近期有許多研究顯示 Wnt 訊息傳遞途徑 (Wnt signaling pathway) 會參與癌症幹細胞 (cancer stem cells) 維持其特性及形成之過程。Secreted frizzled-related protein (SFRP) 家族由五個分泌性醣蛋白成員組成 (SFRP1、SFRP2、SFRP3、SFRP4、SFRP5)，並在 Wnt 訊息傳遞途徑中扮演負向調控者的角色。因 SFRPs 其 N 端序列具有與 Frizzled 相似的半胱胺酸富含區 (cysteine rich domain, CRD)，使 SFRPs 可以和 Wnt 結合，進而阻礙 Wnt 和 Frizzled 之交互作用，抑制 Wnt 訊息傳遞途徑之活化，然而目前仍不清楚 SFRPs 是否只會扮演細胞外調控者的角色，或者可以透過其他機制調節 Wnt 訊息傳遞途徑。

　　本論文的研究發現，在過度表現 SFRPs 的 HM20 細胞中，SFRPs 不但可以進入細胞核內，並且可以進一步與 β-catenin 結合；透過免疫共沉澱法發現： SFRPs 與 β-catenin 結合的區域和 T-cell factor (TCF)/Lymphoid enhancer-binding factor (LEF) 與 β-catenin 的結合位置非常相近，因而進一步偵測 TCF4 和 β-catenin 在細胞核內的結合程度。結果說明：雖然 β-catenin 可以累積在過度表現 SFRPs 的 HM20 細胞核內，但是 SFRP1、SFRP2、SFRP4 和 SFRP5 卻明顯抑制 TCF4 和 β-catenin 的結合作用，反之 SFRP3 則會促進 TCF4 和 β-catenin 的結合。隨後，為了測試 SFRPs 是否除了做為細胞外調控者之外，也可以利用細胞核內與 β-catenin 之結合而調控 Wnt 訊息傳遞的轉錄活性及生物性功能，利用 glycogen synthase kinase 3 beta (GSK3β) 專一性抑制劑誘導 Wnt 訊息傳遞活化後，我們發現 SFRP1、SFRP2、SFRP4 和 SFRP5 仍然可以顯著的抑制 β-catenin/TCF 轉錄的活性和癌症幹細胞形成球體 (sphere-forming) 的能力，而 SFRP3 則會增強由 β-catenin/TCF 所調控的轉錄活性及促進癌症幹細胞球體的形成。綜合以上的研究結果，我們提供了不同於傳統的觀點：SFRPs 於 Wnt 訊息傳遞路徑中不只扮演細胞外拮抗劑的角色，SFRPs 也可以在細胞內藉由和 β-catenin 結合，進而影響 TCF4 和 β-catenin 的交互作用，藉此調控 Wnt 訊息傳遞路徑，並更進一步地影響腫瘤形成的能力。

　　Recent researches suggest the Wnt signaling pathway is involved in maintaining the characteristics and population of cancer stem cells. Secreted frizzled-related protein (SFRP) family is composed of five secreted glycoproteins (SFRP1 to SFRP5) which play modulators in the Wnt signaling pathway. SFRPs contain the Frizzled-like cysteine-rich domain (CRD) at the N-terminus which interacts with Wnt ligands, resulting in the prevention of Wnt ligand-Frizzled receptor interaction and the Wnt signaling pathway activation. However, little is known about whether SFRPs only act as extracellular modulators or could be through another mechanism to regulate the Wnt signaling pathway.

　　In our study, we found that SFRPs could translocate into the nucleus and associate with β-catenin in SFRPs-expressing cells. The interaction domain mapping indicated that the binding region of β-catenin for SFRPs was similar to that for T-cell factor (TCF)/Lymphoid enhancer-binding factor (LEF). Thus, we further investigated the interaction of TCF4 and β-catenin in the nucleus by co-immunoprecipitation. Interestingly, although β-catenin accumulated in the nucleus of SFRPs-expressing cells, SFRP1, SFRP2, SFRP4 and SFRP5 could significantly suppress TCF4 binding β-catenin compared with Mock cells, whereas SFRP3 could promote the recruitment of β-catenin to TCF4. After that, a glycogen synthase kinase 3 beta (GSK3β) specific inhibitor was used to activate the intracellular Wnt signaling cascade and test whether SFRPs could regulate the transcriptional activity of β-catenin/TCF and consequent biological functions beyond acting as extracellular modulators. We found that SFRP1, SFRP2, SFRP4 and SFRP5 significantly inhibited the transcriptional activity of β-catenin/TCF and sphere formation ability after GSK3β specific inhibitor treatment, whereas SFRP3 obviously enhanced the β-catenin/TCF-mediated transcriptional activity and sphere formation ability. Taken together, in addition to the role of extracellular modulators, SFRPs can also regulate the Wnt signaling via associating with β-catenin to modulate the interaction of TCF4 and β-catenin in the nucleus, which impacts on Wnt/β-catenin-elicited the tumor-initiating ability. To all of above, we provide a novel mechanism of SFRPs regulating the Wnt signaling at the intracellular level, which challenges the rule of SFRPs acting as extracellular modulators in the Wnt signaling pathway.

