2- 3年後，消失的腫瘤會再度復發(relapse)；復發的腫瘤不再倚賴男性荷爾蒙，成為非男性荷爾蒙依賴型前列腺癌(androgen- independent prostate cancer)。攝護腺癌症復發後病人約可再存活2- 3年，這種復發的癌症至今仍沒有有效的治療方式。
LXR (liver X receptor) 調節著體內膽固醇、脂肪酸以及葡萄糖的恆定，而人工合成的T0901317 LXR致效劑對於促進LXR活性有很好的效果。最近的研究顯示T0901317可以有效地降低前列腺癌細胞PC- 3 和DU145 的增生及細胞週期的發展；本實驗利用LXR致效劑 T0901317 (LXR agonist) 對非男性荷爾蒙依賴型前列腺癌細胞PC- 3及DU145進行處理，從細胞遷移(migration)、侵入(invasion) 等細胞實驗及利用體內成像系統(in vivo imaging system)判斷並推論LXR致效劑 T0901317 是否能有效的抑制前列腺癌細胞的轉移(metastasis)。經過T0901317 96小時投藥過後，PC- 3與DU145前列腺癌細胞的遷移（migration）和侵入（invasion）能力有大幅的下降；除此之外，相同的結果也在動物實驗中得到應証，經過10 週T0901317餵食（gavage）之裸鼠其體內PC- 3轉移訊號相較於未治療組小鼠明顯降低。此外，進一步的利用高通量西方點墨微陣列(Micro-Western Array)及免疫組織染色(immunohistochemistry) 確定細胞內與轉移相關之蛋白質變化量；我們發現T0901317的作用會降低與細胞增生以及 EMT(epithelial- to -mesenchymal transition) 相關蛋白質的活性，像是Akt、NFκB、 c-Myc和snail。從細胞及動物實驗中的結果顯示出T0901317其有效的轉移抑制能力；未來將會進一步的分析癌症指標中與癌症轉移（metastasis）相關的因子，以期得到T0901317作用機制，並提供進一步開發腫瘤藥物的重要資訊。

Prostate cancer (PCa) is the second most frequently diagnosed cancer of men and the fifth most common cancer overall in the world. Surgeries, such as radical prostatectomy or transurethral resection of the prostate (TURP), are often successful for organ-confined prostate cancer. However, approximately 15% to 30% of patients with localized disease receiving surgery develop relapsed tumors within 5 to 10 years. Most of these patients subsequently show poor therapeutic outcome. More than 80% of patients died from PCa developed bone metastases. Liver X receptors (LXRs) belong to the nuclear receptor superfamily and LXR regulates the homeostasis of cholesterol, fatty acids and glucose. We previously reported that LXR agonist treatment suppresses proliferation of PCa cells via induction of G1 cell cycle arrest. In this study, we demonstrated that LXR agonist T0901317 treatment suppressed migration and invasion of PC-3 and DU-145 human prostate cancer cells. Metastasis of PC-3 xenografts in nude mice was reduced by oral administration of T0901317. Micro-Western Array (MWA) is an antibody-based modified reverse phase array composes of a GeSim Nanoplotter arrayer, a GE multiphor, and a Licor Odyssey scanner. Micro-Western Array allows detecting protein expression level or phosphorylation status change of 96-384 different antibodies in 6-15 samples simultaneously. Micro-Western Array study and immunohistochemistry (IHC) staining indicated that T0901317 treatment reduced signaling proteins involved in regulation of cell proliferation and epithelial-to-mesenchymal transition (EMT), including Akt, NFκB, c-Myc, and snail. Our observations suggested that LXR agonists may be a potential candidate for prevention and inhibition of prostate cancer metastasis.

