Title Page
Contents
Abstract 13
I. INTRODUCTION 15
1. Estrogen Receptor Alpha (ERα) 15
A. Estrogen Receptor in Breast Cancer 15
B. The regulation of ERα Expression 19
2. Vimentin 19
A. Vimentin in Breast Cancer 19
B. The structure and regulation of Vimentin 21
3. RING finger protein 208 (RNF208) 23
4. The stream of the study 25
II. MATERIAL AND METHOD 25
1. Cell culture and reagents. 26
2. Plasmids. 27
3. Generation of stable cell lines. 28
4. RNA extraction, RT-PCR and real-time qRT-PCR. 29
5. RNA sequencing. 29
6. Chromatin immunoprecipitation. 30
7. CRISPR genome editing. 30
8. Human breast cancer tissue microarray and immunohistochemistry. 31
9. In vivo tumor formation and lung metastasis. 32
10. Immunoprecipitation and immunoblot analysis. 33
11. Cross-linking for protein interaction analysis. 34
12. In vitro ubiquitination assay. 35
13. Luciferase assay. 35
14. Cell migration and invasion assays. 36
15. Wound healing assay. 37
16. Statistics and reproducibility. 37
III. RESULTS 38
PART I. The Expression of RNF208 in Breast Cancer 38
1. RNF208 is significantly underexpressed in aggressive TNBC. 38
2. E2-ERα signaling induces RNF208 expression at the transcriptional level in luminal breast cancer. 44
3. ERα regulates RNF208 expression through the transcriptional activation of the RNF208 promoter. 47
4. Re-expression of ERα upregulate the RNF208 level in TNBC cells. 51
PART II. The Biological Function of RNF208 54
1. Overexpression of RNF208 reduces tumorigenesis in TNBC cells. 54
2. Overexpression of RNF208 reduces the lung metastasis of highly metastatic breast cancer cells. 57
PART III. The Regulatory Mechanism of RNF208 to Prevent TNBC Metastasis. 62
1. Overexpression of RNF208 decreases the stability of Vimentin protein. 62
2. RNF208 binds to Vimentin protein. 65
3. RNF208 induces the Vimentin protein degradation by facilitating K27-linked polyubiquitination. 68
4. Expression level of RNF208 is inversely correlated with expression of Vimentin in breast cancer patients. 71
5. Binding domain in between RNF208 and Vimentin interaction. 74
6. Activity of RNF208 E3 ligase is required for the degradation of Vimentin. 77
7. Vimentin degradation through RNF208 mediated ubiquitination is important to prevent TNBC metastasis. 80
8. RNF208 specifically targets the phosphorylation of Vimentin at the Ser39 residue. 84
IV. DISCUSSION 91
V. CONCLUSION 97
REFERENCE 102
국문요약 108
Fig. 1. ER signaling pathway. 16
Fig. 2. Schematic representation of hormone treatment action in... 18
Fig. 3. The role of Vimentin in cancer 20
Fig. 4. Cytoplasmic IF assembly and reorganization in response... 22
Fig. 5. The ubiquitin system 23
Fig. 6. The RING finger domain 24
Fig. 7. The expression of RNF208 in breast cancer cell lines. 39
Fig. 8. The expression of RNF208 in breast cancer patients. 42
Fig. 9. RNF208 expression was transcriptionally activated by ERα in... 45
Fig. 10. ERα binding sites within the RNF208 promoter in luminal breast... 49
Fig. 11. Underexpression of RNF208 is not associated with DNA methylation... 52
Fig. 12. RNF208 overexpression reduces the tumor growth. 55
Fig. 13. RNF208 overexpression reduces lung metastasis. 58
Fig. 14. Loss of RNF208 does not influence aggressive phenotype-induced... 60
Fig. 15. RNF208 degrades Vimentin protein by inducing proteasomal... 63
Fig. 16. The interaction between RNF208 and Vimentin. 66
Fig. 17. RNF208 degreases the Vimentin protein stability via K27 ubiquitin-... 69
Fig. 18. RNF208 expression is inversely correlated with Vimentin expression... 72
Fig. 19. RING domain of RNF208 specifically interacts with head domain of... 75
Fig. 20. Activity of the RNF208 E3 ligase is required for the... 78
Fig. 21. RNF208 suppresses metastasis by targeting the Lys97 residue of... 82
Fig. 22. RNF208 degrades the soluble form of Vimentin by recognizing the... 86
Fig. 23. RNF208 overexpression decreases the cell migration by targeting... 89
Fig. 24. Proposed models demonstrating the function of RNF208 as a novel... 98
Fig. 25. Proposed models demonstrating the function of RNF208 as a novel... 100