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研究生：	黃韻芹 Huang, Yun-Chin
論文名稱：	CTMP在三陰性乳癌中扮演的角色 The role of CTMP in triple negative breast cancer
指導教授：	呂佩融 Lu, Pei-Jung
學位類別：	碩士 Master
系所名稱：	醫學院 - 臨床醫學研究所 Institute of Clinical Medicine
論文出版年：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	90
中文關鍵詞：	三陰性乳癌、癌轉移、羧基末端調節蛋白
外文關鍵詞：	triple-negative breast cancer, metastasis, Carboxyl-terminal modulator protein
相關次數：	點閱：177 下載：0
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在本篇研究中，我們將會分成兩部分探討 CTMP 的角色。三陰性乳癌佔所有乳癌的 15%- 20%因三陰性乳癌缺少雌激素受體、孕酮受體和人類表皮生長因子受體 2 且伴隨著高轉移能力以及缺乏有效的預測性生物標劑和靶向治療造成三陰性乳癌患者相比於其他乳癌明顯降低了存活率。因此發展出更多有效的治療策略對三陰性患者來說是重要的。先前的研究指出，Carboxyl-terminal modulator protein (CTMP)能夠刺激 Akt 的磷酸化進而提高 AKT 的活性造成細胞的增生。在此研究中，我們首先發現 CTMP 與三陰性乳癌的早期復發和轉移相關。因此我們利用三陰性乳癌細胞研究體外和體內 CTMP 在轉移中的功能。在抑制 CTMP 表現時顯著降低了細胞遷移、侵襲能力以及異種移植小鼠模型中的癌轉移。此外，過表現 CTMP 則顯著促進體內細胞遷移、侵襲和轉移。再者，AKT 的顯性抑制突變和 AKT 抑製劑消除了 CTMP 所促進的細胞侵襲能力。根據這些結果，我們認為 CTMP 可以促進三陰性乳腺癌的轉移。我們的結果能夠增加對 CTMP 介導的癌轉移和潛在機制的認識並能為三陰性乳癌患者提供新的治療策略和潛在的預後標誌。而在第二部分中，我們將研究 CTMP 的生物學角色。我們通過蛋白質鑑定分析評估出 32 種可能與 CTMP 有相互作用的潛在蛋白質。基於可能和 CTMP 潛在功能相關的 TXLNA、RPL3、RPL4 和 COPE 將首先藉由 GST 融合蛋白沉降技術和共免疫沉澱測定進行交互作用的分析。

In this research, we studied two parts about the role of CTMP. First part, we identified CTMP in triple negative breast cancer metastasis. Triple negative breast cancer (TNBC) accompanies high grade and high metastatic ability and a lack of predictive biomarkers and target therapy. TNBC is defined as an ack of ER, PR, and HER2 receptors and accounts for 15-20% of all breast cancers. Metastases are more aggressive in TNBC compared to non- TNBC and dramatically decrease patients’ survival. Due to the inefficiency of therapeutic response, improving therapeutic strategy is emerging for TNBC patients. Carboxyl- terminal modulator protein (CTMP) can induce AKT phosphorylation leading to increase AKT activity and promotes cell proliferation. In this study, we first found that CTMP was correlated with early recurrence and metastasis in TNBC. Next, CTMP was manipulated in TNBC cell lines to investigate the functional role in metastasis in vitro and in vivo. CTMP knockdown significantly inhibited cell migration, invasion ability, and inhibited metastasis in the xenograft mice model. In addition, overexpression CTMP significantly promoted cell migration, invasion, and metastasis in vivo. Furthermore, CTMP promoting cell invasion ability was abolished by AKT dominant negative and AKT inhibitor treatment. According to these results, we suggested that CTMP could promote metastasis in triple negative breast cancer. Our results may expand knowledge of CTMP-mediated metastasis and underlying mechanism to provide new therapeutic strategy and a potential prognosis marker for TNBC patients. In the second part, we investigated the biological role of CTMP. We evaluated 32 potential protein which could interact with CTMP by Protein Identification analysis. TXLNA, RPL3, RPL4, and COPE were selected for the first run evaluation due to the potential function of CTMP by GST pull down assay and co-immunoprecipitation assay.