[Catalog]
Express …………………………………………………………………………………………………………………………………… I
Abstract ……………………………………………………………………………………………………………………………… II
Catalog ………………………………………………………………………………………………………………………………… VI
Figure catalog ………………………………………………………………………………………………………… VIII
1. Introduction ……………………………………………………………………………………………………………… 1
2. Experimental procedure …………………………………………………………………………………… 5
3. Materials and methods ……………………………………………………………………………………… 6
3-1 Material ………………………………………………………………………………………………………………… 6
3-2 Cell culture ……………………………………………………………………………………………………… 6
3-2-1 Cell line …………………………………………………………………………………………………… 6
3-2-2 Cell thawing …………………………………………………………………………………………… 8
3-2-3 Cell frozen ……………………………………………………………………………………………… 9
3-3 Xenografts in Athymic mice ………………………………………………………………… 9
3-3-1 Isoflurane anesthesia machine …………………………………………… 10
3-3-2 In vivo bioluminescence imaging system …………………………………………… 10
3-4 Western blot analysis …………………………………………………………………………… 11
3-4-1 Protein extraction ………………………………………………………………………… 11
3-4-2 Protein concentration test …………………………………………………… 12
3-4-3 SDS- page electrophoresis ……………………………………………………… 12
3-4-4 Western- blotting …………………………………………………………………………… 13
3-5 Micro- western array ……………………………………………………………………………… 14
3-5-1 Sample preparation ………………………………………………………………………… 14
3-5-2 Gel fabrication ………………………………………………………………………………… 14
3-5-3 Microarraying ……………………………………………………………………………………… 16
3-6 Transwell invasion assay …………………………………………………………………… 19
3-7 Transwell migration assay ………………………………………………………………… 20
3-8 Immunohistochemistry …………………………………………………………………………………… 21
4. Results ………………………………………………………………………………………………………………………… 22
4-1 Treatment with T0901317 LXR Agonist Suppressed Migration and Invasion of Prostate Cancer Cells ……………………… 22
4-2 T0901317 reduced effectively metastasis of PC- 3 prostate cancer xenografts in nude mice …………………………………………… 23
4-3 T0901317 Treatment affected Signaling Proteins involved in Regulation of Cancer Cell Proliferation and Metastasis ………………………………………………………………………………………………………………………… 24
5. Discussion ………………………………………………………………………………………………………………… 26
6. Figure/ Supplemental figure ………………………………………………………… 29/ 39
7. Reference …………………………………………………………………………………………………………………… 48
[Figure catalog]
Figure 1. Migration and invasion of PC- 3 and DU145 cell line after treated with T0901316 ……………………………………………………………… 29
Figure 2. Gelatinolytic results on SDS- page which shows the MMP activity …………………………………………………………………………………………………………………… 30
Figure 3. Migration of PC- 3Luc cell line after treated with T0901317 ……………………………………………………………………………………………………………………………… 31
Figure 4. Distribution of PC- 3Luc Xenografts shown in vivo bioluminescence imaging system …………………………………………………………………… 32
Figure 5. The diversification of mice body weight ………………… 33
Figure 6. Hematoxylin and Eosin (HE) stain of mice intestine, lung, and prostate ……………………………………………………………………… 34
Figure 7. The results of micro- western array in heat map form ………………………………………………………………………………………………………………………………………… 35
Figure 8. Immunohistochemistry (IHC) stain of nude mice intestine …………………………………………………………………………………………………………………………… 36
Figure 9. Immunohistochemistry (IHC) stain of nude mice lung ……………………………………………………………………………………………………………………………………………………… 37
Figure 10. TRAF6 antibody IHC stain in nude mice lung andintestine section ……………………………………………………………………………………………… 38
Supplemental figure 1. IHC stain of LXRantibody in nude mice lung section ……………………………………………………………………………………………………… 39
Supplemental figure 2. IHC stain of LXRantibody in nude mice intestine section ………………………………………………………………………………………… 39
Supplemental figure 3. Proliferation of PC- 3 cell line after treated with T0901317 …………………………………………………………………………… 40
Supplemental figure 4. Myd88 antibody IHC stain in nude mice lung and intestine section ……………………………………………………………………………… 41
Supplemental figure 5. Luciferase antibody IHC stain in nude mice intestine section ………………………………………………………………………………………… 42
Supplemental figure 6. Luciferase antibody IHC stain in nude mice lung section ……………………………………………………………………………………………………… 42
Supplemental figure 7. Signaling pathway of LXR agonist T09091317 schematic diagram …………………………………………………………………………… 43
Supplemental list 8. The antibodies list for MWA and IHC ……………………………………………………………………………………………………………………………………………………… 44

