||NDP kinase A 需要藉由磷酸轉移酶活性調節神經母細胞瘤之侵入性
||The phosphotransferase activity is required for NDP kinase A mediated neuroblastoma invasiveness
||Institute of Molecular Medicine
腫瘤轉移造成約90% 癌症病人的死亡。而第一個被發現與腫瘤轉移相關的基因為nm23-H1，其所轉譯的蛋白質為NDP kinase A (NDPK-A)。此蛋白質在一些癌症例如乳癌是扮演一個抑制腫瘤轉移的角色，然而在神經母細胞瘤(Neuroblastoma)中其扮演的則為促進腫瘤轉移的角色。在我們實驗室先前的研究中發現，在有腫瘤轉移的神經母細胞瘤病人檢體中發現有14~30 %的病人其nm23-H1基因有過度表現並且有S120G的突變產生。這些與腫瘤轉移相關的NDPK-A之改變在人類NB69神經母細胞瘤細胞中，會增加細胞的侵入性並且會阻礙神經的分化。除此之外，在異種移植的老鼠實驗中這些NDPK-A的改變也會促進神經母細胞瘤的腫瘤轉移。而NDPK-A最被廣而所知的的功能為磷酸轉移酶，其在生物體內主要用於核酸的代謝並參與G 蛋白的訊息傳遞。然而，目前對於NDPK-A此酵素活性是否影響神經母細胞瘤的細胞侵入性還是未知的。與NDPK-AWT相比，NDPK-AS120G及NDPK-AH118F已知其分別的磷酸轉移酶酵素活性為50%及0% 。在內源性NDPK-A表現量低的NB69 細胞中，與載體轉染的控制組相比，穩定表現過量NDPK-A及NDPK-AS120G使得NB69細胞的在wound healing assay中移動能力有顯著增加；相反的，表現缺乏磷酸轉移酶酵素活性的NDPK-AH118F突變卻降低了NB69 細胞的移動能力。然而與NB69 細胞不同，另一株人類神經母細胞瘤SH-SY5Y 細胞表現內源性的NDPK-A。與控制組相比，當shRNA降低NDPK-A的表現時，SH-SY5Y細胞在wound healing assay中的移動能力則顯著的下降。維他命A酸(Retinoic acid) 可誘導神經細胞的分化並且在臨床上使用來治療神經母細胞瘤。經由維他命A酸誘導分化後，SH-SY5Y 細胞steady-state NDPK-A的表現量下降。然而表現過量NDPK-A的SH-SY5Y細胞其經由維他命A酸誘導分化後，神經分化的程度增加。相反的，表現NDPK-AS120G及NDPK-AH118F的SH-SY5Y細胞則無誘導神經分化的現象產生。除此之外，使用shRNA降低NDPK-A內源性表現的SH-SY5Y細胞其神經分化的現象則被抑制。面對腫瘤周圍環境氧氣及養分的缺乏，細胞的存活與否對於腫瘤轉移是重要且必須的。我們發現在異常表現NDPK-AH118F及 降低NDPK-A的表現時，細胞在降低細胞培養液中血清濃度模擬養分缺乏的條件下其細胞存活率是有顯著的下降。使用SH-SY5Y細胞經由shRNA篩選後，我們找出有八個可能參與在NDPK-A所調控之維他命A酸誘導神經分化的基因，而這些候選基因必須再進行進一步的驗證。我們的研究指出NDPK-A所調控的神經母細胞瘤的細胞移轉、存活及神經分化是需要其磷酸轉移酶的酵素活性。因此，NDPK-A的磷酸轉移酶活性及其候選基因所調控的神經分化可能可做為治療轉移性神經母細胞瘤病人的標靶。關鍵詞: 神經母細胞瘤; 神經分化; 腫瘤轉移; 維他命A酸; shRNA篩選
Neuroblastoma is the most common extracranial tumor in childhood, which arises from neural crest cells that fail to differentiate into the sympathetic nervous system. Similar to other cancers, neuroblastoma metastasis is a major cause of patient deaths. The first identified metastasis-associated gene is nm23-H1, which encodes for NDP kinase A (NDPK-A). This protein functions as a metastasis suppressor in certain cancers such as breast carcinoma, while acting as a metastasis promoter in other cancers such as neuroblastoma. Our and other labs have detected nm23-H1 gene amplification and S120G mutation of NDPK-A (NDPK-AS120G) in 14-30% of patients with advanced neuroblastoma. These metastasis-associated NDPK-A alterations increase cell invasiveness and abrogate neuronal differentiation of human neuroblastoma NB69 cells, as well as promote neuroblastoma metastasis in a xenograft mouse model. A well-known function of NDPK-A is the phosphotransferase activity, which is essential for nucleic acid metabolism and G protein signaling. However, it is currently unclear whether this enzymatic activity of NDPK-A contributes to neuroblastoma cell invasiveness. Relative to the wild type, NDPK-AS120G and NDPK-AH118F mutants are known to display ~50% and zero phosphotransferase activity in vitro, respectively. NB69-derived stable transfectants expressing ectopic wild type and NDPK-AS120G increased the migration ability, relative to the vector-transfected control, in the wound healing assay. In contrast, NDPK-AH118F mutant that lacks the phosphotransferase activity significantly decreased such ability. Unlike NB69 cells, human neuroblastoma SH-SY5Y cells expressed endogenous NDPK-A. Knockdown of NDPK-A expression by specific shRNAs decreased the wound-healing ability of SH-SY5Y cells, relative to the control. Retinoic acid (RA) has been used for treating neuroblastoma by inducing neuronal differentiation. Upon RA induction, the steady-state NDPK-A level decreased slightly in SH-SY5Y cells. Although ectopic expression of wild type NDPK-A enhanced RA-induced neuronal differentiation of SH-SY5Y cells, ectopic NDPK-AS120G or NDPK-AH118F mutant failed to do so. Furthermore, knockdown