腫瘤胚胎基因在胚胎或胎兒中表現，而正常成人組織中表現量低或是不表現，但腫瘤組織中又再度表現。Wnt/β-catenin 傳遞路徑在胚胎發育、細胞增生、細胞分化、細胞再生、細胞自我更新及腫瘤生成過程中扮演著重要的角色。先前研究都指出異常活化此路徑及其下游的標靶基因會造成許多癌症，其中包含了肝癌。肝癌是全球最常見的惡性腫瘤之ㄧ，也是第四大癌症導致死亡。目前已有多個 Wnt/β-catenin 標靶基因被發現在腫瘤生成中扮演關鍵角色，如 c-myc，cyclin D1 等。因此，透過鑑定Wnt/β-catenin 標靶基因去了解其所調控的腫瘤生成是很重要的議題。在過去實驗室建立了一個結構完整、具擴展性的生物資料庫，簡稱 Bio-database。我們將此Biodatabase運用來尋找有潛力的 Wnt/β-catenin 標靶基因，其中一個為Zinc Finger Protein 496 (ZNF496) 。所以本篇研究主要是在探討ZNF496 是否是Wnt/β-catenin signaling pathway 的target gene，另外也探討ZNF496 在肝癌中所扮演的角色及功能。首先利用 Lithium chloride 和 Wnt-3A 條件培養液去活化Wnt/β-catenin 傳遞路徑，再經由半定量 RT-PCR 發現 ZNF496 的 mRNA 表現量會增加。進一步利用染色質免疫沉澱法 (ChIP) 、螢光素酶偵測法 (luciferase assays) 、突變法 (mutation assays )證實 β-catenin/TCF complex 會結合於 ZNF496 的 promoter 位置。結果顯示ZNF496為有潛力是Wnt/β-catenin 的target gene。除此之外，我們發現 ZNF496 會在肝癌腫
瘤組織大量表現。而overexpression 實驗結果顯示為 ZNF496 會促進幹細胞特性、上皮-間質轉化 (EMT) 及 細胞增生，而使用 XAV939 (Wnt/β-catenin 傳遞路徑的抑制劑) 可以抑制ZNF496 的表現。

Misregulation of the Wnt/β-catenin signaling pathway occurred in various human cancers including liver cancer. In our laboratory, we developed a combined bioinformatics and experimental approach to find a potential target gene of the Wnt/β-catenin signaling pathway. One of these candidate genes is Zinc Finger Protein 496 (ZNF496). ChIP assay, luciferase assay and mutation assay suggested that the TCF binding site of ZNF496 promoter was critical for β-catenin /TCF dependent regulation. In this study, we investigate the function and characteristics of ZNF496 in human HCC cell lines. The results showed that overexpression of ZNF496 could promote stemness, EMT and cell proliferation. XAV939 which is the Wnt/β-catenin specific inhibitor could inhibit the expression of the target genes of Wnt/β-catenin, such as c-myc, cyclin D1 and ZNF496. These results indicate that ZNF496 may be a potential target gene of Wnt/β-catenin pathway and may play important roles in HCC.

1 Komiya, Y. & Habas, R. Wnt signal transduction pathways. Organogenesis 4, 68-75 (2008).
2 Teo, J.-L. & Kahn, M. The Wnt signaling pathway in cellular proliferation and differentiation: a tale of two coactivators. Advanced drug delivery reviews 62,
1149-1155 (2010).
3 Van Camp, J., Beckers, S., Zegers, D. & Van Hul, W. Wnt signaling and the control of human stem cell fate. Stem Cell Reviews and Reports 10, 207-229 (2014).
4 Rao, T. P. & Kühl, M. An updated overview on Wnt signaling pathways a prelude for more. Circulation research 106, 1798-1806 (2010).
5 Polakis, P. Wnt signaling and cancer. Genes & development 14, 1837-1851 (2000).
6 Kühl, M., Sheldahl, L. C., Park, M., Miller, J. R. & Moon, R. T. The Wnt/Ca 2+ pathway: a new vertebrate Wnt signaling pathway takes shape. Trends in genetics
16, 279-283 (2000).
7 Blagodatski, A., Poteryaev, D. & Katanaev, V. L. Targeting the Wnt pathways for therapies. Molecular and cellular therapies 2, 1 (2014).
8 Benhamouche, S. et al. Apc tumor suppressor gene is the “zonation-keeper” of mouse liver. Developmental cell 10, 759-770 (2006).
9 Anastas, J. N. & Moon, R. T. WNT signalling pathways as therapeutic targets in cancer. Nature Reviews Cancer 13, 11-26 (2013).
10 Giles, R. H., van Es, J. H. & Clevers, H. Caught up in a Wnt storm: Wnt signaling in cancer. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer 1653, 1-24
(2003).
11 Laurent–Puig, P. et al. Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis. Gastroenterology 120, 1763-1773 (2001).
12 Provost, E. et al. Functional correlates of mutation of the Asp32 and Gly34 residues of beta-catenin. Oncogene 24, 2667-2676 (2005).
13 Sareddy, G. R., Kesanakurti, D., Kirti, P. B. & Babu, P. P. Nonsteroidal antiinflammatory drugs diclofenac and celecoxib attenuates Wnt/β-catenin/Tcf signaling pathway in human glioblastoma cells. Neurochemical research 38, 2313-2322 (2013).
14 Zimmerman, Z. F., Moon, R. T. & Chien, A. J. Targeting Wnt pathways in disease. Cold Spring Harbor perspectives in biology 4, a008086 (2012).
