腫瘤轉移一直是癌症病患死亡的主因。在人類中發現核苷二磷酸激酶 A (Nucleotide diphosphate kinase A, NDPK-A) 是第一個發現與癌症轉移有相關的，並且是由 nm23-H1 基因所編碼。在小鼠的黑色素瘤以及人類的乳腺癌之中知道核苷二磷酸激酶 A 扮演著抑制腫瘤轉移的角色。相反的，在我們實驗室發現核苷二磷酸激酶 A在兒童神經母細胞瘤中是促進腫瘤的轉移。除了高量表現外，我們實驗室發現在21%轉移的兒童神經母細胞瘤病患中核苷二磷酸激酶 A有 S120G 突變。核苷二磷酸激酶 A 的高量表現與突變使得神經的發育受到阻礙也增加兒童神經母細胞瘤的侵犯性以及在小鼠上腫瘤轉移的能力。到目前為止核苷二磷酸激酶 A 對轉移的機制還尚不清楚。在本研究中，蛋白質 Y ( Protein Y) 被發現到與核苷二磷酸激酶 A有交互作用。蛋白質 Y 在 HeLa 以及 HEK293T 細胞株中的位置在核仁。然而，在人類兒童神經母細胞瘤細胞株 (NB69) 中蛋白質 Y卻不在於核仁。在NB69細胞中外源性蛋白質 Y 會回到核仁的位置，暗示著在NB69細胞株之中蛋白質Y可能有突變或是缺失。我建立異種移植斑馬魚模式平台發現，野生型與S120G突變的核苷二磷酸激酶 A會促進NB69的侵入性，此結果與已往老鼠的實驗相似。然而，抑制了核苷二磷酸激酶 A ，即H118F突變會降低約40%的NB69 侵入性。此外，在班馬魚模式中野生型外源性蛋白質Y不會影響由NDPK-A 所引起的細胞侵入能力。這個發現說明蛋白質Y與核苷二磷酸激酶 A 有交互作用，但可能不參與在由核苷二磷酸激酶 A 調控的腫瘤轉移。

Metastasis remains as a major cause of death in cancer patients. Nucleotide diphosphate kinase A (abbreviated as NDP kinase A or NDPK-A), encoded by the nm23-H1 gene, is the first metastasis-associated protein identified in humans. NDPK-A acts as a metastasis suppressor in murine melanoma and human breast carcinoma. In contrast, our lab reported that NDPK-A functions as a metastasis promoter in neuroblastoma. In addition to overexpression, our lab has detected the S120G mutation of NDPK-A in 21% of patients with advanced stages of neuroblastoma. These NDPK-A alterations not only abrogate neuronal differentiation and increase invasiveness of neuroblastoma cells, but also promote tumor metastasis in mice. To date, the molecular mechanism of NDPK-A in metastasis is unclear. Previusly, our lab has identifies Protein Y as an NDPK-A interacting protein based on the yeast two-hybrid system. In this study, Protein Y was localized to the nucleolus of human cervical cancer HeLa and human embryonic kidney HEK293T cells. However, such nucleolar localization was not present in human neuroblastoma NB69 cells. Ectopic Protein Y was able to restore its nucleolar localization in NB69 cells. However, deletion of the N-terminus and not C-terminus of Protein Y abrogated its nucelolar localization in NB69 cells. Etoposide or serum deprivation did not significantly affect the subcellular localization of Protein Y and its deletion mutants in NB69 derivaties that expressed ectopic NDPK-A. The interaction of Protein Y and NDPK-A was confirmed by immuno-precipitation experiment. I further established a xenotransplant zebrafish model and found that expression of ectopic wild-type and S120G mutation of NDPK-A promoted the invasiveness of NB69 cells in zebrafish, similar to that reported in xenograft mice. When the phosphotransferase activity of NDPK-A was inactivated by the H118F mutation, NB69 cells reduced their invasiveness by ~40% in xenotransplated fish. However, ectopic expression of Protein Y did not affect NDPK-A mediated invasiveness of NB69 cells in zebrafish. These findings indicate that Protein Y interacted with NDPK-A, but might not participate in NDPK-A mediated tumor metastasis.

1. Brodeur GM, Sawada T, and T. Y, Neuroblastoma. Amsterdam: Elsevier Science B.V., 2000.
2. Alvarado, C.S., et al., Natural history and biology of stage A neuroblastoma: a Pediatric Oncology Group Study. J Pediatr Hematol Oncol, 2000. 22(3): p. 197-205.
3. Perez, C.A., et al., Biologic variables in the outcome of stages I and II neuroblastoma treated with surgery as primary therapy: a children's cancer group study. J Clin Oncol, 2000. 18(1): p. 18-26.
4. Schramm, A., et al., Prediction of clinical outcome and biological characterization of neuroblastoma by expression profiling. Oncogene, 2005. 24(53): p. 7902-12.
