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研究生: 陳永富
Yung-Fu Chen
論文名稱: Wnt-EGFR訊息傳遞途徑動力學計算研究暨抑制劑效應模擬
Modeling of Kinetics of the Wnt-EGFR Signaling Pathway and Inhibitor Effects on its Kinetic Behaviors
指導教授: 孫英傑
Sun, Ying-Chieh
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2012
畢業學年度: 100
語文別: 中文
論文頁數: 91
中文關鍵詞: 訊息傳遞路徑抑制劑模擬
英文關鍵詞: signaling pathway, inhibitor, modeling
論文種類: 學術論文
相關次數: 點閱:103下載:3
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  • Wnt-EGFR 訊息傳遞途徑是已知與細胞增殖、分化和凋亡有很大的相
    關性。在許多癌症裡,發現這些訊息傳遞路徑的異常情形。我們利用電
    腦模擬的方法,計算此兩個訊息傳遞路徑的動力學模型,來幫助我們了
    解這些訊息傳遞路徑的幾個效應。
    以現有的模型為基礎,根據此訊息傳遞途徑的動力學效應,以及相
    關的實驗數據來擴充模型。首先,我們增加一條強度合理的負回饋路徑
    到兩個不同模型的EGFR 路徑中,當ERKpp 對Raf-1 抑制方程式中K1
    值在0.01-10 的區間時,計算結果顯示,此路徑對兩個不同模型中的
    ERKpp 濃度有不同效應。路徑中含有Braf 的模型,此負回饋路徑在模型
    中無法扮演有效抑制ERKpp 濃度的角色。反之路徑中沒有Braf 的模型,
    此負回饋路徑可以有效使ERKpp 濃度表現降低。第二,加入EGFR 與
    Wnt 路徑之間的交談(crosstalk)反應路徑,若為正回饋路徑,當β-catenin
    無過度表達時,其促使未知因子Y 的濃度大小對於ERKpp 活化濃度扮演
    開關效應(switch-like)的行為。若為負回饋路徑,因較強的負回饋效應,
    使得Wnt 訊號刺激期間的ERKpp 活化濃度有大幅度震盪的行為,Wnt
    訊號結束後ERKpp 濃度迅速降回低點。
    iii
    此外,我們添加蛋白激酶抑制劑,探討其對磷酸化ERK 表現的效應。
    以Wnt 訊號短暫刺激後,以及β-catenin 過度表達造成的ERKpp 濃度失
    調時,其濃度被抑制的差異,比較抑制效果。加入單一抑制劑對多個蛋
    白激酶同時抑制,其模擬結果顯示,同時對多個蛋白激酶抑制且抑制強
    度相同時,因為數個被抑制者會競爭抑制劑的濃度,所以其抑制效果比
    單一抑制劑對Raf-1 單獨抑制時差,效應差距最大時,減少的ERKpp 濃
    度為單獨抑制Raf-1 時的1/6 倍。單一抑制劑單獨抑制Raf-1 為較佳抑制
    效果,可以利用模擬結果探討抑制劑選擇性問題。
    加入多個抑制劑時,兩個抑制劑分別抑制Raf-1 及MEK 時,對Raf-1
    與MEK的抑制劑兩者濃度比例大於1.5 倍時(濃度總合同單一抑制劑的濃
    度時),比起單一抑制劑同時抑制Raf-1 與MEK 且強度相同時,有較好的
    抑制效果使ERKpp 表現有明顯的降低。因此兩個抑制劑分別抑制Raf-1
    及MEK,且增加Raf-1 抑制劑的濃度時有較佳的抑制效果。
    這些結果使我們加深了解兩訊息傳遞路徑,同時對未來多目標蛋白
    激酶抑制劑的設計是有幫助的。

    The Wnt and EGFR signaling pathways are known to relate to cell proliferation, differentiation, and apoptosis. Deregulation of these signaling
    pathways were found in various kinds of cancers. Toward better
    understanding of these two pathways, we used computer modeling method to
    model kinetics of these pathways.
    Based on currently available models, we expanded the model in order to
    include more effects in kinetics of these pathways and correlate with available
    experimental data. First, we added a negative feedback loop on the EGFR
    pathway which has inhibition effect on Raf-1 by ERKpp. When the K1 value
    of Raf-1 inhibition reaction was set in the range of 0.01 to 10, the
    computations gave that addition of this loop results in two different effects in
    the two models we used. The negative feedback loop has a little effect on
    ERKpp level in the model which includes Braf. In contrast, the negative
    feedback loop makes the level of phosphorylated ERK go down in the model
    without Braf. Second, a crosstalk between EGFR and Wnt pathways was
    added and kinetic modeling gave that:in the case this is a positive feedback
    loop, it induces the switch-like behavior of ERKpp expression by varying the
    concentration of the added factor Y when the β -catenin was not
    overexpressed. In the case it is a negative feedback loop, due to the stronger
    v
    negative feedback effect, that during Wnt signal stimulate the concentration of
    ERKpp has an oscillation behavior with larger amplitude during the period of
    Wnt signal stimulation. After that, concentration of ERKpp falls back to low
    level quickly.