of endogenous NDPK-A expression reduced RA-induced neuronal differentiation in SH-SY5Y cells. Cell survival is essential for tumor metastasis, and we found that ectopic expression of NDPK-AH118F mutant in NB69 or knockdown of NDPK-A expression in SH-SY5Y dramatically decreased cell survival upon serum deprivation. After a shRNA screen in SH-SY5Y derivatives, eight candidate genes were identified to involve in NDPK-A mediated neuronal differentiation upon RA induction. Further validation of these candidate genes is warranted. Our findings suggest that the phosphotransferase activity is required for NDPK-A mediated neuroblastoma cell migration, survival and neuronal differentiation. Therefore, the phosphotransferase activity of NDPK-A and candidate genes involved in NDPK-A mediated neuronal differentiation might be therapeutic targets for treating patients with metastatic neuroblastoma.
Keywords: neuroblastoma; neuronal differentiation; metastasis; retinoic acid; shRNA screen
TABLE OF CONTENTS
TABLE OF CONTENTS VII
I. INTRODUCTION 1
I. 1 Tumor metastasis 1
I. 2 Neuroblastoma 1
I. 3 Human nm23 / NDP kinases 2
I. 4 Hypothesis 4
Ⅱ. MATERIALS AND METHODS 5
Ⅱ.1 Cell culture 5
Ⅱ.2 Transient transfection and lentiviral transduction 5
Ⅱ.3 Wound healing assay 6
Ⅱ.4 Neurite outgrowth analysis 6
Ⅱ.5 Western blot analysis 7
Ⅱ.6 Cell morphology and motility determined by time-lapse microscopy 7
Ⅱ.7 The MTT Assay 8
Ⅱ.8 A shRNA screen of potential NDPK-A targets involved in neurite outgrowth 8
Ⅲ. RESULTS 10
Ⅲ-1 The phosphotransferase activity is required for NDPK-A mediated NB69 cell migration 10
Ⅲ-2 Knockdown of NDPK-A expression reduces the migration of SH-SY5Y cells 11
Ⅲ-3 The steady-state level of NDPKA is reduced during retinoic acid induced neuronal differentiation of SH-SY5Y cells. 12
Ⅲ-4 NDPK-A with a full phosphotransferase activity enhances retinoic acid induced neuronal differentiation of SH-SY5Y cells 12
Ⅲ-5 NDPK-A increases the survival of neuroblastoma cells upon serum deprivation 13
Ⅲ-6 Screen of potential candidates involved NDPK-A mediated neuronal differentiation 14
Ⅲ-7 Candidate genes involved in NDPK-A mediated neuronal differentiation in SH-SY5Y cells 14
Ⅳ. DISCUSSION 16
Ⅴ. FIGURES 20
Figure 1. Loss of phosphotransferase activity reduced NDPK-A mediated migration of human neuroblastoma NB69 cells. 21
Figure 2. Knockdown of NDPK-A reduced the migration of human neuroblastoma SH-SY5Y cells. 22
Figure 3. The steady-state level of NDPKA is reduced during retinoic acid (RA)-induced neuronal differentiation of SH-SY5Y cells. 24
Figure 4. The phosphate transferase activity of NDPK-A is required for retinoic acid induced neuronal differentiation of SH-SY5Y cells. 25
Figure 5. NDPK-A is required for cell survival upon serum deprivation. 27
Figure 6. Selected potential targets of NDPK-A for the shRNA screen. 28
Figure 7. A procedure for automatic screening of potential targets of NDPK-A involved in RA-induced neuronal differentiation. 29
Figure 8. Graphical outputs of a 20-plate siRNA screen in SH-V cells as analyzed with the robust SSMD method with GUItars. 30
Figure 9. Graphical outputs of a 10-plate siRNA screen in SH-W cells as analyzed with the robust SSMD method with GUItars. 32
Ⅵ. TABLES 34
Table 1. Established SH-SY5Y-derived transfectants expressing ectopic NDPK-A variants. 34
Table 2. Gene candidates involved in RA-induced neuronal differentiation of SH-V cells. 35
Table 3. Gene candidates involved in RA-induced neuronal differentiation of SH-W cells expressing ectopic NDPK-A. 37
Table 4. Gene candidates involved in NDPK-A mediated neuronal differentiation of SH-SY5Y cells upon RA induction. 38
