Pin1 為 PPIase (peptidyl-prolyl isomerase) 家族的異構酶，可以特異性的辨認某些被磷酸化的蛋白 Ser/Thr-Pro motifs,並且引起此 motif 的構型在 cis 以及 trans 之間的轉換，進而去影響蛋白功能。許多參與細胞週期調控、DNA 損傷反應、轉錄調節和神經生長的蛋白，例如 Cyclin D1, β-catenin, p53 都會被 Pin1 辨認而影響。多形性膠質母細胞瘤 (Glioblastoma multiforme, 簡稱 GBM) 在原發性腦瘤中是最常見且惡性程度最高的一個類型。CD133 被視為癌症幹細胞的重要標誌之一，且表現 CD133 的膠質瘤細胞被認為在腫瘤的復發以及對放射性的抵抗性扮演著重要的角色。所以我們研究 Pin1的表現對於神經膠質瘤細胞對於放線抵抗性以及幹標誌基因 CD133 和 Sox2 蛋白表現的影響。
實驗結果顯示，Pin1 在表現 CD133 的膠質瘤細胞 (S1 和 S1R1) 中表現量較 GBM8401 細胞高。而在 GBM 細胞中 Knockdown Pin1 的表現量會增加細胞對放射線的敏感性，而外加入 Pin1 至 GBM 細胞中則會降低細胞對放射線的敏感性。在表現 CD133 的膠質瘤細胞 (S1 和 S1R1) 中，降低 Pin1 的表現會降低一些幹細胞標誌基因的表現例如 CD133, Sox2 等。而將 Pin1 knockdown 也會降低 β-catenin 的表現，而活化或抑制 β-catenin 會影響 Sox2 的蛋白表現但 Pin1 的蛋白表現則不受到影響。由我們的結果建議，Pin1 可能作用於 β-catenin 的上游進而影響 Sox2 的表現。除此之外，利用免疫共沉澱的方法，我們的結果顯示 Pin1 可能藉由直接與 Sox2 蛋白的互相作用來影響其蛋白穩定度。

Pin1, a peptidyl-prolyl isomerase, specifically recognizes the phosphorylated Ser/Thr-Pro motifs and induces cis/trans isomerization of proline amide bonds contained within the target proteins, such conformational changes have profound effects on the function of Pin1 substrates. Several phosphoproteins involved in cell cycle control, DNA damage responses, transcriptional regulation and neuronal survival including Cyclin D1, β-catenin, p53 are recognized and affected by Pin1. Glioblastoma multiforme (GBM), the most common primary brain tumor, is rapidly fatal. CD133 has been considered as an important marker for cancer stem-like cells, and CD133-expressing glioma cells are known to play a key role in tumor recovery and radioresistance. Here, we investigated the effects of Pin1 expression on radiosensitivity and CD133 and Sox2 expression in Glioblastoma cells.
We showed that enhanced Pin1 expression occurred in CD133-expressing glioblastoma cells (S1 and S1R1) compared to GBM cells. Knockdown of Pin1 in GBM cells increased their sensitivities to radiation, whereas over-expression of Pin1 reduced radiosensitivity. Knockdown of Pin1 decreased several stemness gene expression including CD133, Sox2 etc. Pin1 knockdown also reduced β-catenin expression, where activation or inhibition of β-catenin affected the Sox2 protein expression but not Pin1 expression. This suggests that Pin1 acted at upstream of β-catenin and subsequently affected Sox2 expression. On the other hand, by co-immunoprecipitation assay, we showed that Pin1 might directly interact with Sox2 protein and altered its stability.

