p53的R248Q突變改變了高分化漿液性卵巢癌中的分子運輸和標靶藥物反應

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研究生：	蔡暟耘 Tsai, Kai-Yun
論文名稱：	p53的R248Q突變改變了高分化漿液性卵巢癌中的分子運輸和標靶藥物反應 R248Q mutation of p53 alters molecular trafficking and targeted drug responses in high-grade serous ovarian carcinoma
指導教授：	莊永仁 Chuang, Yung-Jen
口試委員:	詹鴻霖林玉俊
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：	43
中文關鍵詞：	p53 、表皮生長因子受體（EGFR）、Akt 、MDM2 、複合式療法
外文關鍵詞：	p53, epidermal growth factor receptor (EGFR), Akt, mouse double minute 2 homolog (MDM2), combination therapy
相關次數：	點閱：3 下載：0
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腫瘤抑制蛋白p53及其調節因子的功能異常是人類癌症的常見標誌。具體而言，高達96％的高分化漿液性卵巢癌（high-grade serous ovarian carcinoma, HGSOC）可檢測到p53突變。此外，突變的p53可能透過表皮生長因子受體（EGFR）信號傳導的持續激活而導致致癌的「功能獲得」表現型。在這項研究中，我們調查了p53突變是否會改變HGSOC中EGFR相關的信號傳導和其治療策略。我們選擇了p53 R248Q突變體（p53R248Q）用於本研究，該突變體在HGSOC中的突變頻率是最高的。在本研究中，我們發現當p53R248Q瞬時過表達時，Akt（EGFR下游信號傳導的關鍵分子）的磷酸化會顯著增加。透過免疫細胞化學染色分析進一步顯示，在p53R248Q過表達後，幾種Akt依賴性的介質分子在細胞內以獨特的模式易位。過去我們已經證明，gefitinib和JNJ-26854165對EGFR和MDM2-p53途徑的聯合抑制在p53突變的HGSOC細胞中發揮強烈的協同致死作用。接著本研究的分析顯示，在這種組合抑制下，EGFR和MDM2的細胞內易位模式將被破壞。此外，當我們進一步比較不同p53狀態細胞對gefitinib和JNJ-26854165的藥物反應時，我們發現在過表達p53R248Q的細胞中對單一藥物或組合抑制治療的敏感性也發生了改變。綜合以上所述，我們的研究結果證明，p53的R248Q突變導致信號傳導、分子運輸和藥物反應的顯著變化，這可能有助於促進我們對p53和癌症生物學的理解，並改善HGSOC的治療策略。

Dysfunctions of the tumor suppressor p53 and its regulators are common hallmarks of human cancers. Specifically, p53 mutations are detected in up to 96% of high-grade serous ovarian carcinoma (HGSOC). Moreover, mutant p53 may cause oncogenic gain-of-function phenotypes in sustained activation of epidermal growth factor receptor (EGFR) signaling. In this study, we investigated whether p53 mutation could affect EGFR-related signaling and therapeutic strategies in HGSOC. We selected the p53 R248Q mutant (p53R248Q), which has the highest mutation frequency in ovarian cancer, for this study. In this study, we showed that the phosphorylation of Akt, a critical molecule of EGFR downstream signaling, increased significantly when p53R248Q was transiently overexpressed. Immunocytochemistry analysis further showed that upon p53R248Q overexpression, several Akt-dependent mediators translocate in unique patterns within the cell. Previously, we demonstrated that the combined inhibition of EGFR and MDM2-p53 pathways by gefitinib and JNJ-26854165 exerts a strong synergistic lethal effect on p53-mutated HGSOC cells. Subsequent analysis revealed that under this combined inhibition, the cytonuclear trafficking of EGFR and MDM2 is disrupted. Moreover, when we further compared gefitinib and JNJ-26854165 responses in different p53 status cells, we found that the sensitivity to the single- or combined-inhibition treatments is also altered in p53R248Q-overexpressing cells. In summary, our findings suggest that the R248Q mutation of p53 causes significant changes in signaling transduction, molecular trafficking and drug responses, which might help to advance our understanding of p53 and cancer biology and improve therapeutic strategies for HGSOC.