Chapter 1　Introduction 6
1.1 Lung cancer 6
1.2 The Wnt signaling pathway 7
1.3 The family of secreted frizzled-related proteins 8
1.4 Cancer stem cells 10
Chapter 2　Materials and methods 12
2.1 Functional fractionation of cancer cells by invasive assays 12
2.2 Stably transfected cells and cell culture 12
2.3 Western blotting (Immunoblotting) 13
2.4 Nuclear/Cytosolic fractions isolation 13
2.5 Co-immunoprecipitation (Co-IP) 14
2.6 Transfection 15
2.7 TOP luciferase reporter assay 15
2.8 Sphere formation assay 15
2.9 Sphere limiting dilution assay 16
2.10 Statistical analysis 16
Chapter 3　Results 17
3.1 SFRPs translocate into the nucleus and associate with β-catenin 17
3.2 Mapping of SFRPs and β-catenin interaction domains 18
3.3 The association of SFRPs with β-catenin modulates TCF4 recruitment 19
3.4 SFRPs regulate the transcriptional activity of β-catenin/TCF beyond acting as extracellular modulators 20
3.5 Nuclear SFRPs are potent modulators of Wnt/β-catenin-elicited promotion of the cancer stem cell phenotype 22
Chapter 4　Discussion 24
References 29
Figures and legends 34

1. Minna, J.D., J.A. Roth, and A.F. Gazdar, Focus on lung cancer. Cancer Cell, 2002. 1(1): p. 49-52.
2. Siegel, R., D. Naishadham, and A. Jemal, Cancer statistics, 2013. CA Cancer J Clin, 2013. 63(1): p. 11-30.
3. Ettinger, D.S., et al., Non-small cell lung cancer. J Natl Compr Canc Netw, 2012. 10(10): p. 1236-71.
4. Pacheco-Pinedo, E.C., et al., Wnt/beta-catenin signaling accelerates mouse lung tumorigenesis by imposing an embryonic distal progenitor phenotype on lung epithelium. J Clin Invest, 2011. 121(5): p. 1935-45.
5. Lemjabbar-Alaoui, H., et al., Wnt and Hedgehog are critical mediators of cigarette smoke-induced lung cancer. PLoS One, 2006. 1: p. e93.
6. Ohgaki, H., et al., APC mutations are infrequent but present in human lung cancer. Cancer Lett, 2004. 207(2): p. 197-203.
7. Mazieres, J., et al., Wnt signaling in lung cancer. Cancer Lett, 2005. 222(1): p. 1-10.
8. Uematsu, K., et al., Activation of the Wnt pathway in non small cell lung cancer: evidence of dishevelled overexpression. Oncogene, 2003. 22(46): p. 7218-21.
9. Logan, C.Y. and R. Nusse, The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol, 2004. 20: p. 781-810.
10. Bhanot, P., et al., A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature, 1996. 382(6588): p. 225-30.
11. Wehrli, M., et al., arrow encodes an LDL-receptor-related protein essential for Wingless signalling. Nature, 2000. 407(6803): p. 527-30.
12. Zeng, X., et al., A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation. Nature, 2005. 438(7069): p. 873-7.
13. Mao, J., et al., Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway. Mol Cell, 2001. 7(4): p. 801-9.
14. Wong, H.C., et al., Direct binding of the PDZ domain of Dishevelled to a conserved internal sequence in the C-terminal region of Frizzled. Mol Cell, 2003. 12(5): p. 1251-60.
15. He, X., et al., LDL receptor-related proteins 5 and 6 in Wnt/beta-catenin signaling: arrows point the way. Development, 2004. 131(8): p. 1663-77.
16. Li, V.S., et al., Wnt signaling through inhibition of beta-catenin degradation in an intact Axin1 complex. Cell, 2012. 149(6): p. 1245-56.
