Title Page
Abstract
Contents
1. Introduction 11
2. Materials and Methods 22
2.1. Cell lines and tumor specimens 22
2.2. Reagents and antibodies 23
2.3. Cell viability assay 24
2.4. Plasmid and siRNA transfection 24
2.5. Dual-Luciferase® Reporter assay 25
2.6. RNA extraction and quantitative real-time PCR 26
2.7. Western blotting analysis and Phos-tagTM gel preparation 27
2.8. Cycloheximide chase assay 28
2.9. Proteasome inhibition analysis 28
2.10. Ubiquitination assay 29
2.11. Nuclear-cytoplasmic protein fractionation 29
2.12. Immunocytochemistry 30
2.13. Statistical analysis 31
3. Results 32
3.1. Tolfenamic acid inhibits cancer cell growth and YAP/TAZ target gene expression 32
3.2. Tolfenamic acid attenuates YAP/TAZ protein level 37
3.3. Protein level change of NAG-1 and YAP/TAZ by Tolfenamic acid are independent 42
3.4. YAP/TAZ degradation by Tolfenamic acid occurs through proteasomal degradation pathway 44
3.5. Tolfenamic acid increases YAP/TAZ phosphorylation 46
3.6. Tolfenamic acid promotes ubiquitination of YAP/TAZ 50
3.7. Cytoplasmic localization of YAP/TAZ is increased by Tolfenamic acid treatment 53
4. Discussion 57
5. References 62
국문 초록 70
Table 1. The number of deaths for leading causes of death, US, 2015-2020. 11
Figure 1. The Functions of Hippo pathway. 15
Figure 2. Hippo signaling pathway and main regulators. 16
Figure 3. The Hippo signaling pathway in tissue homeostasis, regeneration, and cancer. 17
Figure 4. Inhibition of YAP/TAZ transcriptional coactivators by LATS1/2. 18
Figure 5. Regulatory domains of the Hippo pathway effector proteins YAP and TAZ. 19
Figure 6. TA inhibits human cancer cell growth. (A-D) Cell viability assay of SW480 (A), HCT116 (B), LoVo (C), and BCPAP (D) cells... 34
Figure 7. TA attenuates luciferase activity of YAP/TAZ-responsive TEAD-luciferase reporter. Luciferase assay of HCT116 cells... 35
Figure 8. TA lowers YAP/TAZ target gene expression. (A, B) Quantitative reverse transcription PCR (qRT-PCR) results of LoVo... 36
Figure 9. TA decreases YAP/TAZ protein level. (A) Immunoblotting of YAP/TAZ and NAG-1 in SW480 cells treated with various... 39
Figure 10. TA decreases YAP/TAZ protein level. (A) Immunoblotting of flag-tagged YAP and TAZ in HCT116 cells transfected with flag-... 40
Figure 11. TAZ is highly expressed in human thyroid tumor tissues. Immunoblotting of YAP/TAZ and NAG-1 in normal (N) tissues (n=... 41
Figure 12. NAG-1 induction and YAP/TAZ reduction by TA occur in independent pathway. (A) Immunoblotting of YAP/TAZ and NAG-1... 43
Figure 13. TA promotes YAP/TAZ degradation by proteasomal degradation pathway. (A, B) Cycloheximide chase assay of SW480... 45
Figure 14. TA increases YAP/TAZ phosphorylation. (A, B) The phosphorylation status of YAP (A) and TAZ (B) was measured on a... 48
Figure 15. TA increases YAP/TAZ ubiquitination. (A) Immunoblotting of YAP/TAZ and pan 14-3-3 in LoVo and SW480... 51
Figure 16. TA increases cytoplasmic localization of YAP/TAZ. (A) Nuclear-cytoplasmic fractionation in LoVo, SW480, and HCT116... 54
Figure 17. Schematic diagram of YAP/TAZ protein localization and degradation affected by TA treatment. 56