Abstract    i
中文摘要    ii
Table of Contents    iii
List of Abbreviations    vi
1. Introduction    1
1.1. Ovarian cancer and TP53 mutation    1
1.2. The relationship between mutant p53 and Akt signaling    1
1.3. Akt-dependent molecular trafficking    2
1.4. Clinical challenges of ovarian cancer    2
1.5. Synergistic effect of combined EGFR and MDM2 inhibition    3
1.6. Aim of this study    3
2. Experimental procedures    4
2.1. Cell culture    4
2.2. Drugs and reagents    4
2.3. Gene knockdown and overexpression    4
2.4. Western blot analysis    5
2.5. Immunocytochemistry (ICC)    5
2.6. Cell viability assay    6
2.7. Analysis of combination index    6
2.8. Statistics    7
3. Results    8
3.1. R248Q mutation of p53 amplified p-Akt signaling in ovarian cancer cells    8
3.2. p53R248Q overexpression and EGF stimulation resulted in similar cytonuclear trafficking of Akt, EGFR, MDM2, and FOXO3a    9
3.3. Combined blockade by gefitinib and JNJ attenuated EGFR and MDM2 cytonuclear trafficking    10
3.4. R248Q mutation of p53 increased the sensitivity of EGFR and MDM2 inhibitors    11
3.5. R248Q mutation of p53 decreased the synergistic lethal effect of gefitinib and JNJ    12
3.6. MAPK and p21 regulated the effects of single and combined treatment of gefitinib and JNJ    13
4. Discussion    15
4.1. Summary    15
4.2. Comparison of the mechanisms of enhanced Akt by p53R248Q in different cancers    15
4.3. Interpretation of how the Akt-dependent cytonuclear trafficking works and its clinical significance    15
4.4. The relationship between Akt-dependent cytonuclear trafficking, synergistic effect, and p53 status    17
4.5. Hypothetical models for synergistic lethal effects of gefitinib and JNJ in cells with different p53 statuses    18
5. Acknowledgments    19
6. References    20
7. TABLES    23
Table 1. IC50 values of OVCAR3 cells with different p53 status to EGFR and/or MDM2 inhibitors    23
Table 2. Combination index (CI) values and synergism grading of combined JNJ and gefitinib treatment at different effective doses (ED50, ED75 and ED90)    24
Table 3. Antibodies list    25
8. FIGURES    26
Figure 1. p53R248Q resulted in amplification of p-Akt.    26
Figure 2. p53R248Q overexpression and EGF stimulation altered the intracellular localization patterns of Akt, EGFR, MDM2 and FOXO3a.    30
Figure 3. Cytonuclear trafficking of EGFR and MDM2 would be disrupted under combined inhibition of EGFR and MDM2 by gefitinib and JNJ.    32
Figure 4. Overexpression of p53R248Q in OVCAR3 cells increased sensitivity to JNJ or gefitinib.    33
Figure 5. Synergistic effects of JNJ and gefitinib on OVCAR3 cells with different p53 statuses are shown in Fa-CI plots and isobolograms.    34
Figure 6. MAPK and p21 were involved in regulating the cell signaling responses to single or combined treatments of gefitinib and JNJ.    36
Figure 7. Hypothetical models to integrate molecular trafficking and synergistic lethal effects of gefitinib and JNJ with different p53 statuses    39
9. Supplementary data    40
Supplementary Figure 1. Time-dependent cytonuclear trafficking patterns of EGFR and MDM2    40
Supplementary Figure 2. Dose-response curves of OVCAR3 cells with different p53 statuses under inhibition with JNJ and/or gefitinib    41
Supplementary Figure 3. Quantification graphs of immunoblots in Figure 6    42
Supplementary Figure 4. Plasmid map of p53 for overexpression    43
                                

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