17. Schuijers, J., et al., Wnt-induced transcriptional activation is exclusively mediated by TCF/LEF. EMBO J, 2014. 33(2): p. 146-56.
18. Kawano, Y. and R. Kypta, Secreted antagonists of the Wnt signalling pathway. J Cell Sci, 2003. 116(Pt 13): p. 2627-34.
19. Bienz, M. and H. Clevers, Linking colorectal cancer to Wnt signaling. Cell, 2000. 103(2): p. 311-20.
20. Terry, K., et al., Sfrp-1 and sfrp-2 are expressed in overlapping and distinct domains during chick development. Mech Dev, 2000. 97(1-2): p. 177-82.
21. Leimeister, C., A. Bach, and M. Gessler, Developmental expression patterns of mouse sFRP genes encoding members of the secreted frizzled related protein family. Mech Dev, 1998. 75(1-2): p. 29-42.
22. Rattner, A., et al., A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors. Proc Natl Acad Sci U S A, 1997. 94(7): p. 2859-63.
23. Jones, S.E. and C. Jomary, Secreted Frizzled-related proteins: searching for relationships and patterns. Bioessays, 2002. 24(9): p. 811-20.
24. Bafico, A., et al., Interaction of frizzled related protein (FRP) with Wnt ligands and the frizzled receptor suggests alternative mechanisms for FRP inhibition of Wnt signaling. J Biol Chem, 1999. 274(23): p. 16180-7.
25. Fukui, T., et al., Transcriptional silencing of secreted frizzled related protein 1 (SFRP 1) by promoter hypermethylation in non-small-cell lung cancer. Oncogene, 2005. 24(41): p. 6323-7.
26. Lo, P.K., et al., Epigenetic suppression of secreted frizzled related protein 1 (SFRP1) expression in human breast cancer. Cancer Biol Ther, 2006. 5(3): p. 281-6.
27. Shih, Y.L., et al., Promoter methylation of the secreted frizzled-related protein 1 gene SFRP1 is frequent in hepatocellular carcinoma. Cancer, 2006. 107(3): p. 579-90.
28. Saini, S., et al., Functional significance of secreted Frizzled-related protein 1 in metastatic renal cell carcinomas. Cancer Res, 2009. 69(17): p. 6815-22.
29. Fukuhara, K., et al., Secreted frizzled related protein 1 is overexpressed in uterine leiomyomas, associated with a high estrogenic environment and unrelated to proliferative activity. J Clin Endocrinol Metab, 2002. 87(4): p. 1729-36.
30. Uren, A., et al., Secreted frizzled-related protein-1 binds directly to Wingless and is a biphasic modulator of Wnt signaling. J Biol Chem, 2000. 275(6): p. 4374-82.
31. Cheng, Y.Y., et al., Frequent epigenetic inactivation of secreted frizzled-related protein 2 (SFRP2) by promoter methylation in human gastric cancer. Br J Cancer, 2007. 97(7): p. 895-901.
32. Lin, Y.W., et al., Promoter methylation of SFRP3 is frequent in hepatocellular carcinoma. Dis Markers, 2014. 2014: p. 351863.
33. He, B., et al., Secreted frizzled-related protein 4 is silenced by hypermethylation and induces apoptosis in beta-catenin-deficient human mesothelioma cells. Cancer Res, 2005. 65(3): p. 743-8.
34. Veeck, J., et al., Epigenetic inactivation of the secreted frizzled-related protein-5 (SFRP5) gene in human breast cancer is associated with unfavorable prognosis. Carcinogenesis, 2008. 29(5): p. 991-8.
35. Yamamura, S., et al., Oncogenic functions of secreted Frizzled-related protein 2 in human renal cancer. Mol Cancer Ther, 2010. 9(6): p. 1680-7.
36. Clevers, H., The cancer stem cell: premises, promises and challenges. Nat Med, 2011. 17(3): p. 313-9.
37. Lapidot, T., et al., A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature, 1994. 367(6464): p. 645-8.
38. Bonnet, D. and J.E. Dick, Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med, 1997. 3(7): p. 730-7.
39. Al-Hajj, M., et al., Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A, 2003. 100(7): p. 3983-8.
40. Visvader, J.E. and G.J. Lindeman, Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer, 2008. 8(10): p. 755-68.
41. Reya, T., et al., Stem cells, cancer, and cancer stem cells. Nature, 2001. 414(6859): p. 105-11.
42. Dean, M., T. Fojo, and S. Bates, Tumour stem cells and drug resistance. Nat Rev Cancer, 2005. 5(4): p. 275-84.
43. Reya, T. and H. Clevers, Wnt signalling in stem cells and cancer. Nature, 2005. 434(7035): p. 843-50.
44. Lustig, B. and J. Behrens, The Wnt signaling pathway and its role in tumor development. J Cancer Res Clin Oncol, 2003. 129(4): p. 199-221.
