探討標靶治療在高分化漿液性卵巢癌中的協同致死作用

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研究生：	鍾怡娟 Chung, I-Jiuan.
論文名稱：	探討標靶治療在高分化漿液性卵巢癌中的協同致死作用 Determining Cooperative Lethal Effect of Targeted therapy in High-Grade Serous Ovarian Carcinoma
指導教授：	莊永仁 Chuang, Yung-Jen
口試委員:	詹鴻霖張姓治
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2019
畢業學年度：	107
語文別：	英文
論文頁數：	48
中文關鍵詞：	卵巢癌、協同致死作用、TP53 、標靶治療
外文關鍵詞：	Ovarian cancer, Cooperative lethal effect, TP53, Targeted therapy
相關次數：	點閱：3 下載：0
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基因體分析顯示，腫瘤抑制因子p53及其調節因子的功能喪失或突變，是人類癌症的共同特徵。例如，在高分化漿液性卵巢癌（high-grade serous ovarian carcinoma, HGSOC）中，有將近96%的腫瘤，p53都有突變的現象。在所有的p53調節蛋白中，MDM2（具有介導p53的泛素化和各種致癌活性）被發現在漿液性卵巢癌中高度表達。另一方面，在9%-62%的人類卵巢癌中，也檢測到EGFR蛋白的過度表達，並與卵巢癌患者的預後不良與抗藥性有關。因此，有鑑於發展HGSOC的精準癌症治療，本研究旨在測試：以複合抗癌藥物方式，聯合抑制EGFR和MDM2兩個分子靶標，是否可以增強治療卵巢癌的療效。
在先前的研究中，我們證實JNJ和Gefitinib的聯合治療對p53突變的HGSOC細胞可發揮最強的協同抑制作用，這表示MDM2 / EGFR的協同抑制治療，在p53突變的卵巢癌患者中有潛在性的臨床應用。在本研究中，我們再次驗證EGFR抑制劑（Gefitinib）和MDM2抑制劑（JNJ）的聯合抑制作用，對於不同p53狀態的HGSOC細胞的影響。我們旨在確認這兩個分子靶點對HGSOC增殖的重要性及協同作用。
我們接下來探討了MDM2和EGFR之間的關係，希望揭示其協同抑制的可能機制。我們發現，在EGF刺激下，p53突變細胞的MDM2蛋白表達，會迅速且大量增加。有趣的是，免疫細胞化學染色法顯示，在EGF刺激後，細胞內的EGFR和MDM2會移位，這意味著EGFR和MDM2的移位可能參與了協同抑制作用。進一步分析顯示，經過JNJ和Gefitinib聯合抑制後，EGFR和MDM2移位程度，會受到抑制。我們接下來驗證了EGFR下游信號傳導途徑，以確定哪些因子參與了JNJ和Gefitinib的協同抑制作用。我們的數據表示，JNJ和Gefitinib的聯合抑制，確實是透過EGFR下游信號傳導途徑實現協同抑制的作用。
總結來說，我們的研究結果顯示，在JNJ和Gefitinib協同抑制期間，細胞內分子運輸可能受到影響，這指出了針對此特性發展治療HGSOC中具有特定p53突變的可能性，並且可能有助於修正我們對其他具有特定p53狀態的癌症的理解。