Abstract 3
Chinese Abstract 4
Introduction 5
Glioblastoma Multiforme 5
Pin1 6
CD133 7
Sox2 9
Specific Aims 10
Materials and Methods 11
Results 22
Knockdown and over-expression of Pin1 in GBM cells affect their sensitivities to radiation 22
The effects of Pin1 knockdown on growth curve, protein expression and cell cycle distribution in S1 and S1R1 cells 23
The effects of Pin1 knockdown on sphere formation ability in S1、S1R1 cells 23
The effects of Pin1 knockdown on stem cell markers in S1、S1R1 cells 24
The effects of knockdown or over-expression of Sox2 on growth curve and Pin1 protein expression in S1 cells 24
Pin1 affects the level of β-catenin protein expression in S1 cells 25
β-catenin affects the level of Sox2 protein expression in S1 cells 26
The effect of over-expression β-catenin on Sox2 promoter activity 26
Pin1 may directly interact with Sox2 in S1 cells 27
Discussion 28
References 31
Figures 38
Appendix 49

1. DeAngelis, L. 2001. Brain tumors. N Engl J Med 344:114-123.
2. Davis, F., Kupelian, V., Freels, S., McCarthy, B., and Surawicz, T. 2001. Prevalence estimates for primary brain tumors in the United States by behavior and major histology groups. Neuro Oncol 3:152-158.
3. Beauchesne, P. 2002. Promising survival and concomitant radiation plus temozolomide followed by adjuvant temozolomide. J Clin Oncol 20:3180-3181; author reply 3181.
4. Stupp, R., and Baumert, B. 2003. Promises and controversies in the management of low-grade glioma. Ann Oncol 14:1695-1696.
5. Krex, D., Klink, B., Hartmann, C., von Deimling, A., Pietsch, T., Simon, M., Sabel, M., Steinbach, J., Heese, O., Reifenberger, G., et al. 2007. Long-term survival with glioblastoma multiforme. Brain 130:2596-2606.
6. Wheeler, C., Black, K., Liu, G., Mazer, M., Zhang, X., Pepkowitz, S., Goldfinger, D., Ng, H., Irvin, D., and Yu, J. 2008. Vaccination elicits correlated immune and clinical responses in glioblastoma multiforme patients. Cancer Res 68:5955-5964.
7. Kleihues, P., and Ohgaki, H. 1999. Primary and secondary glioblastomas: from concept to clinical diagnosis. Neuro Oncol 1:44-51.
8. Kleihues, P., and Sobin, L. 2000. World Health Organization classification of tumors. Cancer 88:2887.
9. Ohgaki, H., Dessen, P., Jourde, B., Horstmann, S., Nishikawa, T., Di Patre, P., Burkhard, C., Schüler, D., Probst-Hensch, N., Maiorka, P., et al. 2004. Genetic pathways to glioblastoma: a population-based study. Cancer Res 64:6892-6899.
10. Reardon, D., Rich, J., Friedman, H., and Bigner, D. 2006. Recent advances in the treatment of malignant astrocytoma. J Clin Oncol 24:1253-1265.
11. Stupp, R., Hegi, M., Gilbert, M., and Chakravarti, A. 2007. Chemoradiotherapy in malignant glioma: standard of care and future directions. J Clin Oncol 25:4127-4136.
12. Norman, D., Stevens, E., Wing, S., Levin, V., and Newton, T. 1978. Quantitative aspects of contrast enhancement in cranial computed tomography. Radiology 129:683-688.
13. Brooks, D., Beaney, R., Lammertsma, A., Leenders, K., Horlock, P., Kensett, M., Marshall, J., Thomas, D., and Jones, T. 1984. Quantitative measurement of blood-brain barrier permeability using rubidium-82 and positron emission tomography. J Cereb Blood Flow Metab 4:535-545.
14. Lefebvre, P., and Laval, F. 1986. Enhancement of O6-methylguanine-DNA-methyltransferase activity induced by various treatments in mammalian cells. Cancer Res 46:5701-5705.
15. Grombacher, T., Mitra, S., and Kaina, B. 1996. Induction of the alkyltransferase (MGMT) gene by DNA damaging agents and the glucocorticoid dexamethasone and comparison with the response of base excision repair genes. Carcinogenesis 17:2329-2336.