Ⅶ. REFERENCES 39
1. Fidler, I. J. The organ microenvironment and cancer metastasis. Differentiation. 2002; 70: 498–505.
2. Folkman, J. How is blood vessel growth regulated in normal and neoplastic tissue ? G.H.A. Clowes memorial award lecture. Cancer Res. 1986; 46: 467–743.
3. Nicolson, G. L. Cancer metastasis: tumour cell and host organ properties important in metastasis to specific secondary sites. Biochem. Biophys. Acta. 1988; 948: 175–224 .
4. Kristjan R. Jessen, Rhona Mirsky. The origin and development of glial cells in peripheral nerves. Nat Rev Neurosci. 2005; 6: 671-682.
5. Howman-Giles R, Shaw PJ, Uren RF, Chung DK. Neuroblastoma and other neuroendocrine tumors. Semin Nucl Med. 2007; 37(4): 286-302.
6. Maris JM, Hogarty MD, Bagatell R, Cohn SL. Neuroblastoma. Lancet. 2007; 369: 2106-2120.
7. Alvarado CS, London WB, Look AT, et al. Natural history and biology of stage A neuroblastoma: a Pediatric Oncology Group study. J Pediatr Hematol Oncol. 2000; 22: 197-205.
8. Perez CA, Matthay KK, Atkinson JB, 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: 18-26.
9. Matthay KK, Villablanca JG, Seeger RC, 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: 1165-73.
10. Nakagawara A, Ohira M: Comprehensive genomics linking between neural development and cancer: neuroblastoma as a model. Cancer Lett. 2004; 204: 213-224
11. Postel, E. H., Berberich, S. J., Flint, S. J., and Ferrone, C. A. Human c-myc transcription factor PuF identified as nm23-H2 nucleoside diphosphate kinase, a candidate suppressor of tumor metastasis, Science. 1993; 261: 478-480.
12. Brodeur GM, Seeger RC, Schwab M, Varmus HE, Bishop JM. Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage. Science 1984; 224(4653):1121-4.
13. Brodeur GM. Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer. 2003; 3: 203-16.
14. Milon, L., Meyer, P., Chiadmi, M., Munier, A., Johansson, M., Karlsson, A., Lascu, I., Capeau, J., Janin, J., and Lacombe, M. L. The human nm23-H4 gene product is a mitochondrial nucleoside diphosphate kinase, J. Biol. Chem. 2000; 75: 4264-14272.
15. Bevilacqua G, Sobel ME, Liotta LA, Steeg PS. Association of low nm23 RNA levels in human primary infiltrating ductal breast carcinomas with lymph node involvement and other histopathological indicators of high metastatic potential. Cancer Res. 1989; 49(18):5185–5190.
16. Nakayama T, Ohtsuru A, Nakao K, Shima M, Nakata K, Watanabe K, Ishii N, Kimura N, Nagataki S. Expression in human hepatocellular carcinoma of nucleoside diphosphate kinase, a homologue of the nm23 gene product. J Natl Cancer Inst. 1992; 84(17):1349–1354.
17. John Gearhart, Evanthia E. Pashos and Megana K. Prasad. Pluripotency Redux — Advances in Stem-Cell Research. N Engl J Med. 2007; 357:1469-1472
18. Facchini LM, Penn LZ. The molecular role of Myc in growth and transformation: recent discoveries lead to new insights. FASEB J. 1998 ;12(9):633-51.