Genomic analysis has revealed that dysfunction of tumor suppressor p53 and its regulators is a common feature of human cancer. For example, p53 mutation is detected in up to 96% of high-grade serous ovarian carcinoma (HGSOC). Among all p53 regulatory proteins, MDM2, which mediates ubiquitination of p53 and various oncogenic activities, has highly expressed in serous ovarian cancer. On the other hand, overexpression of EGFR protein is also detected in 9%-62% of human ovarian cancer, which is associated with poor prognosis and drug resistance in ovarian cancer patients. Thus, in view of developing precision cancer therapy for HGSOC, we aimed to test whether combining two anticancer agents to co-inhibiting EGFR and MDM2 could enhance treatment efficacy for ovarian cancer.
Previously, we have demonstrated that combination therapy of JNJ (JNJ-26854165) and Gefitinib might exert the strongest synergistic inhibition on p53-mutated HGSOC cells (i.e. CAOV3), which suggested the potential clinical application of MDM2/EGFR co-inhibition for ovarian cancer. In the present study, we first re-assessed the combined inhibition of MDM2 by JNJ and EGFR by Gefitinib on selected HGSOC cell lines with different p53 status. This was to validate the observed synergism of these two molecular targets on inhibiting HGSOC proliferation. We next explored the relationship between MDM2 and EGFR to reveal the underlying mechanisms for the cooperative inhibition. We found that, upon EGF stimulation, MDM2 protein expression increased rapidly and transiently in p53 mutant cell lines. Immunocytochemistry analysis further showed that, upon EGF stimulation, EGFR and MDM2 in CAOV3 cells would translocate together, which implied the involvement of molecular trafficking in the cooperative inhibition. Subsequent analysis revealed that, after JNJ and Gefitinib combination treatment, the levels of EGFR and MDM2 translocation would be inhibited. We surveyed the EGFR downstream signaling pathway to identify which factors may relay the synergistic inhibition of JNJ and Gefitinib. We indeed found the combination treatment of JNJ and Gefitinib might achieve synergistic inhibition through one specific downstream mediator of EGFR signaling.
In summary, our findings suggest that the EGFR signaling-dependent trafficking of EGFR and MDM2 in HGSOC with p53 mutation might be altered during synergistic inhibition by JNJ and Gefitinib, which might help to revise our understanding on other cancers with specific p53 status.

Abstract    I
中文摘要    III
Table of Contents    V
1. INTRODUCTION    1
1.1 Ovarian cancer    1
1.2 TP53 mutations are found in up to 96% of ovarian cancer    2
1.3 MDM2 is highly expressed in ovarian cancer    3
1.4 EGFR overexpression in ovarian cancer    4
1.5 Precision cancer therapy    5
1.6 Objectives of this study    6
2. MATERIALS AND METHODS    7
2.1 Cell culture    7
2.2 Drugs and reagents    7
2.3 Western blot analysis    8
2.4 Immunocytochemistry (ICC)    9
2.5 WST cell proliferation assay    9
3. RESULTS    10
3.1 Reconfirm the previous results of synergistic lethal effect    10
3.2 Time differential induction of EGFR and MDM2 after treatment with EGF    10
3.3 Analysis of intracellular EGFR and MDM2 in ovarian cancer cells    12
3.4 MDM2 may associate with downstream molecules of EGFR signaling during cytonuclear
trafficking    13
3.5 EGFR downstream signaling analysis: STAT3, AKT and MAPK expression profiling after
drug treatment    14
3.6 Effect of AKT inhibition on EFGR and MDM2’s cyto-nuclear trafficking    17
3.7 Hypothetical model for synergistic lethal effect of JNJ and Gefitinib in HGSOC with different
p53 status    18
4. CONCLUSION    20
5. DISCUSSION    22
5.1 Synergistic lethal effect was stronger in Mutp53 cells than in Wtp53 cells    22
5.2 The protein expression of p-AKT was significantly inhibited in Wtp53 cells under combined
treatment of JNJ and Gefitinib    22
5.3 Inhibition of AKT may affect nuclear translocation of EGFR    23
6. ACKNOWLEDGEMENTS    25
7. REFERENCES    26
8. FIGURES    29
Figure 1. Dose-response curves under signal and combined inhibition of MDM2 and EGFR in
Mutp53 and Wtp53 HGSOC cell lines.    29
Figure 2. Effect of EGF-induction on phosphorylated EGFR and MDM2 expression in ovarian
cancer cells.    31
Figure 3. Intracellular expression and localization of EGFR and MDM2.    35
Figure 4. Expression of STAT3, AKT and MAPK proteins in various ovarian cancer cell lines
under JNJ and Gefitinib combination treatment.    40
Figure 5. Effect of AKT inhibition on EGFR and MDM2 cytonuclear translocation    43
Figure 6. Hypothetical model for synergistic lethal effect of Gefitinib and JNJ in wild-type p53
(Wtp53) and mutant p53 (Mutp53) HGSOC    44
9. TABLES    46
Table 1. IC 50 values of JNJ and Gefitinib for each ovarian cancer cell lines.    46
Table 2. List of Antibodies.    47
10. ABBREVIATIONS    48

                                

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