16. Singh, S., Clarke, I., Terasaki, M., Bonn, V., Hawkins, C., Squire, J., and Dirks, P. 2003. Identification of a cancer stem cell in human brain tumors. Cancer Res 63:5821-5828.
17. Singh, S., Clarke, I., Hide, T., and Dirks, P. 2004. Cancer stem cells in nervous system tumors. Oncogene 23:7267-7273.
18. Galli, R., Binda, E., Orfanelli, U., Cipelletti, B., Gritti, A., De Vitis, S., Fiocco, R., Foroni, C., Dimeco, F., and Vescovi, A. 2004. Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res 64:7011-7021.
19. Sanai, N., Alvarez-Buylla, A., and Berger, M. 2005. Neural stem cells and the origin of gliomas. N Engl J Med 353:811-822.
20. Dean, M., Fojo, T., and Bates, S. 2005. Tumour stem cells and drug resistance. Nat Rev Cancer 5:275-284.
21. Lu, K., Hanes, S., and Hunter, T. 1996. A human peptidyl-prolyl isomerase essential for regulation of mitosis. Nature 380:544-547.
22. Galat, A. 2003. Peptidylprolyl cis/trans isomerases (immunophilins): biological diversity--targets--functions. Curr Top Med Chem 3:1315-1347.
23. Lu, K., Suizu, F., Zhou, X., Finn, G., Lam, P., and Wulf, G. 2006. Targeting carcinogenesis: a role for the prolyl isomerase Pin1? Mol Carcinog 45:397-402.
24. Yeh, E., and Means, A. 2007. PIN1, the cell cycle and cancer. Nat Rev Cancer 7:381-388.
25. Yaffe, M., Schutkowski, M., Shen, M., Zhou, X., Stukenberg, P., Rahfeld, J., Xu, J., Kuang, J., Kirschner, M., Fischer, G., et al. 1997. Sequence-specific and phosphorylation-dependent proline isomerization: a potential mitotic regulatory mechanism. Science 278:1957-1960.
26. Ranganathan, R., Lu, K., Hunter, T., and Noel, J. 1997. Structural and functional analysis of the mitotic rotamase Pin1 suggests substrate recognition is phosphorylation dependent. Cell 89:875-886.
27. Lu, K., and Zhou, X. 2007. The prolyl isomerase PIN1: a pivotal new twist in phosphorylation signalling and disease. Nat Rev Mol Cell Biol 8:904-916.
28. Wulf, G., Ryo, A., Wulf, G., Lee, S., Niu, T., Petkova, V., and Lu, K. 2001. Pin1 is overexpressed in breast cancer and cooperates with Ras signaling in increasing the transcriptional activity of c-Jun towards cyclin D1. EMBO J 20:3459-3472.
29. Ryo, A., Nakamura, M., Wulf, G., Liou, Y., and Lu, K. 2001. Pin1 regulates turnover and subcellular localization of beta-catenin by inhibiting its interaction with APC. Nat Cell Biol 3:793-801.
30. Liou, Y., Ryo, A., Huang, H., Lu, P., Bronson, R., Fujimori, F., Uchida, T., Hunter, T., and Lu, K. 2002. Loss of Pin1 function in the mouse causes phenotypes resembling cyclin D1-null phenotypes. Proc Natl Acad Sci U S A 99:1335-1340.
31. Ryo, A., Liou, Y., Wulf, G., Nakamura, M., Lee, S., and Lu, K. 2002. PIN1 is an E2F target gene essential for Neu/Ras-induced transformation of mammary epithelial cells. Mol Cell Biol 22:5281-5295.
32. Fujimori, F., Takahashi, K., Uchida, C., and Uchida, T. 1999. Mice lacking Pin1 develop normally, but are defective in entering cell cycle from G(0) arrest. Biochem Biophys Res Commun 265:658-663.