5. Feder, M.K. and F. Gilbert, Clonal evolution in a human neuroblastoma. J Natl Cancer Inst, 1983. 70(6): p. 1051-6.
6. Zaizen, Y., S. Taniguchi, and S. Suita, The role of cellular motility in the invasion of human neuroblastoma cells with or without N-myc amplification and expression. J Pediatr Surg, 1998. 33(12): p. 1765-70.
7. Almgren, M.A., et al., Nucleoside diphosphate kinase A/nm23-H1 promotes metastasis of NB69-derived human neuroblastoma. Mol Cancer Res, 2004. 2(7): p. 387-94.
8. Chang, C.L., et al., Nm23-H1 mutation in neuroblastoma. Nature, 1994. 370(6488): p. 335-6.
9. Brodeur, G.M., Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer, 2003. 3(3): p. 203-16.
10. Nakagawara, A. and M. Ohira, Comprehensive genomics linking between neural development and cancer: neuroblastoma as a model. Cancer Lett, 2004. 204(2): p. 213-24.
11. Berg, P., and Joklik, W. K., Nature (London), 1953. 172: p. 1008–1009.
12. Krebs, H.A., and Hems, R., Biochem. Biophys. Acta., 1953. 12: p. 172–180.
13. Postel, E.H., Multiple biochemical activities of NM23/NDP kinase in gene regulation. J Bioenerg Biomembr, 2003. 35(1): p. 31-40.
14. Heidbuchel, H., et al., Membrane-bound nucleoside diphosphate kinase activity in atrial cells of frog, guinea pig, and human. Circ Res, 1992. 71(4): p. 808-20.
15. Morera, S., et al., Mechanism of phosphate transfer by nucleoside diphosphate kinase: X-ray structures of the phosphohistidine intermediate of the enzymes from Drosophila and Dictyostelium. Biochemistry, 1995. 34(35): p. 11062-70.
16. Postel, E.H., NM23-NDP kinase. Int J Biochem Cell Biol, 1998. 30(12): p. 1291-5.
17. Lacombe, M.L., et al., The human Nm23/nucleoside diphosphate kinases. J Bioenerg Biomembr, 2000. 32(3): p. 247-58.
18. Cervoni, L., et al., Binding of nucleotides to nucleoside diphosphate kinase: a calorimetric study. Biochemistry, 2001. 40(15): p. 4583-9.
19. Freije, J.M., et al., Site-directed mutation of Nm23-H1. Mutations lacking motility suppressive capacity upon transfection are deficient in histidine-dependent protein phosphotransferase pathways in vitro. J Biol Chem, 1997. 272(9): p. 5525-32.
20. Bosnar, M.H., et al., Subcellular localization of A and B Nm23/NDPK subunits. Exp Cell Res, 2004. 298(1): p. 275-84.
21. Lambeth, D.O., et al., Characterization and cloning of a nucleoside-diphosphate kinase targeted to matrix of mitochondria in pigeon. J Biol Chem, 1997. 272(39): p. 24604-11.
22. Steeg, P.S., et al., Evidence for a novel gene associated with low tumor metastatic potential. J Natl Cancer Inst, 1988. 80(3): p. 200-4.
23. Ouatas, T., et al., Basic and translational advances in cancer metastasis: Nm23. J Bioenerg Biomembr, 2003. 35(1): p. 73-9.
24. Aryee, D.N., et al., Variability of nm23-H1/NDPK-A expression in human lymphomas and its relation to tumour aggressiveness. Br J Cancer, 1996. 74(11): p. 1693-8.
25. Oda, Y., et al., Comparison of histological changes and changes in nm23 and c-MET expression between primary and metastatic sites in osteosarcoma: a clinicopathologic and immunohistochemical study. Hum Pathol, 2000. 31(6): p. 709-16.
26. Nakamori, S., et al., Expression of nucleoside diphosphate kinase/nm23 gene product in human pancreatic cancer: an association with lymph node metastasis and tumor invasion. Clin Exp Metastasis, 1993. 11(2): p. 151-8.
27. Henriksson, K.C., et al., A fluorescent orthotopic mouse model for reliable measurement and genetic modulation of human neuroblastoma metastasis. Clin Exp Metastasis, 2004. 21(6): p. 563-70.
28. Wharton, R.P. and A.K. Aggarwal, mRNA regulation by Puf domain proteins. Sci STKE, 2006. 2006(354): p. pe37.
29. Wickens, M., et al., A PUF family portrait: 3'UTR regulation as a way of life. Trends Genet, 2002. 18(3): p. 150-7.
30. Wang, X., et al., Modular recognition of RNA by a human pumilio-homology domain. Cell, 2002. 110(4): p. 501-12.
31. Chritton, J.J. and M. Wickens, Translational repression by PUF proteins in vitro. RNA. 16(6): p. 1217-25.
32. Kuo, M.W., et al., A novel puf-A gene predicted from evolutionary analysis is involved in the development of eyes and primordial germ-cells. PLoS One, 2009. 4(3): p. e4980.