45. Graham, T.A., et al., Crystal structure of a beta-catenin/Tcf complex. Cell, 2000. 103(6): p. 885-96.
46. Porfiri, E., et al., Induction of a beta-catenin-LEF-1 complex by wnt-1 and transforming mutants of beta-catenin. Oncogene, 1997. 15(23): p. 2833-9.
47. Takahashi-Yanaga, F. and M. Kahn, Targeting Wnt signaling: can we safely eradicate cancer stem cells? Clin Cancer Res, 2010. 16(12): p. 3153-62.
48. Cao, L., et al., Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines. BMC Gastroenterol, 2011. 11: p. 71.
49. Anastas, J.N. and R.T. Moon, WNT signalling pathways as therapeutic targets in cancer. Nat Rev Cancer, 2013. 13(1): p. 11-26.
50. Ying, Y. and Q. Tao, Epigenetic disruption of the WNT/beta-catenin signaling pathway in human cancers. Epigenetics, 2009. 4(5): p. 307-12.
51. Suzuki, H., et al., Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer. Nat Genet, 2004. 36(4): p. 417-22.
52. Niehrs, C. and S.P. Acebron, Wnt signaling: multivesicular bodies hold GSK3 captive. Cell, 2010. 143(7): p. 1044-6.
53. McCartney, B.M. and I.S. Nathke, Cell regulation by the Apc protein Apc as master regulator of epithelia. Curr Opin Cell Biol, 2008. 20(2): p. 186-93.
54. Xing, Y., et al., Crystal structure of a beta-catenin/APC complex reveals a critical role for APC phosphorylation in APC function. Mol Cell, 2004. 15(4): p. 523-33.
55. Valenta, T., G. Hausmann, and K. Basler, The many faces and functions of beta-catenin. EMBO J, 2012. 31(12): p. 2714-36.
56. Tago, K., et al., Inhibition of Wnt signaling by ICAT, a novel beta-catenin-interacting protein. Genes Dev, 2000. 14(14): p. 1741-9.
57. Singh, A.M., et al., Chibby, an antagonist of the Wnt/beta-catenin pathway, facilitates cardiomyocyte differentiation of murine embryonic stem cells. Circulation, 2007. 115(5): p. 617-26.
58. Wawrzak, D., et al., Wnt3a binds to several sFRPs in the nanomolar range. Biochem Biophys Res Commun, 2007. 357(4): p. 1119-23.
59. Gan, X.Q., et al., Nuclear Dvl, c-Jun, beta-catenin, and TCF form a complex leading to stabilization of beta-catenin-TCF interaction. J Cell Biol, 2008. 180(6): p. 1087-100.
60. Hirota, M., et al., Smad2 functions as a co-activator of canonical Wnt/beta-catenin signaling pathway independent of Smad4 through histone acetyltransferase activity of p300. Cell Signal, 2008. 20(9): p. 1632-41.
61. Li, C., et al., The roles of Notch3 on the cell proliferation and apoptosis induced by CHIR99021 in NSCLC cell lines: a functional link between Wnt and Notch signaling pathways. PLoS One, 2013. 8(12): p. e84659.
62. Yu, Z., et al., Cancer stem cells. Int J Biochem Cell Biol, 2012. 44(12): p. 2144-51.
63. Holland, J.D., et al., Wnt signaling in stem and cancer stem cells. Curr Opin Cell Biol, 2013. 25(2): p. 254-64.
64. Kanwar, S.S., et al., The Wnt/beta-catenin pathway regulates growth and maintenance of colonospheres. Mol Cancer, 2010. 9: p. 212.
65. Sandberg, C.J., et al., Comparison of glioma stem cells to neural stem cells from the adult human brain identifies dysregulated Wnt- signaling and a fingerprint associated with clinical outcome. Exp Cell Res, 2013. 319(14): p. 2230-43.
66. Bisson, I. and D.M. Prowse, WNT signaling regulates self-renewal and differentiation of prostate cancer cells with stem cell characteristics. Cell Res, 2009. 19(6): p. 683-97.