19. Chi V. Dang. c-Myc Target Genes Involved in Cell Growth, Apoptosis, and Metabolism. Mol. Cell. Biol. 1999;19: 1-11.
20. Dang CV, Resar LM, Emison E, Kim S, Li Q, Prescott JE, Wonsey D, Zeller K. Function of the c-Myc oncogenic transcription factor. Exp Cell Res. 1999; 253(1):63-77.
21. Nau, M. M . , Brooks, B. J . , Battey, J . , Suasville, E. , Gazdar, A . F . , et al. L-myc, a new myc-related gene amplified and expressed in human small cell lung cancer. Nature. 1985; 318:69-73
22. Zimmerman KA, Yancopoulos GD, Collum RG, Smith RK, Kohl NE, Denis KA, Nau MM, Witte ON, Toran-Allerand D, Gee CE. Differential expression of myc family genes during murine development. Nature. 1986; 319(6056): 780-3.
23. Bhatia, K., Huppi, K., Spangler, G., Siwarski, D., Iyer, R., and Magrath, I. Point mutations in the c-Myc transactivation domain are common in Burkitt’s lymphoma and mouse plasmacytomas. Nat. Genet. 1993; 5: 56–61
24. Munier, A., Feral, C., Milon, L., Pinon, V. P., Gyapay, G., Capeau, J., Guellaen, G., and Lacombe, M. L. A new human nm23homologue (nm23-H5) specifically expressed in testis germinal cells. FEBS Lett. 1998; 434: 289-294.
25. Sadek, C. M., Damdimopoulos, A. E., Pelto-Huikko, M., Gustafsson, J. A., Spyrou, G., and Miranda-Vizuete, A. Sptrx-2, a fusion protein composed of one thioredoxin and three tandemly repeated NDP-kinase domains is expressed in human testis germ cells, Genes Cells. 2001; 6: 1077-1090.
26. Mehus, J. G., Deloukas, P., and Lambeth, D. O. NME6: A new member of the nm23/nucleoside diphosphate kinase gene family located on human chromosome 3p21.3. Hum. Gene. 1999; 104: 454-459
27. Nakayama H, Yasui W, Yokozaki H, Tahara E. Reduced expression of nm23 is associated with metastasis of human gastric carcinomas. Jpn J Cancer Res. 1993; 84(2): 184–190.
28. Leone A, Seeger RC, Hong CM, et al. Evidence for nm23 RNA overexpression, DNA amplification and mutation in aggressive childhood neuroblastomas. Oncogene. 1993; 8: 855-65.
29. Hailat N, Keim DR, Melhem RF, 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: 341-5.
30. Oda Y, Naka T, Takeshita M, 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: 709-16.
31. Nakamori S, Ishikawa O, Ohhigashi H, 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: 151-8.
32. Nakamori S, Ishikawa O, Ohigashi H, et al. Clinicopathological features and prognostic significance of nucleoside diphosphate kinase/nm23 gene product in human pancreatic exocrine neoplasms. Int J Pancreatol. 1993; 14: 125-33.
33. Chang CL, Zhu XX, Thoraval DH, et al. NM23-H1 mutation in neuroblastoma. Nature. 1994; 370: 335-6.
34. Mougneau, E., Lemieux, L., Rassoulzadegan, M., and Cuzin, F. Biological activities of v-myc and rearranged c-myc oncogenes in rat fibroblast cells in culture. Proc. Natl. Acad. Sci. USA. 1984; 81: 5758–5762
35. Almgren MA, Henriksson KC, Fujimoto J, Chang CL.Nucleoside diphosphate kinase A/ nm23-H1 promotes metastasis of NB69-derived human neuroblastoma. Mol Cancer Res. 2004; 2: 387-94.
36. Ma, D., McCorkle, J. R., and Kaetzel, D. M. The metastasis suppressor NM23-H1 possesses 3'-5'exonuclease activity, J. Biol. Chem. 2004; 279: 18073-18084.
37. Qingbei Zhang, Joseph R. McCorkle, Marian Novak, Mengmeng Yang and David M. Kaetzel. Metastasis suppressor function of NM23-H1 requires its 3'-5' exonuclease activity. Int. J. Cancer. 2011; 128: 40–50.
38. Wagner PD, Vu ND. Phosphorylation of ATP-citrate lyase by nucleoside diphosphate kinase. J Biol Chem. 1995; 270: 21758–64.
39. Postel EH. NM23-NDP kinase. Int J Biochem Cell Biol. 1998; 30: 1291-1295.
40. Chang CL, Strahler JR, Thoraval DH, Qian MG, Hinderer R, and Hanash SM. A nucleoside diphosphate kinase A (nm23-H1) serine120→glycine substitution in advanced stage neuroblastoma affects enzyme stability and alters protein-protein interaction. Oncogene. 1996; 12: 659-667.