33. Lu, K. 2003. Prolyl isomerase Pin1 as a molecular target for cancer diagnostics and therapeutics. Cancer Cell 4:175-180.
34. Yeh, E., Cunningham, M., Arnold, H., Chasse, D., Monteith, T., Ivaldi, G., Hahn, W., Stukenberg, P., Shenolikar, S., Uchida, T., et al. 2004. A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells. Nat Cell Biol 6:308-318.
35. Yeh, E., Lew, B., and Means, A. 2006. The loss of PIN1 deregulates cyclin E and sensitizes mouse embryo fibroblasts to genomic instability. J Biol Chem 281:241-251.
36. Yin, A., Miraglia, S., Zanjani, E., Almeida-Porada, G., Ogawa, M., Leary, A., Olweus, J., Kearney, J., and Buck, D. 1997. AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 90:5002-5012.
37. Mizrak, D., Brittan, M., and Alison, M. 2008. CD133: molecule of the moment. J Pathol 214:3-9.
38. Singh, S., Hawkins, C., Clarke, I., Squire, J., Bayani, J., Hide, T., Henkelman, R., Cusimano, M., and Dirks, P. 2004. Identification of human brain tumour initiating cells. Nature 432:396-401.
39. Haraguchi, N., Utsunomiya, T., Inoue, H., Tanaka, F., Mimori, K., Barnard, G., and Mori, M. 2006. Characterization of a side population of cancer cells from human gastrointestinal system. Stem Cells 24:506-513.
40. Liu, G., Yuan, X., Zeng, Z., Tunici, P., Ng, H., Abdulkadir, I., Lu, L., Irvin, D., Black, K., and Yu, J. 2006. Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol Cancer 5:67.
41. Ma, S., Lee, T., Zheng, B., Chan, K., and Guan, X. 2008. CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway. Oncogene 27:1749-1758.
42. Bao, S., Wu, Q., McLendon, R., Hao, Y., Shi, Q., Hjelmeland, A., Dewhirst, M., Bigner, D., and Rich, J. 2006. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444:756-760.
43. Beier, D., Hau, P., Proescholdt, M., Lohmeier, A., Wischhusen, J., Oefner, P., Aigner, L., Brawanski, A., Bogdahn, U., and Beier, C. 2007. CD133(+) and CD133(-) glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles. Cancer Res 67:4010-4015.
44. Chiou, S., Kao, C., Chen, Y., Chien, C., Hung, S., Lo, J., Chen, Y., Ku, H., Hsu, M., and Wong, T. 2008. Identification of CD133-positive radioresistant cells in atypical teratoid/rhabdoid tumor. PLoS One 3:e2090.
45. Chang, C., Hsu, C., Yung, M., Chen, K., Tzao, C., Wu, W., Chou, H., Lee, Y., Lu, K., Chiou, S., et al. 2009. Enhanced radiosensitivity and radiation-induced apoptosis in glioma CD133-positive cells by knockdown of SirT1 expression. Biochem Biophys Res Commun 380:236-242.
46. Denny, P., Swift, S., Brand, N., Dabhade, N., Barton, P., and Ashworth, A. 1992. A conserved family of genes related to the testis determining gene, SRY. Nucleic Acids Res 20:2887.
47. Kamachi, Y., Uchikawa, M., and Kondoh, H. 2000. Pairing SOX off: with partners in the regulation of embryonic development. Trends Genet 16:182-187.
48. Takahashi, K., and Yamanaka, S. 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663-676.
49. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861-872.
50. Graham, V., Khudyakov, J., Ellis, P., and Pevny, L. 2003. SOX2 functions to maintain neural progenitor identity. Neuron 39:749-765.
51. Ferri, A., Cavallaro, M., Braida, D., Di Cristofano, A., Canta, A., Vezzani, A., Ottolenghi, S., Pandolfi, P., Sala, M., DeBiasi, S., et al. 2004. Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain. Development 131:3805-3819.
