Title Page
ABSTRACT
Contents
LIST OF ABBREVIATIONS 14
INTRODUCTION 17
Ⅰ. SAMHD1 17
1. Functions of SAMHD1 17
2. Localization of SAMHD1 20
3. SAMHD1 in cancer 21
Ⅱ. Renal cell carcinoma 25
Ⅲ. Cancer cell migration 28
1. Epithelial-mesenchymal transition (EMT) 28
2. Focal adhesion Kinase (FAK) 31
3. Rho GTPase activity 34
4. Lamellipodia 38
5. Endocytosis 40
PURPOSE OF THIS STUDY 43
MATERIALS AND METHODS 44
1. Cell culture and reagents 44
2. siRNA and plasmid construction 44
3. Transfection and stable cell line construction 45
4. Antibodies 45
5. Immunoblotting and immunoprecipitation 46
6. G-LISA based small GTPase activity measurement 47
7. Wound healing assay 47
8. Transwell migration and invasion assay 48
9. RNA isolation and mRNA analysis 48
10. Immunocytochemistry 48
11. Immunohistochemistry 49
12. The Cancer Genome Atlas (TCGA) transcriptome data analysis 50
13. Survival analysis for RCC subtypes 51
14. ccRCC clinical specimen collection 51
15. Statistical analysis 52
RESULTS 53
1. High expression of SAMHD1 is associated with poor prognosis in ccRCC 53
2. SAHMD1 potentiates migration and invasion of ccRCC cells 64
3. SAMHD1 enhances ccRCC cell mesenchymal characteristics to increase migration capacity 71
4. SAMHD1 promotes lamellipodia formation in ccRCC cells 77
5. SAMHD1 increases small GTPase activities, especially of Rac1 activity 84
6. SAMHD1 bind with cortactin to promotes endocytosis 92
7. SAMHD1 facilitates ccRCC cell migration via endocytosis 103
8. SAMHD1 activates endosomal FAK signaling 111
DISCUSSION 123
REFERENCES 129
국문초록 142
Table 1. SAMHD1 mutations in RCC. 63
Figure 1. Structure of SAMHD1 and its functions as dNTPase. 19
Figure 2. SAMHD1 mutation in various types of cancer. 23
Figure 3. SAMHD1 hydrolyzes nucleoside analogs. 24
Figure 4. Histological subtypes of RCC. 27
Figure 5. The progression of EMT. 30
Figure 6. FAK mediates focal adhesion formation. 33
Figure 7. Activation and inactivation cycles of Rho GTPases 36
Figure 8. Rho GTPases in the reorganization of cytoskeleton. 37
Figure 9. Cortactin mediates lamellipodia formation. 39
Figure 10. Endocytosis promotes cancer cell migration. 41
Figure 11. The process of endocytosis and recycling. 42
Figure 12. Analysis of SAMHD1 mRNA expression in various cancer types. 55
Figure 13. Analysis of SAMHD1 mRNA expression by histological RCC subtypes. 56
Figure 14. Analysis of SAMHD1 mRNA expression in paired tissues by RCC subtypes 57
Figure 15. Survival analysis of SAMHD1 in ccRCC patients. 58
Figure 16. Survival analysis of SAMHD1 in PRCC patients. 59
Figure 17. Survival analysis of SAMHD1 in chRCC patients. 60
Figure 18. Histological analysis of SAMHD1 expression in ccRCC patient tissues. 61
Figure 19. SAMHD1 protein expression in ccRCC patients. 62
Figure 20. Comparison of basal SAMHD1 level in ccRCC cell lines. 65
Figure 21. SAMHD1 expression on cell proliferation 66
Figure 22. Wound healing assay with SAMHD1 knockdown. 67
Figure 23. Wound healing assay with SAMHD1 overexpression. 68
Figure 24. Transwell migration assay analysis with SAMHD1 knockdown and overexpression. 69
Figure 25. Invasion assay analysis with SAMHD1 knockdown and overexpression. 70
Figure 26. SAMHD1 changes the cell morphology to the mesenchymal characteristics. 72
Figure 27. Analysis of EMT-related gene expressions with SAMHD1 knockdown in ccRCC cell lines. 73
Figure 28. Analysis of EMT-related gene expressions with SAMHD1 overexpression in ccRCC cell lines. 74
Figure 29. Analysis of EMT-related gene from ccRCC from TCGA dataset. 75
Figure 30. E-cadherin expression in 20 paired of ccRCC patients. 76
Figure 31. GO analysis in cellular component subcategory. 79
Figure 32. GSEA analysis related to cytoskeletal change. 80
Figure 33. Immunofluorescence analysis for paxillin. 81
Figure 34. SAMHD1 knockdown impaired lamellipodium formation. 82
Figure 35. SAMHD1 contributes to lamellipodium development. 83
Figure 36. GO analysis for molecular function category. 86
Figure 37. GSEA analysis related to GTPase bindings. 87
Figure 38. SAMHD1 facilitates Rac1 activity. 88
Figure 39. SAMHD1 enhances cdc42 activity. 89
Figure 40. SAMHD1 mediated cell migration is inhibited by Rac1 inactivation. 90
Figure 41. Inhibition of Rac1 suppresses SAMHD1-induced cell migration. 91
Figure 42. GSEA analysis related to Endocytosis. 94
Figure 43. EEA1 is decreased in SAMHD1 downregulated cells. 95
Figure 44. SAMHD1 facilitates EEA1 vesicle formation. 96
Figure 45. Localization of SAMHD1 in the nucleus and few in the cytoplasm and cell membrane. 97
Figure 46. SAMHD1 mainly localizes in the nucleus and partially in the cytoplasm and edge of membrane. 98
Figure 47. SAMHD1 colocalizes with cortactin in the cytoplasm and in the lamellipodia. 99
Figure 48. Exogenous and endogenous SAMHD1 binds with cortactin. 100
Figure 49. SAMHD1 increases cortactin phosphorylation. 101
Figure 50. SAMHD1 and phosphorylated-cortactin are increased in ccRCC patient tumor tissues. 102
Figure 51. SAMHD1 increases and colocalizes with EEA1. 105
Figure 52. SAMHD1-mediated endosome complex formation is sustained by endocytosis inhibition. 106
Figure 53. Cortactin phosphorylation contributes to SAMHD1-mediated endosomal complex formation. 107
Figure 54. The ability of SAMHD1-mediated cell migration is inhibited by dynasore treatment. 108
Figure 55. SAMHD1-induced endocytosis promotes ccRCC cell migration. 109
Figure 56. Rac1 is downstream of endocytosis signaling. 110
Figure 57. SAMHD1 activates FAK phosphorylation. 113
Figure 58. SAMHD1-mediated endocytosis is required for FAK phosphorylation. 114
Figure 59. EEA1 induced by SAMHD1 is impaired by FAK inactivation. 115
Figure 60. Inhibition of FAK reduces the SAMHD1-mediated endosomal complex formation. 116
Figure 61. SAMHD1 mediated FAK signaling is necessary for cortactin phosphorylation. 117
Figure 62. SAMHD1-mediated cell migration was sustained by FAK inhibition. 118
Figure 63. SAMHD1-induced endosomal FAK signaling promotes ccRCC cell migration. 119
Figure 64. Expression of SAMHD1 and phosphorylated-FAK are increased in ccRCC patients' samples. 120
Figure 65. Expression of phosphorylated-SAMHD1 in ccRCC patients' samples. 121
Figure 66. SAMHD1 promotes ccRCC metastasis through endosomal FAK signaling activation. 122