Title Page
Contents
ABBREVIATION 16
ABSTRACT 18
1. Introduction 22
1.1. Breast cancer and its characteristics 22
1.1.1. Breast cancer and its subtypes 22
1.1.2. Migration, invasion, and metastasis of breast cancer cells 26
1.2. Roles of NDRG2 in cancer 28
1.3. EHF: molecular basis and roles in cancers 30
1.3.1. Members and structures of ETS family 30
1.3.2. Roles of EHF in cancer 32
1.4. STAT3 signaling pathway and its relevance to cancers 34
1.5. Mechanisms and functions of cellular senescence 35
1.5.1. What is senescence? 35
1.5.2. Cause of senescence 37
2. Materials and Methods 38
2.1. Cell culture and reagents 38
2.2. Mice and tumor models 38
2.3. Plasmid transfection and overexpression of NDRG2 and EHF genes in MDA-MB-231 cells 39
2.4. Determination of cell growth using IncuCyte ZOOM®[이미지참조] 40
2.5. MTT assay 40
2.6. RNA isolation and quantitative real-time RT-PCR 40
2.7. Knockdown of transcription factors by small interfering RNA (siRNA) 41
2.8. Western blot 42
2.9. Wound healing assay 42
2.10. Transwell assay 43
2.11. Senescence-associated β-gal assay 44
2.12. PI/Annexin V apoptosis assay 44
2.13. Survival analysis 45
2.14. Dataset analysis 45
2.15. Statistical analysis 46
3. Results 48
3.1. NDRG2 gene expression is positively correlated with EHF gene expression in breast cancer 48
3.2. NDRG2 gene expression is positively correlated with EHF gene expression in TNBC cell lines 52
3.3. EHF inhibited cell growth and promoted paclitaxel-induced apoptosis 56
3.4. EHF induced senescence of MDA-MB-231 cells 60
3.5. EHF inhibited migration in tumor cells 62
3.6. EHF expression is correlated with EMT markers 64
3.7. EHF inhibited the phosphorylation of STAT3 69
3.8. EHF exhibited tumor suppressive role in mouse mammary tumor cells 74
3.9. EHF inhibited tumor progression in vivo 82
3.10. EHF expression is correlated with infiltration of immune cells 87
3.11. EHF overexpression suppressed pulmonary nodule formation 93
3.12. EHF expression did not affect tumor sizes, but inhibited metastasis of breast cancer cells in nude mice 97
3.13. EHF prolonged survival of breast cancer patients 101
3.14. Low expression of EHF is associated with the high pathological T and N stages 106
3.15. Rosiglitazone induced EHF expression in breast cancer cells 109
3.16. EHF inhibits breast cancer progression by regulating senescence, EMT, and the STAT3 signaling pathway. 111
4. Discussion 113
5. References 122
ABSTRACT IN KOREAN 136
Table 1. Quantitative PCR primer sequences 47
Figure 1. Subtypes of breast cancer. 25
Figure 2. The metastatic process. 27
Figure 3. Various processes targeted by NDRG2. 29
Figure 4. The plasmid map of pCMV-Tag-2B and pCMV6-Entry vector. 39
Figure 5. A positive correlation between NDRG2 and EHF gene expression in breast cancer patients and breast cancer cell lines. 49
Figure 6. Expression of NDRG2 and EHF in various subtypes of breast cancer. 50
Figure 7. Expression of NDRG2 and EHF in FNA biopsies from breast cancer patients. 51
Figure 8. Expression of NDRG2 and EHF in four TNBC cell lines. 53
Figure 9. Expression of NDRG2 and EHF in MDA-MB-231-wt, -mock, and -NDRG2 cells. 54
Figure 10. Expression of NDRG2 and EHF in 4T1-mock and 4T1-NDRG2 cells. 55
Figure 11. Relative cell confluence of MDA-MB-231 and MDA-MB-453 cells. 57
Figure 12. Stable overexpression of EHF and relative cell confluence of MDA-MB-231-mock and MDA-MB-231-EHF cells. 58
Figure 13. Paclitaxel-induced apoptosis of stable MDA-MB-231-mock and MDA-MB-231-EHF cells. 59
Figure 14. EHF induced the cellular senescence of MDA-MB-231 cells. 61
Figure 15. EHF suppressed the migration and invasion ability of MDA-MB-231 cells. 63
Figure 16. Heatmap showing the correlation between EHF and EMT markers. 65
Figure 17. Effect of EHF overexpression on EMT markers in stable MDA-MB-231 cells. 66
Figure 18. Effect of transient EHF overexpression on EMT markers in MDA-MB-231 and MDA-MB-453 cells. 67
Figure 19. Effect of knockdown of EHF on EMT markers in MDA-MB-231-EHF cells. 68
Figure 20. EHF overexpression suppressed the phosphorylation of STAT3 in MDA-MB-231 and MDA-MB-453 cells. 71
Figure 21. Overexpression of EHF suppressed the phosphorylation of STAT3. 72
Figure 22. Correlation between EHF, IL-6, and IL-6 receptor expression. 73
Figure 23. Stable overexpression of EHF and growth retardation of 4T1-EHF cells. 76
Figure 24. Overexpression of EHF induced the cellular senescence. 77
Figure 25. Wound healing assay of 4T1 cells. 78
Figure 26. Transwell assay of 4T1-mock and 4T1-EHF cells. 79
Figure 27. Doxorubicin-induced apoptosis of 4T1 cells. 80
Figure 28. Cancer patients' response to doxorubicin according to EHF expression. 81
Figure 29. Scheme and the graphs showing the effect of EHF overexpression on tumor volume and weight. 83
Figure 30. The spleen length and weight of Balb/c mice subcutaneously injected with 4T1 tumor cells. 84
Figure 31. EHF tumors obtained from mice in vivo showed decreased expression of Slug and phospho-STAT3. 85
Figure 32. The SA-β-gal staining of mock and EHF tumors. 86
Figure 33. Regulatory T cell infiltration according to EHF expression in breast cancer. 88
Figure 34. MDSCs infiltration according to EHF expression in breast cancer. 89
Figure 35. CD8⁺ T cell infiltration according to EHF expression in breast cancer. 90
Figure 36. CD4⁺ T cell infiltration according to EHF expression in breast cancer. 91
Figure 37. CD3⁺ T cell infiltration in 4T1-mock and 4T1-EHF primary tumor. 92
Figure 38. EHF expression suppressed formation of pulmonary nodules. 94
Figure 39. Intravenous injection of 4T1-EHF attenuated formation of pulmonary nodules. 95
Figure 40. Relative EHF mRNA expression in 4T1 cells and explants. 96
Figure 41. EHF overexpression did not affect breast tumor growth in nude mice. 98
Figure 42. EHF did not affect the weight and length of spleens in nude mice. 99
Figure 43. EHF suppressed metastasis of breast cancer in nude mice. 100
Figure 44. EHF expression in normal breast tissue and breast invasive carcinoma. 102
Figure 45. EHF prolonged survival of TNBC patients. 103
Figure 46. Relapse-free survival of TNBC patients with chemotherapy. 104
Figure 47. Disease-free interval of the TCGA breast cancer patients. 105
Figure 48. Pathological T and N stages divided by the median value of EHF expression. 107
Figure 49. Pathological T and N stages divided by the tertile value of EHF expression. 108
Figure 50. Rosiglitazone treatment induced expression of EHF in MDA-MB-231 cells. 110
Figure 51. EHF inhibits breast tumor progression. 112