Wnt-EGFR訊息傳遞途徑動力學計算研究暨抑制劑效應模擬

Abstract

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.