15 Huang, S.-M. A. et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 461, 614-620 (2009).
16 Emami, K. H. et al. A small molecule inhibitor of β-catenin/cyclic AMP response element-binding protein transcription. Proceedings of the National Academy of
Sciences of the United States of America 101, 12682-12687 (2004).
17 Pishvaian, M. J. & Byers, S. W. Biomarkers of WNT signaling. Cancer Biomarkers 3, 263-274 (2007).
18 Severin, S. et al. Antitumor activity of conjugates of the oncofetal protein alphafetoprotein and phthalocyanines in vitro. IUBMB Life 43, 1081-1089 (1997).
19 Liao, B., Hu, Y., Herrick, D. J. & Brewer, G. The RNA-binding protein IMP-3 is a translational activator of insulin-like growth factor II leader-3 mRNA during
proliferation of human K562 leukemia cells. Journal of Biological Chemistry 280, 18517-18524 (2005).
20 Loganath, A. et al. Comparison of AFP and β-hCG levels in infiltrating duct mammary carcinoma at different stages of malignancy. Pathology 20, 275-278(1988).
21 Shimokawa, T. et al. Involvement of the FGF18 gene in colorectal carcinogenesis, as a novel downstream target of the β-catenin/T-cell factor complex. Cancer
research 63, 6116-6120 (2003).
22 Güre, A. O. et al. Serological identification of embryonic neural proteins as highly immunogenic tumor antigens in small cell lung cancer. Proceedings of the National Academy of Sciences 97, 4198-4203 (2000).
23 Hsu, C. et al. Identifying LRRC16B as an oncofetal gene with transforming enhancing capability using a combined bioinformatics and experimental approach.
Oncogene 30, 654-667 (2011).
24 陳怡文. 結合生物資訊暨實驗篩選以尋找 Wnt/β-catenin 傳遞路徑之新穎基因. 成功大學醫學檢驗生物技術學系學位論文, 1-78 (2011).
25 He, T.-C. et al. Identification of c-MYC as a target of the APC pathway. Science 281, 1509-1512 (1998).
26 Higano, K., Takada, R., Ito, F., Takeichi, M. & Takada, S. Cytoskeletal reorganization by soluble Wnt‐3a protein signalling. Genes to Cells 3, 659-670(1998).
27 Reya, T., Morrison, S. J., Clarke, M. F. & Weissman, I. L. Stem cells, cancer, and cancer stem cells. nature 414, 105-111 (2001).
28 Ponti, D. et al. Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer research 65, 5506-5511 (2005).
29 Beachy, P. A., Karhadkar, S. S. & Berman, D. M. Tissue repair and stem cell renewal in carcinogenesis. Nature 432, 324-331 (2004).
30 Stingl, J. & Caldas, C. Molecular heterogeneity of breast carcinomas and the cancer stem cell hypothesis. Nature Reviews Cancer 7, 791-799 (2007).
31 Lin, C.-W. et al. Epithelial cell adhesion molecule regulates tumor initiation and tumorigenesis via activating reprogramming factors and epithelial-mesenchymal transition gene expression in colon cancer. Journal of Biological Chemistry 287, 39449-39459 (2012).
32 Yamashita, T., Budhu, A., Forgues, M. & Wang, X. W. Activation of hepatic stem cell marker EpCAM by Wnt–β-catenin signaling in hepatocellular carcinoma. Cancer research 67, 10831-10839 (2007).
33 Lamouille, S., Xu, J. & Derynck, R. Molecular mechanisms of epithelial– mesenchymal transition. Nature reviews. Molecular cell biology 15, 178 (2014).
34 Galliher, A. J. & Schiemann, W. P. β 3 integrin and Src facilitate transforming growth factor-β mediated induction of epithelial-mesenchymal transition in
mammary epithelial cells. Breast Cancer Research 8, 1 (2006).
35 Jiang, Y. G. et al. Role of Wnt/β‐catenin signaling pathway in epithelialmesenchymal transition of human prostate cancer induced by hypoxia‐inducible factor‐1α. International Journal of Urology 14, 1034-1039 (2007).
36 Kalluri, R. & Weinberg, R. A. The basics of epithelial-mesenchymal transition. The Journal of clinical investigation 119, 1420-1428 (2009).
37 Thiery, J. P., Acloque, H., Huang, R. Y. & Nieto, M. A. Epithelial-mesenchymal transitions in development and disease. Cell 139, 871-890 (2009).
38 Cowin, P., Rowlands, T. M. & Hatsell, S. J. Cadherins and catenins in breast cancer. Current opinion in cell biology 17, 499-508 (2005).
39 Wu, Y. & Zhou, B. P. New insights of epithelial‐mesenchymal transition in cancer metastasis. Acta biochimica et biophysica Sinica 40, 643-650 (2008).
40 Junghans, D., Haas, I. G. & Kemler, R. Mammalian cadherins and protocadherins: about cell death, synapses and processing. Current opinion in cell biology 17, 446-
452 (2005).
41 Li, J. & Zhou, B. P. Activation of β-catenin and Akt pathways by Twist are critical for the maintenance of EMT associated cancer stem cell-like characters. BMC
cancer 11, 1 (2011).
42 Pritchard, C. C. & Grady, W. M. Colorectal cancer molecular biology moves into clinical practice. Gut, gut. 2009.206250 (2010).
43 Golik, M. et al. Differential expression of ruminant ZNF496 variants: Association with quantitative trait locus affecting bovine milk concentration and fertility 1, 2. Journal of dairy science 94, 2092-2102 (2011).
44 Hoshida, Y. et al. Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma. Cancer research 69, 7385-7392
(2009).
45 Cagatay, T. & Ozturk, M. P53 mutation as a source of aberrant beta-catenin accumulation in cancer cells. Oncogene 21, 7971-7980 (2002).