52. Schmitz, M., Temme, A., Senner, V., Ebner, R., Schwind, S., Stevanovic, S., Wehner, R., Schackert, G., Schackert, H., Fussel, M., et al. 2007. Identification of SOX2 as a novel glioma-associated antigen and potential target for T cell-based immunotherapy. Br J Cancer 96:1293-1301.
53. Ben-Porath, I., Thomson, M., Carey, V., Ge, R., Bell, G., Regev, A., and Weinberg, R. 2008. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet 40:499-507.
54. Willert, K., Brown, J., Danenberg, E., Duncan, A., Weissman, I., Reya, T., Yates, J.r., and Nusse, R. 2003. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423:448-452.
55. Haegele, L., Ingold, B., Naumann, H., Tabatabai, G., Ledermann, B., and Brandner, S. 2003. Wnt signalling inhibits neural differentiation of embryonic stem cells by controlling bone morphogenetic protein expression. Mol Cell Neurosci 24:696-708.
56. Sato, N., Meijer, L., Skaltsounis, L., Greengard, P., and Brivanlou, A. 2004. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 10:55-63.
57. Ogawa, K., Nishinakamura, R., Iwamatsu, Y., Shimosato, D., and Niwa, H. 2006. Synergistic action of Wnt and LIF in maintaining pluripotency of mouse ES cells. Biochem Biophys Res Commun 343:159-166.
58. Miyabayashi, T., Teo, J., Yamamoto, M., McMillan, M., Nguyen, C., and Kahn, M. 2007. Wnt/beta-catenin/CBP signaling maintains long-term murine embryonic stem cell pluripotency. Proc Natl Acad Sci U S A 104:5668-5673.
59. 羅智文. 2008. 具放射線抗性之腦腫瘤細胞的特徵與Pin1在其中所參與的角色. 國立陽明大學 醫學生物技術暨檢驗學系暨研究所 碩士論文.
60. 賴麗娟. 2009. 在神經膠質瘤細胞抑制Pin1的表現會加強對放射線的敏感性. 國立陽明大學 醫學生物技術暨檢驗學系暨研究所 碩士論文

61. Lee, W., Yeh, M., Tu, Y., Han, S., and Wang, Y. 1988. Establishment and characterization of a malignant glioma cell line, GBM8401/TSGH,NDMC. J Surg Oncol 38:173-181.
62. Lee, J., Kotliarova, S., Kotliarov, Y., Li, A., Su, Q., Donin, N., Pastorino, S., Purow, B., Christopher, N., Zhang, W., et al. 2006. Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell 9:391-403.
63. Doble, B., and Woodgett, J. 2003. GSK-3: tricks of the trade for a multi-tasking kinase. J Cell Sci 116:1175-1186.
64. Swaney, D., Wenger, C., Thomson, J., and Coon, J. 2009. Human embryonic stem cell phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry. Proc Natl Acad Sci U S A 106:995-1000.
65. Van Hoof, D., Muñoz, J., Braam, S., Pinkse, M., Linding, R., Heck, A., Mummery, C., and Krijgsveld, J. 2009. Phosphorylation dynamics during early differentiation of human embryonic stem cells. Cell Stem Cell 5:214-226.
66. Schechter, C. 1999. Re: Brain and other central nervous system cancers: recent trends in incidence and mortality. J Natl Cancer Inst 91:2050-2051.
67. Jordan, C., Guzman, M., and Noble, M. 2006. Cancer stem cells. N Engl J Med 355:1253-1261.
68. Ryo, A., Liou, Y., Lu, K., and Wulf, G. 2003. Prolyl isomerase Pin1: a catalyst for oncogenesis and a potential therapeutic target in cancer. J Cell Sci 116:773-783.