1. Huggins, C. and C.V. Hodges, Studies on prostatic cancer. The Journal of Urology. 167(2): p. 948-951.
2. Debes, J.D. and D.J. Tindall, Mechanisms of androgen-refractory prostate cancer. N Engl J Med, 2004. 351(15): p. 1488-90.
3. Petrylak, D.P., et al., Docetaxel and estramustine compared with mitoxantrone and prednisone for advanced refractory prostate cancer. N Engl J Med, 2004. 351(15): p. 1513-20.
4. Chuu, C.P., et al., Inhibition of tumor growth and progression of LNCaP prostate cancer cells in athymic mice by androgen and liver X receptor agonist. Cancer Res, 2006. 66(13): p. 6482-6.
5. Zelcer, N. and P. Tontonoz, Liver X receptors as integrators of metabolic and inflammatory signaling. J Clin Invest, 2006. 116(3): p. 607-14.
6. Simons, K. and D. Toomre, Lipid rafts and signal transduction. Nat Rev Mol Cell Biol, 2000. 1(1): p. 31-9.
7. Ettinger, S.L., et al., Dysregulation of Sterol Response Element-Binding Proteins and Downstream Effectors in Prostate Cancer during Progression to Androgen Independence. Cancer Research, 2004. 64(6): p. 2212-2221.
8. Brown, A.J., CHOLESTEROL, STATINS AND CANCER. Clinical and Experimental Pharmacology and Physiology, 2007. 34(3): p. 135-141.
9. Swinnen, J.V., et al., Androgens, lipogenesis and prostate cancer. The Journal of Steroid Biochemistry and Molecular Biology, 2004. 92(4): p. 273-279.
10. Suburu, J. and Y.Q. Chen, Lipids and prostate cancer. Prostaglandins & Other Lipid Mediators, 2012. 98(1–2): p. 1-10.
11. Chuu, C.P., et al., Modulation of liver X receptor signaling as novel therapy for prostate cancer. J Biomed Sci, 2007. 14(5): p. 543-53.
12. Pommier, A.J., et al., Liver x receptors protect from development of prostatic intra-epithelial neoplasia in mice. PLoS Genet, 2013. 9(5): p. e1003483.
13. Guo, D., et al., An LXR Agonist Promotes Glioblastoma Cell Death through Inhibition of an EGFR/AKT/SREBP-1/LDLR–Dependent Pathway. Cancer Discovery, 2011. 1(5): p. 442-456.
14. Yamauchi, Y., et al., Positive feedback loop between PI3K-Akt-mTORC1 signaling and the lipogenic pathway boosts Akt signaling: induction of the lipogenic pathway by a melanoma antigen. Cancer Res, 2011. 71(14): p. 4989-97.
15. Zhang, Y., et al., Kinase AKT controls innate immune cell development and function. Immunology, 2013. 140(2): p. 143-152.
16. Liu, W., et al., Cacalol, a natural sesquiterpene, induces apoptosis in breast cancer cells by modulating Akt-SREBP-FAS signaling pathway. Breast Cancer Research and Treatment, 2011. 128(1): p. 57-68.
17. Luu, W., et al., Akt acutely activates the cholesterogenic transcription factor SREBP-2. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2012. 1823(2): p. 458-464.
