||Protein Y counteracts the enhancing ability of NDPK-A in neuroblastoma cell invasiveness
||Institute of Molecular Medicine
人類的核苷二磷酸激酶A (Nucleotide diphosphate kinase A, NDPK-A) 是第一個被發現與癌症轉移有相關的蛋白質，其由 nm23-H1 基因所編碼。核苷二磷酸激酶 A在小鼠的黑色素瘤以及人類的乳腺癌中扮演著抑制腫瘤轉移的角色。相反的，我們實驗室發現核苷二磷酸激酶 A (Nucleotide diphosphate kinase A, NDPK-A)與其基因變異核苷二磷酸激酶AS120G (NDPK-AS120G)是促進兒童神經母細胞瘤的轉移。到目前為止，核苷二磷酸激酶 A 對腫瘤轉移的機制尚不清楚。我們實驗室最進發現蛋白質Y (Protein Y)與核苷二磷酸激酶A交互作用，但無法與核苷二磷酸激酶AS120G交互作用。在本研究發現在三株神經母細胞瘤之人類細胞株中，意料發現蛋白質Y的mRNA突變產生stop codon因而表現不正常的蛋白質Y。進一步過量表現蛋白質Y在神經母細胞瘤NB69 中，同時表達核苷二磷酸激酶 A或是變異核苷二磷酸激酶AS120G 進行實驗。
Trans-well試驗中，核苷二磷酸激酶 A顯著提高細胞在二度空間的遷移能力，但蛋白質 Y大幅抑制此能力，而兩者則互相增減此能力。但是蛋白質 Y並不抑制變異核苷二磷酸激酶AS120G 遷移能力。在clonogenic試驗中，核苷二磷酸激酶A顯著提高兒童神經母細胞瘤的colony formation能力，但是蛋白質Y則降低此能力，而兩者則彼此增減此能力。當knockdown 蛋白質Y 發現部分回復侵襲轉移能力，相似現象發現在當蛋白質Y的N端被刪除後，僅缺乏N端的蛋白質Y則喪失抑制核苷二磷酸激酶A之能力。並且在斑馬魚 transplantation model 發現到，蛋白質Y不能抑制核苷二磷酸激酶AS120G神經母細胞瘤的侵襲能力。本研究在斑馬魚的發育過程中，利用whole mount in-situ hybridization 發現到nme2b.2 與蛋白質Y 似乎並不參與在斑馬魚神經分化成交感神經的過程。總結以上結果，蛋白質Y具有抑制核苷二磷酸激酶 A 所提高的侵襲轉移能力，但對於核苷二磷酸激酶AS120G 並不能有效的抑制。並且推斷三株神經母細胞瘤可能因蛋白質Y無法正常表現，因而無法抑制核苷二磷酸激酶A對腫瘤轉移的促進能力。
Nucleotide diphosphate kinase A (NDPK-A) is encoded by the nm23-H1/nme1 gene, and acts as a metastasis promoter in neuroblastoma. Overexpression or S120G mutation of NDPK-A found in advanced neuroblastoma increases both invasive and metastatic potential of neuroblastoma cells. However, the molecular mechanism by which NDPK-A promotes neuroblastoma metastasis remains poorly understood. Our lab has found a novel protein, termed Protein Y, that interacted with NDPK-A, but not NDPK-AS120G. In this study, non-sense mutations of the Protein Y mRNA were found in human neuroblastoma NB69, IMR32 and SH-SY5Y cell lines. In stable transducents established from NB69 cells, ectopic expression of NDPK-A or NDPK-AS120G increased cell migration by more than two folds, whereas ectopic expression of Protein Y decreased cell migration by 47% in the trans-well assay. Co-expression of Protein Y completely abolished the migration-enhancing ability of NDPK-A. In contrast, Protein Y displayed little effect on the migration-enhancing ability of NDPK-AS120G. In the clonogenic assay, Protein Y reduced the colony number by 17%, whereas NDPK-A or NDPK-AS120G increased the colony number by 20-25%. Co-expressed Protein Y almost completely abolished the colony-enhancing ability of NDPK-A, but not NDPK-AS120G. While knocking down Protein Y expression partially restored the invasiveness-enhancing ability of NDPK-A, deletion of the N-terminus abolished the ability of Protein Y to counteract the invasiveness-enhancing ability of NDPK-A and, to a lesser degree, NDPK-AS120G. In a zebrafish transplantation model, Protein Y did not seem to affect the extravasation ability of NB69 cells, nor suppress the extravasation-enhancing ability of NDPK-AS120G. By whole mount in-situ hybridization, zebrafish NDPK-A and Protein Y orthologs, namely nme2b.2 and Protein Y, did not appear to participate in the differentiation of neural crest into sympathetic neurons, relevant to neuroblastoma. In conclusion, Protein Y counteracted the ability of NDPK-A and, to a lesser degree, NDPK-AS120G in promoting the invasiveness of NB69 cells. However, such a counteracting ability of Protein Y is likely abrogated by mutation-caused truncations, as detected in its mRNA in human neuroblastoma cell lines examined.
