Title Page
Contents
LIST OF ABBREVIATIONS 15
ABSTRACT 16
Ⅰ. INTRODUCTION 18
1. Cancer stem cells 18
1.1. CSC concept 18
1.2. CSC markers 19
1.3. CSC properties 20
1.4. Intrinsic factors of CSC development 22
2. HIF signaling in cancer 23
2.1. HIFs and their regulatory components 23
2.2. Target genes activation by HIFs 26
2.3. Activation of HIFs in cancer 28
2.4. Differential roles of HIF-1α and HIF-2α in cancer 31
2.5. HIF-2α in clear-cell renal cell carcinoma 33
3. NRF2 signaling in cancer 34
3.1. KEAP1-NRF2 system 34
3.2. Activation of NRF2 in cancer 37
3.3. NRF2 and CSC phenotypes 40
3.4. NRF2 and hypoxia 42
4. Association of HIFs and NRF2 signaling in CSC phenotypes 45
5. Purpose of the study 48
Ⅱ. MATERIALS AND METHODS 49
1. Chemical and reagents 49
2. Plasmids 50
3. Cell culture 50
4. Transfection of siRNA, microRNA mimic and inhibitor 52
5. Cell counting 52
6. WST assay 53
7. Immunoblot analysis 53
8. Total RNA isolation and quantitative PCR (qPCR) analysis 54
9. Isolation of nuclear fraction 55
10. Soft agar colony formation assay 56
11. Transwell migration assay 56
12. Sphere culture 57
13. Animal experiments 57
14. Luciferase reporter assay 59
15. HIF-2α promoter analysis 60
16. Clinical analysis using public datasets 60
17. Statistical analysis 61
Ⅲ. RESULTS 63
1. NRF2 level is associated with HIF-2α induced cancer stem cell phenotypes under chronic hypoxia 63
Summary 64
1.1. Chronic hypoxia induces aggressive phenotypes and HIF-2α accumulation in colorectal cancer cells 66
1.2. HIF-2α mediates CSC-like properties under chronic hypoxic condition 68
1.3. NRF2-silencing suppresses HIF-2α elevation under chronic hypoxic condition 73
1.4. NRF2-silencing suppresses CSC-like properties in chronic hypoxic environment 76
1.5. NRF2 inhibitor brusatol suppresses CSC-like properties in chronic hypoxic environment 80
1.6. NRF2 levels is positively correlated with HIF-2α level 81
1.7. NRF2-silencing-induced miR-181a-2-3p inhibits HIF-2α elevation under chronic hypoxia 85
1.8. NRF2-silencing-induced miR-181a-2-3p suppresses hypoxia-induced CSC-like properties 88
1.9. miR-181a-2-mediated HIF-2α inhibition suppresses CSC-like properties in NRF2-silenced colorectal cancer cells 91
2. NRF2 regulates HIF-2α-mediated cancer stem-like phenotypes in clear-cell renal cell carcinoma 94
Summary 95
2.1. Upregulation of HIF-2α and stem cell markers in ccRCC is accompanied by increase of NRF2 97
2.2. HIF-2α silencing reduces the stem-cell like properties in ccRCC 101
2.3. NRF2-knockdown suppresses HIF-2α-induced stem cell-like phenotypes of ccRCC 106
2.4. NRF2 directly regulates HIF-2α expression 112
2.5. Inhibitors of NRF2 reduce HIF-2α and subsequent CSC-like properties 114
Ⅳ. DISCUSSION 119
Ⅴ. CONCLUSION 132
REFERENCES 133
APPENDICES 147
Appendix 1. Declaration of Permission 147
Appendix 2. Permission Letter 148
Appendix 3. List of Publication and Presentation Award 150
국문초록 155
Table 1. Cell lines used in this study 51
Table 2. Sequences of silencing RNAs 52
Table 3. Primer sequences for qPCR analysis of human genes and miRNAs 55
Table 4. Common microRNAs, which are upregulated (〉1.5 fold) both in HCT116 and HT29, following a stable silencing of NRF2. Hypoxia-related target... 87
Ⅰ. INTRODUCTION 12
Figure 1. Cancer stem cells properties 21
Figure 2. Structure and regulatory component of HIFs 24
Figure 3. Mechanism of target gene activation by HIFα under hypoxia 27
Figure 4. Specific roles of HIF-1α and HIF-2α in tumors 33
Figure 5. Regulation of KEAP1-NRF2 system in basal and oxidative stress condition 35
Figure 6. NRF2 activation in cancer 39
Figure 7. NRF2 induces cancer stem cell properties 42
Ⅲ. RESULTS 12