18. Fu, W., et al., LXR Agonist Regulates the Carcinogenesis of PCa via the SOCS3 Pathway. Cellular Physiology and Biochemistry, 2014. 33(1): p. 195-204.
19. Akira, S. and K. Takeda, Toll-like receptor signalling. Nat Rev Immunol, 2004. 4(7): p. 499-511.
20. Naugler, W.E., et al., Gender Disparity in Liver Cancer Due to Sex Differences in MyD88-Dependent IL-6 Production. Science, 2007. 317(5834): p. 121-124.
21. Rakoff-Nahoum, S. and R. Medzhitov, Regulation of spontaneous intestinal tumorigenesis through the adaptor protein MyD88. Science, 2007. 317(5834): p. 124-7.
22. Liang, B., et al., Myeloid Differentiation Factor 88 Promotes Growth and Metastasis of Human Hepatocellular Carcinoma. Clinical Cancer Research, 2013. 19(11): p. 2905-2916.
23. Jia, R.J., et al., Enhanced myeloid differentiation factor 88 promotes tumor metastasis via induction of epithelial-mesenchymal transition in human hepatocellular carcinoma. Cell Death Dis, 2014. 5: p. e1103.
24. Zhou, B.P., et al., Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition. Nat Cell Biol, 2004. 6(10): p. 931-40.
25. Bachelder, R.E., et al., Glycogen synthase kinase-3 is an endogenous inhibitor of Snail transcription: implications for the epithelial-mesenchymal transition. J Cell Biol, 2005. 168(1): p. 29-33.
26. Rigoglou, S. and A.G. Papavassiliou, The NF-κB signalling pathway in osteoarthritis. The International Journal of Biochemistry & Cell Biology, 2013. 45(11): p. 2580-2584.
27. Mori, K., et al., DU145 human prostate cancer cells express functional receptor activator of NFkappaB: new insights in the prostate cancer bone metastasis process. Bone, 2007. 40(4): p. 981-90.
28. Barbera, M.J., et al., Regulation of Snail transcription during epithelial to mesenchymal transition of tumor cells. Oncogene, 2004. 23(44): p. 7345-54.
29. Mak, P., et al., ERβ Impedes Prostate Cancer EMT by Destabilizing HIF-1α and Inhibiting VEGF-Mediated Snail Nuclear Localization: Implications for Gleason Grading. Cancer Cell, 2010. 17(4): p. 319-332.
30. Cano, A., et al., The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol, 2000. 2(2): p. 76-83.
31. Wen, Y.-C., et al., Snail as a potential marker for predicting the recurrence of prostate cancer in patients at stage T2 after radical prostatectomy. Clinica Chimica Acta, 2014. 431(0): p. 169-173.
32. Wang, Y., et al., The Role of Snail in EMT and Tumorigenesis. Curr Cancer Drug Targets, 2013. 13(9): p. 963-72.
33. Fukuchi, J., et al., Antiproliferative Effect of Liver X Receptor Agonists on LNCaP Human Prostate Cancer Cells. Cancer Research, 2004. 64(21): p. 7686-7689.
34. McCawley, L.J. and L.M. Matrisian, Matrix metalloproteinases: they're not just for matrix anymore! Current Opinion in Cell Biology, 2001. 13(5): p. 534-540.
35. Chantrain, C.F., et al., Stromal Matrix Metalloproteinase-9 Regulates the Vascular Architecture in Neuroblastoma by Promoting Pericyte Recruitment. Cancer Research, 2004. 64(5): p. 1675-1686.
36. Nelson, A.R., et al., Matrix Metalloproteinases: Biologic Activity and Clinical Implications. Journal of Clinical Oncology, 2000. 18(5): p. 1135.
37. Krycer, J.R. and A.J. Brown, Cholesterol accumulation in prostate cancer: A classic observation from a modern perspective. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 2013. 1835(2): p. 219-229.