69. Nakashima, M., Meirmanov, S., Naruke, Y., Kondo, H., Saenko, V., Rogounovitch, T., Shimizu-Yoshida, Y., Takamura, N., Namba, H., Ito, M., et al. 2004. Cyclin D1 overexpression in thyroid tumours from a radio-contaminated area and its correlation with Pin1 and aberrant beta-catenin expression. J Pathol 202:446-455.
70. Fukuchi, M., Fukai, Y., Kimura, H., Sohda, M., Miyazaki, T., Nakajima, M., Masuda, N., Tsukada, K., Kato, H., and Kuwano, H. 2006. Prolyl isomerase Pin1 expression predicts prognosis in patients with esophageal squamous cell carcinoma and correlates with cyclinD1 expression. Int J Oncol 29:329-334.
71. Ryo, A., Hirai, A., Nishi, M., Liou, Y., Perrem, K., Lin, S., Hirano, H., Lee, S., and Aoki, I. 2007. A suppressive role of the prolyl isomerase Pin1 in cellular apoptosis mediated by the death-associated protein Daxx. J Biol Chem 282:36671-36681.
72. Saunders, L., and Verdin, E. 2007. Sirtuins: critical regulators at the crossroads between cancer and aging. Oncogene 26:5489-5504.
73. Potente, M., Ghaeni, L., Baldessari, D., Mostoslavsky, R., Rossig, L., Dequiedt, F., Haendeler, J., Mione, M., Dejana, E., Alt, F., et al. 2007. SIRT1 controls endothelial angiogenic functions during vascular growth. Genes Dev 21:2644-2658.
74. Network, C.G.A.R. 2008. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 455:1061-1068.
75. Hong, H., Takahashi, K., Ichisaka, T., Aoi, T., Kanagawa, O., Nakagawa, M., Okita, K., and Yamanaka, S. 2009. Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature 460:1132-1135.
76. Dreesen, O., and Brivanlou, A. 2007. Signaling pathways in cancer and embryonic stem cells. Stem Cell Rev 3:7-17.
77. Ryo, A., Suizu, F., Yoshida, Y., Perrem, K., Liou, Y., Wulf, G., Rottapel, R., Yamaoka, S., and Lu, K. 2003. Regulation of NF-kappaB signaling by Pin1-dependent prolyl isomerization and ubiquitin-mediated proteolysis of p65/RelA. Mol Cell 12:1413-1426.
78. Liao, Y., Wei, Y., Zhou, X., Yang, J., Dai, C., Chen, Y., Agarwal, N., Sarbassov, D., Shi, D., Yu, D., et al. 2009. Peptidyl-prolyl cis/trans isomerase Pin1 is critical for the regulation of PKB/Akt stability and activation phosphorylation. Oncogene 28:2436-2445.
79. Nakano, A., Koinuma, D., Miyazawa, K., Uchida, T., Saitoh, M., Kawabata, M., Hanai, J., Akiyama, H., Abe, M., Miyazono, K., et al. 2009. Pin1 down-regulates transforming growth factor-beta (TGF-beta) signaling by inducing degradation of Smad proteins. J Biol Chem 284:6109-6115.
80. Matsuura, I., Chiang, K., Lai, C., He, D., Wang, G., Ramkumar, R., Uchida, T., Ryo, A., Lu, K., and Liu, F. 2010. Pin1 promotes transforming growth factor-beta-induced migration and invasion. J Biol Chem 285:1754-1764.
81. Ikushima, H., Todo, T., Ino, Y., Takahashi, M., Miyazawa, K., and Miyazono, K. 2009. Autocrine TGF-beta signaling maintains tumorigenicity of glioma-initiating cells through Sry-related HMG-box factors. Cell Stem Cell 5:504-514.
82. Ge, Y., Zhou, F., Chen, H., Cui, C., Liu, D., Li, Q., Yang, Z., Wu, G., Sun, S., Gu, J., et al. 2010. Sox2 is translationally activated by eukaryotic initiation factor 4E in human glioma-initiating cells. Biochem Biophys Res Commun.