33. Edwards, T.A., et al., Structure of Pumilio reveals similarity between RNA and peptide binding motifs. Cell, 2001. 105(2): p. 281-9.
34. Gupta, Y.K., et al., Structures of human Pumilio with noncognate RNAs reveal molecular mechanisms for binding promiscuity. Structure, 2008. 16(4): p. 549-57.
35. Galgano, A., et al., Comparative analysis of mRNA targets for human PUF-family proteins suggests extensive interaction with the miRNA regulatory system. PLoS One, 2008. 3(9): p. e3164.
36. Cheong, C.G. and T.M. Hall, Engineering RNA sequence specificity of Pumilio repeats. Proc Natl Acad Sci U S A, 2006. 103(37): p. 13635-9.
37. Van Etten, J., et al., Human Pumilio proteins recruit multiple deadenylases to efficiently repress messenger RNAs. J Biol Chem. 287(43): p. 36370-83.
38. Brickner, A.G., et al., The immunogenicity of a new human minor histocompatibility antigen results from differential antigen processing. J Exp Med, 2001. 193(2): p. 195-206.
39. Chang, H.Y., et al., hPuf-A/KIAA0020 modulates PARP-1 cleavage upon genotoxic stress. Cancer Res, 2011. 71(3): p. 1126-34.
40. Fan, C.C., et al., Upregulated hPuf-A promotes breast cancer tumorigenesis. Tumour Biol, 2013. 171(3): p. 1126-34.
41. Lee, L.M., et al., The fate of human malignant melanoma cells transplanted into zebrafish embryos: assessment of migration and cell division in the absence of tumor formation. Dev Dyn, 2005. 233(4): p. 1560-70.
42. Haldi, M., et al., Human melanoma cells transplanted into zebrafish proliferate, migrate, produce melanin, form masses and stimulate angiogenesis in zebrafish. Angiogenesis, 2006. 9(3): p. 139-51.
43. Jo, D.H., et al., Orthotopic transplantation of retinoblastoma cells into vitreous cavity of zebrafish for screening of anticancer drugs. Mol Cancer. 12: p. 71.
44. Salerno, M., et al., Inhibition of signal transduction by the nm23 metastasis suppressor: possible mechanisms. Clin Exp Metastasis, 2003. 20(1): p. 3-10.
45. Marino, N., J.C. Marshall, and P.S. Steeg, Protein-protein interactions: a mechanism regulating the anti-metastatic properties of Nm23-H1. Naunyn Schmiedebergs Arch Pharmacol. 384(4-5): p. 351-62.
46. Matthay, K.K., et al., Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. Children's Cancer Group. N Engl J Med, 1999. 341(16): p. 1165-73.
47. Martin, T.D., et al., Ral and Rheb GTPase activating proteins integrate mTOR and GTPase signaling in aging, autophagy, and tumor cell invasion. Mol Cell. 53(2): p. 209-20.
48. Hailat, N., et al., High levels of p19/nm23 protein in neuroblastoma are associated with advanced stage disease and with N-myc gene amplification. J Clin Invest, 1991. 88(1): p. 341-5.
49. Leone, A., et al., Evidence for nm23 RNA overexpression, DNA amplification and mutation in aggressive childhood neuroblastomas. Oncogene, 1993. 8(4): p. 855-65.
50. Raska, I., P.J. Shaw, and D. Cmarko, Structure and function of the nucleolus in the spotlight. Curr Opin Cell Biol, 2006. 18(3): p. 325-34.
51. Corkery, D.P., G. Dellaire, and J.N. Berman, Leukaemia xenotransplantation in zebrafish--chemotherapy response assay in vivo. Br J Haematol. 153(6): p. 786-9.
52. Mohseny, A.B., P.C. Hogendoorn, and A.M. Cleton-Jansen, Osteosarcoma models: from cell lines to zebrafish. Sarcoma. 2012: p. 417271.
53. Yang, X.J., et al., A novel zebrafish xenotransplantation model for study of glioma stem cell invasion. PLoS One. 8(4): p. e61801.
54. Teng, Y., et al., Evaluating human cancer cell metastasis in zebrafish. BMC Cancer. 13: p. 453.
55. Bansal, N., et al., Enrichment of human prostate cancer cells with tumor initiating properties in mouse and zebrafish xenografts by differential adhesion. Prostate. 74(2): p. 187-200.
56. Drabsch, Y., et al., Transforming growth factor-beta signalling controls human breast cancer metastasis in a zebrafish xenograft model. Breast Cancer Res. 15(6): p. R106.