中文摘要 I 
Abstract II
致謝  III 
Content IV 
Chapter I. Introduction  1 
1.1 Breast cancer 1
1.2 Metastasis  2
1.3 Carboxyl-terminal modulator protein (CTMP)  3 
1.4 Significance  4 
Chapter II. Materials and Methods 5 
2-1. Cell culture 5 
2-2. Clinical specimens  5 
2-3. Plasmid generation 6 
2-4. DNA transfection  6 
2-5. Lentivirus production and infection  7 
2-6. Establishment of CTMP overexpression/knockdown cell line 7 
2-7. RNA extraction  8 
2-8. Reverse Transcription  8 
2-9. Quantitative real-time polymerase chain reaction (qPCR)  9 
2-10. Western blotting 9 
2-11. Migration and invasion assays  10 
2-12. MTT assay  10 
2-13. Drug resistance  11 
2-14. Animal studies 11 
2-15. Hematoxylin and eosin (H&E) staining 12 
2-16. Immunohistochemistry (IHC)  12 
2-17. Immunofluorescence (IF) 13 
2-18. Nuclear and cytoplasm extraction  14 
2-19 Immunoprecipitation (IP)  14 
2-20. Protein induction  14 
2-21. Statistical analysis  15 
Chapter III. Results 16 
Part I: CTMP promotes metastasis through AKT signaling pathway in triple negative breast cancer 16 
3-1. High CTMP is correlated with recurrence in triple negative breast cancer 16 
3-2. CTMP promotes TNBC metastasis in vitro and in vivo 17 
3-3. CTMP overexpression enhances mesenchymal-related genes expression and increases cisplatin resistant in TNBC cells  21 
3-4. CTMP promotes TNBC cells invasion ability through AKT pathway  22 
Part II: Investigation the biological role of CTMP in triple negative breast cancer  25 
Chapter IV. Discussion and Conclusion 27 
Figures and Tables 31 
Figure 1. High CTMP had a tendency of disease-free survival correlation in TNBC patients 32 
Figure 2. The scoring of CTMP in TNBC tissue microarray.  34 
Figure 3. High CTMP was correlated with disease-free survival in TNBC patients.  35 
Figure 4. CTMP expression was increased in high invasive cells.  37 
Figure 5. High CTMP expression in highly metastatic cells promoted metastasis in vivo. 38 
Figure 6. CTMP highly expressed in distant organ metastasis sites. 39 
Figure 7. The expression of CTMP in TNBC cells.  40 
Figure 8. CTMP promotes cell migration and invasion in TNBC cells.  41 
Figure 9. Knockdown CTMP inhibit cell migration and invasion in TNBC cells. 42 
Figure 10. MDA-MB-231 CTMP knockdown cells were no significantly difference of tumor growth and metastasis ability in vivo.  43 
Figure 11. MDA-MB-231 CTMP knockdown cells were no significantly difference of distant organ metastasis ability in vivo. . 44 
Figure 12. Down-regulating CTMP expression inhibited metastasis in vivo.  46 
Figure 13. MDA-MB-231 CTMP knockdown cells decreased distant organ metastasis 
ability by tail-vein injection in vivo. . 48 
Figure 14. Down-regulating CTMP expression decreased the bone erosion rate in vivo. 49 
Figure 15. Down-regulating CTMP expression inhibits bone metastasis in vivo.  50 
Figure 16. Overexpression CTMP promoted cells proliferation in TNBC cells. 51 
Figure 17. CTMP was upregulated expression of mesenchymal-related genes and drug resistance-related genes in BT-549 cells.  52 
Figure 18. CTMP did not change sphere formation ability and number in MDA-MB- 231 and BT-549 cells. 53 
Figure 19. CTMP promoted cisplatin resistance.  54 
Figure 20. The RNA sequencing analysis in CTMP overexpression compared with MDA-MB-231 parental.  57 
Figure 21. CTMP was highly expressed in metastatic cells and induced AKT phosphorylation.  58 
Figure 22. CTMP promoting cells invasion ability are reduced by blocking the AKT kinase activity.  59 
Figure 23. CTMP promoting cells invasion ability are reduced after AKT inhibitor IV treatment. . 61 
Figure 24. The endogenous CTMP expression in TNBC cells. 62 
Figure 25. The endogenous CTMP expression in non-TNBC cells.  63 
Figure 26. The localization of endogenous CTMP was expressed in cytosol and nucleolus in TNBC cells. 64 
Figure 27. Endogenous CTMP expressed in cytoplasm and nucleus. 65 
Figure 28. No CTMP interacting RNAs were isolated by immunoprecipitated assay. 66 
Figure 29. Identification of CTMP-interacting protein.  67 
Figure 30. Validation of the candidates which interact with CTMP through Protein Identification. 68 
Figure 31. Classification of the candidates which interact with CTMP through Protein Identification. 69 
Figure 32. Induction of different fragments in CTMP.  70 
Figure 33. Purification of different fragments in CTMP. 72 
Figure 34. Validation of CTMP-interacting proteins by co-immunoprecipitation. 75 
Table 1. High CTMP is correlated with recurrence and metastasis in TNBC patients. 76 
Table 2. CTMP mediated metastasis did not have organ tropism in TNBC. 77 
Table 3 List of the gene-specific primer sequences used in the qPCR assays: 78 
Table 4. List of antibodies used in the study. 81 
References  82 
Appendix  87 
Appendix 1. The expression of AKT and CTMP. 88 
Appendix 2. Reduction of theioesterase activity of CTMP decreased cells invasion ability. 90
                                    

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2026-09-13公開

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