41. Postel EH and Ferrone CA. Nucleoside diphosphate kinase enzyme activity of NM23-H2/PuF is not required for its DNA binding and in vitro transcriptional functions. J Biol Chem. 1994; 269: 8627-8630.
42. Natascia Marino, Jean-Claude Marshall, and Patricia S. Steeg. Protein-protein interactions: a mechanism regulating the anti-metastatic properties of Nm23-H1. Naunyn-Schmiedeberg"s Arch Pharmacol. 2011; 384: 351–362.
43. Masamitsu Tanaka, Riuko Ohashi,Ritsuko Nakamura, Kazuya Shinmura, Takaharu Kamo, Ryuichi Sakai and Haruhiko Sugimura. Tiam1 mediates neurite outgrowth induced by ephrin-B1 and EphA2. EMBO J. 2004; 23: 1075–1088
44. Murakami M, Meneses PI, Knight JS, Lan K, Kaul R, Verma SC, Robertson ES. Nm23-H1 modulates the activity of the guanine exchange factor Dbl-1. Int J Canc. 2008; 123(3): 500–510.
45. Marino N, Marshall J-C and Steeg PS. Protein–protein interactions: a mechanism regulating the anti-metastatic properties of Nm23-H1. Naunyn-Schmiedeberg's Arch Pharmacol. 2011; 384: 351–362
46. Feder MK, Gilbert F. Clonal evolution in a human neuroblastoma. J Natl Cancer Inst. 1983; 70: 1050-1056.
47. Almgren MA, Henriksson KC, Fujimoto J, and Chang CL. Nucleoside diphosphate kinase A/nm23-H1 promotes metastasis of NB69-derived human neuroblastoma. Mol Cancer Res. 2004; 2: 387-94.
48. Otsuki Y, Tanaka M, Yoshii S, et al. Tumor metastasis suppressor nm23H1 regulates Rac1 GTPase by interaction with Tiam1. Proc Natl Acad Sci USA. 2001; 98:4385-90.
49. Habets GG, Scholtes EH, Zuydgeest D, et al. Identification of an invasioninducing gene, Tiam-1, that encodes a protein with homology to GDP-GTP exchangers for Rho-like proteins. Cell. 1994;77:537-49.
50. Eve-Ellen Govek, Sarah E. Newey, and Linda Van Aelst. The role of the Rho GTPases in neuronal development. Genes Dev. 2005; 19:1–49
51. Steeg PS, Bevilacqua G, Kopper L, Thorgeirsson UP, Talmadge JE, Liotta LA, et al. Evidence for a novel gene associated with low tumor metastatic potential. J Natl Cancer Inst. 1988; 80: 200-4.
52. Biggs J, Tripoulas N, Hersperger E, Dearolf C, Shearn A. Analysis of the lethal interaction between the prune and Killer of prune mutations of Drosophila. Genes Dev. 1988 ; 2: 1333–1343.
53. MacDonald, N., Freije, J., Stracke, M., Manrow, R., and Steeg, P. Site directed mutagenesis of nm23-H1: mutation of proline 96 or serine 120 abrogates its motility inhibitory activity upon transfection into human breast carcinoma cells. J Biol Chem. 1996; 271: 25107–25116.
54. Reynolds CP, Lemons RS. Retinoid therapy of childhood cancer. Hematol Oncol Clin North Am. 2001; 15: 867-910.
55. Pahlman S, Ruusala AI, Abrahamsson L, Mattsson ME, Esscher T. Retinoic acid-induced differentiation of cultured human neuroblastoma cells: a comparison with phorbolester- induced differentiation. Cell Differ. 1984; 14: 135–44.
56. Backer MV, Kamel N, Sandoval C, et al. Overexpression of NM23-1 enhances responsiveness of IMR-32 human neuroblastoma cells to differentiation stimuli. Anticancer Res. 2000; 20: 1743-9.
57. Veas-Perez de Tudela M1, Delgado-Esteban M, Cuende J, Bolaños JP, Almeida A.. Human neuroblastoma cells with MYCN amplification are selectively resistant to oxidative stress by transcriptionally up-regulating glutamate cysteine ligase. J Neurochem. 2010; 113(4): 819-25.
58. Goktug AN, Ong SS, Chen T. GUItars: A GUI Tool for Analysis of High-Throughput RNA Interference Screening Data. PLoS ONE. 2012; 7(11): e49386.
59. Zhang XD, A new method with flexible and balanced control of false negatives and false positives for hit selection in RNA interference high-throughput screening assays. J Biomol Screen. 2007; 12: 645–655.