    In addition, we investigated effect of adding kinase inhibitor(s) on the
    level of phosphorylated ERK (ERKpp) under the condition of β-catenin
    overexpression plus undergoing a wnt signal transient stimulation. In the case
    of adding one kinase inhibitor, the modeling gave that:kinase inhibitor of
    multiple-targets of the same strength have less inhibitory effect than inhibitor
    of singlet-target of Raf-1 kinase. Reduction of concentration of ERKpp was as
    small as to 1/6 fold only compared with the case of singlet-target in the
    examined model. Furthermore, we investigate effects of multiple-target
    inhibitors inhibiting Raf-1 and MEK kinases. It was found that in the case the
    ratio of Raf-1 inhibitor concentration to MEK inhibitor concentration is larger
    than 1.5, the inhibitor effect is better than one inhibitor of multiple-target with
    the same inhibition strengths. These results deepen our understanding of these
    two pathways and should be useful for future multiple-target kinase inhibitor
    design.

    致謝 i 中文摘要 ii ABSTRACT iv 目錄(CONTENTS) vi 圖目錄 (LIST OF FIGURES) viii 表目錄 (LIST OF TABLES) xi Chapter 1 緒論(Introduction) 1 1.1 前言 (Preface) 2 1.2 Wnt pathway 4 1.3 EGFR pathway 8 1.4 回饋效應(Feedback Loop) 13 1.5 研究目標 (Research Objective) 15 Chapter 2 實驗方法 (Experimental Methods) 18 2.1 實驗理論 (Theories) 19 2.1.1 軟體介紹 (Software Introduction) 21 2.1.2 參數探討(Parameter Scan) 24 2.1.3 控制係數介紹(Control Coefficient) 26 2.1.4 回饋機制介紹 (Feedback Loop) 27 2.2 模型檢測/再現 (Model Reproduction) 28 Chapter 3 結果與討論 (Results and Discussions) 37 3.1 ERK-Raf負回饋效應在較大模型中的表現 38 3.2 模型外加新迴圈效應 47 3.3 模擬單一抑制劑進入訊息傳遞路徑 56 3.4 模擬多個抑制劑同時進入訊息傳遞路徑 79 Chapter 4 結論 (Conclusion) 84 Chapter 5 附錄與參考文獻(Appendix & Reference) 87

    1 Sancho, E., Batlle, E. & Clevers, H. Live and let die in the intestinal epithelium. Curr. Opin. Cell Biol. 15, 763-770, (2003).
    2 Almeida, M., Han, L., Bellido, T., Manolagas, S. C. & Kousteni, S. Wnt proteins prevent apoptosis of both uncommitted osteoblast progenitors and differentiated osteoblasts by beta-catenin-dependent and -independent signaling cascades involving Src/ERK and phosphatidylinositol 3-kinase/AKT. J. Biol. Chem. 280, 41342-41351, (2005).
    3 Thomas, S. M. et al. Cross-talk between G protein-coupled receptor and epidermal growth factor receptor signaling pathways contributes to growth and invasion of head and neck squamous cell carcinoma. Cancer Research 66, 11831-11839, (2006).
    4 Kolch, W. Coordinating ERK/MAPK signalling through scaffolds and inhibitors. Nature Reviews Molecular Cell Biology 6, 827-837, (2005).
    5 Fang, J. Y. & Richardson, B. C. The MAPK signalling pathways and colorectal cancer. Lancet Oncol. 6, 322-327, (2005).
    6 Downward, J. Targeting ras signalling pathways in cancer therapy. Nat. Rev. Cancer 3, 11-22, (2003).
    7 Jho, E. H. et al. Wnt/beta-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway. Mol. Cell. Biol. 22, 1172-1183, (2002).
    8 Leung, J. Y. et al. Activation of AXIN2 expression by beta-catenin-T cell factor - A feedback repressor pathway regulating Wnt signaling. J. Biol. Chem. 277, 21657-21665, (2002).
    9 Katanaev, V. L., Ponzielli, R., Semeriva, M. & Tomlinson, A. Trimeric G protein-dependent frizzled signaling in Drosophila. Cell 120, 111-122, (2005).
    10 Nusse, R. Cell biology - Relays at the membrane. Nature 438, 747-749, (2005).
    11 Paez, J. G. et al. EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science 304, 1497-1500, (2004).
    12 Ung, C. Y. et al. Simulation of the regulation of EGFR endocytosis and EGFR-ERK signaling by endophilin-mediated RhoA-EGFR crosstalk. FEBS Lett. 582, 2283-2290, (2008).
    13 Orton, R. J. et al. Computational modelling of the receptor-tyrosine-kinase-activated MAPK pathway. Biochem. J. 392, 249-261, (2005).
    14 De Falco, V. et al. RET/papillary thyroid carcinoma oncogenic signaling through the Rap1 small GTPase. Cancer Research 67, 381-390, (2007).
    15 Fu, Z. et al. Metastasis suppressor gene Raf kinase inhibitor protein (RKIP) is a novel prognostic marker in prostate cancer. Prostate 66, 248-256, (2006).