1 Introduction 1
1.1 Tumor metastasis 1
1.2 Neuroblastoma 1-2
1.3 NDPK-A 2
1.4 The Puf family 2-3
1.5 Protein Y 3
1.6 Zebrafish and applications 3-4
1.7 Zebrafish orthologs of human NDPK-A and Protein Y 4
2 Objectives & Specific Aims 5
3 Materials and Methods 6
3.1 Cell lines and culture 6
3.2 Generation of stable transducents that express NDPK-A and/or Protein Y variants 6-7
3.3 Knockdown of Protein Y expression in stNB-W cells 7
3.4 Immunoblotting 7-8
3.5 Reverse transcription (RT)-PCR 8-9
3.6 Genomic DNA extraction and amplification 9
3.7 The MTT assay 9
3.8 Cell proliferation rate determination 10
3.9 The clonogenic assay 10
3.10 Wound healing and trans-well assays 10-11
3.11 Zebrafish maintenance 11
3.12 Cloning and generating zebrafish Protein Y and nme2b2 probes 11-12
3.13 Whole mount in-situ hybridization (WISH) 12
3.14 Zebrafish transplantation 12-13
3.15 Immunocytochemistry 13
4.1 N-terminus deletion of Protein Y does not affect its stability in human neuroblastoma NB69 cells 14
4.2 Altered subcellular location of Protein Y in certain human neuroblastoma cell lines 14-15
4.3 The effects of Protein Y and/or NDPK-A variants on the migration of NB69 derivatives 15-16
4.4 The effects of Protein Y and/or NDPK-A variants on the survival of NB69 derivatives 16-17
4.5 The effects of Protein Y and /or NDPK-A variants on human neuroblastoma cell proliferation 18
4.6 Mutations occur in Protein Y mRNA in human neuroblastoma cell lines 18-19
4.7 Knockdown of Protein Y expression decrease the inhibition of cell colony formation and migration ability 19-20
4.8 Effects of Protein Y on the invasiveness of human neuroblastoma NB69 cells in a zebrafish transplantation model 20
4.9 Spatial-temporal expression of zebrafish orthologs of human NDPK-A and Protein Y during early development 21
5 Discussion 22-24
6 References 25-27
7 Table 1. The doubling time of NB69 derivatives expressing NDPK-A and/or Protein Y variants 28
8.1 Figure 1 29-30
8.2 Figure 2 31
8.3 Figure 3 32
8.4 Figure 4 33
8.5 Figure 5 34-35
8.6 Figure 6 36
8.7 Figure 7 37
8.8 Figure 8 38-39
8.9 Figure 9 40
8.10 Figure 10 41-42
8.11 Figure 11 43-44
8.12 Figure 12 45-47
9.1 Appendix 1. List of antibodies used in this study 48
9.2 Appendix 2. List of buffers for whole mount in-situ hybridization 48
9.3 Appendix 3. PCR primers for amplifying exons 2, 3, 4, 5 and 18 in the human Protein Y gene 49
9.4 Appendix 4. Sequence alignment of RT-PCR products of Protein Y mRNA in five human cancer cell lines 50-51
9.5 Appendix 5. Alignment the total RT-PCR translation amino acid sequences of the five human cancer cell Protein Y 52-54
9.6 Appendix 6. Sequence alignment of Protein Y mRNA after cloning of RT-PCR products in four human cell lines 55-58
9.7 Appendix 7. IMR32 Protein Y mRNA 5’ region direct sequencing PCR product and two different TA clone construct sequencing found mutation 59
9.8 Appendix 8. Sequence alignment of exons 2, 3, 4 and 18 in Protein Y gene in four human cell lines 60
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