Figure 8. HIF-2α is increased following prolonged hypoxia. 67
Figure 9. Inhibition of HIF-2α decrease CSC markers and cell growth in prolonged hypoxia. 69
Figure 10. HIF-2α mediates cancer stem cells (CSCs)-like properties under prolonged hypoxia. 70
Figure 11. HIF-2α siRNA #2 shows reduction of CSC markers and phenotypes under prolonged hypoxia. 71
Figure 12. Reduce mRNA level of CSC markers in HIF-2α-silenced HT29 cells. 72
Figure 13. HIF-1α expression has less effect on CSC phenotypes under chronic hypoxia. 73
Figure 14. NRF2 silencing suppresses prolonged hypoxia-induced HIF-2α elevation. 74
Figure 15. NRF2 silencing suppresses HIF-2α-induced CSC markers. 75
Figure 16. NRF2 silenced-HT29 cells show reduced HIF-2α-induced CSC markers. 76
Figure 17. NRF2 mediates prolonged hypoxia-induced CSC-like properties. 77
Figure 18. HIF-2α introduction in NRF2-silenced cells restores CSC phenotypes. 78
Figure 19. NRF2-silencing impairs tumor growth in vivo. 79
Figure 20. Brusatol inhibits NRF2 signaling in HCT116 cells. 80
Figure 21. NRF2 inhibition by brusatol repressed HIF-2α induced-CSC-like properties. 81
Figure 22. NRF2 level is positively associated with HIF-2α levels. 82
Figure 23. KEAP1-knockdown cells increase HIF-2α levels and CSC phenotypes in chronic hypoxia. 83
Figure 24. Activation of NRF2 by bardoxolone alters HIF-2α level and migration in prolonged hypoxia. 84
Figure 25. Identification of miR-181a-2 and miR-2278 to regulate HIF-2α in NRF2-silenced cancer cells. 86
Figure 26. Involvement of miR-181a-2-3p in HIF-2α regulation in NRF2-silenced cancer cells. 88
Figure 27. Level of miR-181a-2 is negatively associated with NRF2 and cancer HIF-2α. 89
Figure 28. miR-181a-2 suppresses HIF-2α-mediated CSC properties. 90
Figure 29. Inhibition of miR-181a-2 restores CSC markers expression in NRF2-silenced cancer cells. 92
Figure 30. miR-181a-2 mediates the suppressive effect of NRF2-silencing on hypoxia-induced CSC properties. 93
Figure 31. Clinical correlation of HIF-2α, cancer stem-like markers and NRF2 target genes in ccRCC. 98
Figure 32. Transcript levels of HIF-2α, NRF2, and their target genes in renal carcinoma cell lines panel. 99
Figure 33. Protein level of HIF and NRF2 signaling in renal carcinoma cell lines panel. 100
Figure 34. HIF-2α silencing suppresses stem cell-like phenotypes in A498 cell lines. 102
Figure 35. HIF-2α silencing suppresses renal cancer phenotypes in 786-O cell lines. 103
Figure 36. Silencing of HIF-2α represses renal cancer phenotypes in A704 cell lines. 104
Figure 37. HIF-2α silencing has minimal effect on UMRC-2 phenotypes. 106
Figure 38. NRF2 knockdown inhibits HIF-2α and cancer stem cell markers. 107
Figure 39. Knockdown of NRF2 suppresses HIF-2α mediated cancer stem-like phenotypes. 108
Figure 40. NRF2 silencing suppresses HIF-2α mediated cancer phenotypes in 786-O cells. 109
Figure 41. Silencing of NRF2 inhibits HIF-2α mediated cancer phenotypes in A704 cells. 110
Figure 42. NRF2 silencing suppresses HIF-2α and stem cell-like phenotypes in UMRC-2 cells. 111
Figure 43. NRF2 level mediates HIF-2α promoter activity. 112
Figure 44. NRF2 positively regulates HIF-2α through antioxidant response element. 114
Figure 45. NRF2 inhibitors reduce HIF-2α-mediated cancer stem-like phenotypes in A498 cell lines. 116
Figure 46. Pharmacological inhibition of NRF2 suppresses HIF-2α-induced CSC phenotypes in A704 cells. 117
Ⅳ. DISCUSSION 14
Figure 47. A schematic diagram of the effect of NRF2 levels on the prolonged hypoxia-induced HIF-2α activation and CSC-like properties 122
Figure 48. A schematic diagram of the positive regulation of HIF-2α by NRF2 in chronic hypoxic environment and VHL-mutated ccRCC. 131