    16 Li, H. Z. et al. Effects of Raf kinase inhibitor protein expression on metastasis and progression of human epithelial ovarian cancer. Mol. Cancer Res. 6, 917-928, (2008).
    17 Shin, S.-Y. et al. Functional Roles of Multiple Feedback Loops in Extracellular Signal-Regulated Kinase and Wnt Signaling Pathways That Regulate Epithelial-Mesenchymal Transition. Cancer Research 70, 6715-6724, 2010).
    18 Shin, S.-Y. et al. Positive- and negative-feedback regulations coordinate the dynamic behavior of the Ras-Raf-MEK-ERK signal transduction pathway. Journal of Cell Science 122, 425-435, (2009).
    19 Garofalo, R. S. et al. Severe diabetes, age-dependent loss of adipose tissue, and mild growth deficiency in mice lacking Akt2/PKB beta. J. Clin. Invest. 112, 197-208, (2003).
    20 Yang, Z. Z. et al. Physiological functions of protein kinase B/Akt. Biochem. Soc. Trans. 32, 350-354, (2004).
    21 Faratian, D. et al. Systems Biology Reveals New Strategies for Personalizing Cancer Medicine and Confirms the Role of PTEN in Resistance to Trastuzumab. Cancer Research 69, 6713-6720, (2009).
    22 Kumar, N., Afeyan, R., Kim, H. D. & Lauffenburger, D. A. Multipathway model enables prediction of kinase inhibitor cross-talk effects on migration of Her2-overexpressing mammary epithelial cells. Molecular Pharmacology 73, 1668-1678, (2008).
    23 Orton, R. et al. Computational modelling of cancerous mutations in the EGFR/ERK signalling pathway. BMC Systems Biology 3, 100 (2009).
    24 Kim, D., Rath, O., Kolch, W. & Cho, K. H. A hidden oncogenic positive feedback loop caused by crosstalk between Wnt and ERK Pathways. Oncogene 26, 4571-4579, (2007).
    25 Sturm, O. E. et al. The Mammalian MAPK/ERK Pathway Exhibits Properties of a Negative Feedback Amplifier. Science Signaling 3,
    (2010).
    26 Vera, J. et al. Investigating dynamics of inhibitory and feedback loops in ERK signalling using power-law models. Molecular BioSystems 6 (2010).
    27 Avraham, R. & Yarden, Y. Feedback regulation of EGFR signalling: decision making by early and delayed loops. Nature Reviews Molecular Cell Biology 12, 104-117, (2011).
    28 Naruo, Y. et al. Epidermal growth factor receptor mutation in combination with expression of MIG6 alters gefitinib sensitivity. BMC Systems Biology 5,(2011).
    29 Back, T. & Schwefel, H. P. An Overview of Evolutionary Algorithms for Parameter Optimization. Evol. Comput. 1, 1-23, (1993).
    30 Zobeley, J., Lebiedz, D., Kammerer, J., Ishmurzin, A. & Kummer, U. A new time-dependent complexity reduction method for biochemical systems. Transactions on Computational Systems Biology I, 90-110 (2005).
    31 Hoops, S. et al. COPASI- A COmplex PAthway SImulator. Bioinformatics 22, 3067-3074, (2006).
    32 Lion, S. et al. An extension to the metabolic control theory taking into account correlations between enzyme concentrations. Eur. J. Biochem. 271, 4375-4391, (2004).
    33 Gibson, M. A. & Bruck, J. Efficient exact stochastic simulation of chemical systems with many species and many channels. J. Phys. Chem. A 104, 1876-1889, (2000).
    34 Tyson, J. J., Chen, K. C. & Novak, B. Sniffers, buzzers, toggles and blinkers: dynamics of regulatory and signaling pathways in the cell. Curr. Opin. Cell Biol. 15, 221-231, (2003).
    35 Brown, K. S. et al. The statistical mechanics of complex signaling networks: nerve growth factor signaling. Phys. Biol. 1, 184-195, (2004).
    36 Park, S., Yeung, M. L., Beach, S., Shields, J. M. & Yeung, K. C. RKIP downregulates B-Raf kinase activity in melanoma cancer cells. Oncogene 24, 3535-3540, (2005).
    37 Zaravinos, A., Bizakis, J. & Spandidos, D. A. RKIP and BRAF aberrations in human nasal polyps and the adjacent turbinate mucosae. Cancer Letters 264, 288-298, (2008).
    38 Burack, W. R. & Sturgill, T. W. The activating dual phosphorylation of MAPK by MEK is nonprocessive. Biochemistry 36, 5929-5933, (1997).
    39 Davies, S. P., Reddy, H., Caivano, M. & Cohen, P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351, 95-105, (2000).
    40 Schoeberl, B. et al. Therapeutically Targeting ErbB3: A Key Node in Ligand-Induced Activation of the ErbB Receptor-PI3K Axis. Science Signaling 2, (2009).
    41 Gould, A. E. et al. Design and Optimization of Potent and Orally Bioavailable Tetrahydronaphthalene Raf Inhibitors. J. Med. Chem. 54, 1836-1846, (2011).

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