38. Friedl, P. and K. Wolf, Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer, 2003. 3(5): p. 362-74.
39. Tontonoz, P. and D.J. Mangelsdorf, Liver X receptor signaling pathways in cardiovascular disease. Mol Endocrinol, 2003. 17(6): p. 985-93.
40. Trasino, S.E., Y.S. Kim, and T.T.Y. Wang, Ligand, receptor, and cell type–dependent regulation of ABCA1 and ABCG1 mRNA in prostate cancer epithelial cells. Molecular Cancer Therapeutics, 2009. 8(7): p. 1934-1945.
41. Bensinger, S.J., et al., LXR signaling couples sterol metabolism to proliferation in the acquired immune response. Cell, 2008. 134(1): p. 97-111.
42. Wang, N., et al., LXR-Induced Redistribution of ABCG1 to Plasma Membrane in Macrophages Enhances Cholesterol Mass Efflux to HDL. Arteriosclerosis, Thrombosis, and Vascular Biology, 2006. 26(6): p. 1310-1316.
43. Klucken, J., et al., ABCG1 (ABC8), the human homolog of the Drosophila white gene, is a regulator of macrophage cholesterol and phospholipid transport. Proc Natl Acad Sci U S A, 2000. 97(2): p. 817-22.
44. Rough, J.J., et al., Anti-proliferative effect of LXR agonist T0901317 in ovarian carcinoma cells. J Ovarian Res, 2010. 3: p. 13.
45. Nieva, C., et al., The lipid phenotype of breast cancer cells characterized by Raman microspectroscopy: towards a stratification of malignancy. PLoS One, 2012. 7(10): p. e46456.
46. Bouraoui, Y., et al., Profile of NF-kappaBp(65/NFkappaBp50) among prostate specific antigen sera levels in prostatic pathologies. Pathol Biol (Paris), 2012. 60(5): p. 301-5.
47. Lee, Shuet T., et al., Context-Specific Regulation of NF-κB Target Gene Expression by EZH2 in Breast Cancers. Molecular Cell, 2011. 43(5): p. 798-810.
48. Ling, J. and R. Kumar, Crosstalk between NFkB and glucocorticoid signaling: a potential target of breast cancer therapy. Cancer Lett, 2012. 322(2): p. 119-26.
49. Hers, I., E.E. Vincent, and J.M. Tavaré, Akt signalling in health and disease. Cellular Signalling, 2011. 23(10): p. 1515-1527.
50. Murtola, T.J., et al., The importance of LDL and cholesterol metabolism for prostate epithelial cell growth. PLoS One, 2012. 7(6): p. e39445.
51. Ni, J., et al., Role of the EpCAM (CD326) in prostate cancer metastasis and progression. Cancer Metastasis Rev, 2012. 31(3-4): p. 779-91.
52. Went, P.T.H., et al., Frequent EpCam protein expression in human carcinomas. Human Pathology, 2004. 35(1): p. 122-128.
53. Ni, J., et al., Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer metastasis and chemo/radioresistance via the PI3K/Akt/mTOR signaling pathway. The International Journal of Biochemistry & Cell Biology, 2013. 45(12): p. 2736-2748.
54. Ladu, S., et al., E2F1 Inhibits c-Myc-Driven Apoptosis via PIK3CA/Akt/mTOR and COX-2 in a Mouse Model of Human Liver Cancer. Gastroenterology, 2008. 135(4): p. 1322-1332.
55. Wang, H., et al., Stabilization of Snail through AKT/GSK-3β signaling pathway is required for TNF-α-induced epithelial–mesenchymal transition in prostate cancer PC3 cells. European Journal of Pharmacology, 2013. 714(1–3): p. 48-55.
56. Shibata, N. and C.K. Glass, Regulation of macrophage function in inflammation and atherosclerosis. J Lipid Res, 2009. 50 Suppl: p. S277-81.
57. Ogawa, S., et al., Molecular determinants of crosstalk between nuclear receptors and toll-like receptors. Cell, 2005. 122(5): p. 707-21.