- 學位論文
分泌型Frizzled相關蛋白1和3對Wnt訊號傳導路徑調控癌細胞的幹細胞特性和腫瘤新生能力中所造成的影響
The Effect of Secreted Frizzled-related Protein 1 and 3 on the Wnt Signaling in Modulating Stemness and Tumor-initiating Abilities of Cancer Cells
摘要
根據我們的研究結果發現，sFRP蛋白主要是依靠位在N端的cysteine-rich domain (CRD) 與Wnt3a做結合，而C端netrin-related (NTR) domain則是能與β-catenin做結合。在Luciferase reporter assay所得到的實驗結果證實，大量表現sFRP1全長蛋白能將TopFlash的活性顯著地抑制到幾乎和基準值一樣低，而sFRP1 N端和sFRP1 C端的剪切型蛋白也能抑制TopFlash的活性。在sFRP3全長蛋白持續表現的細胞株中，其TCF/β-catenin轉錄活性是被增強的，然而sFRP3 N端和sFRP3 C端的剪切型蛋白卻是高度抑制TopFlash的活性的。另外，我們實驗結果發現持續表現sFRP1及sFRP3全長蛋白會拮抗Wnt3a活化LRP6 co-receptor，使其LRP6 co-receptor磷酸化水平降低。而我們也觀察到經由剪切後的sFRP蛋白也會調控Wnt3a活化LRP6 co-receptor，存在sFRP3 N端的剪切型蛋白會抑制LRP6 co-receptor磷酸化水平，而sFRP1 C端的剪切型蛋白能夠提升LRP6 co-receptor磷酸化水平。在LRP6訊號下游的glycogen synthase kinase-3β (GSK-3β) 活性亦會受到sFRP蛋白的調控，表現sFRP1全長蛋白會抑制GSK-3β磷酸化，反之表現sFRP3全長蛋白會促進GSK-3β磷酸化。我們也觀察到存在sFRP1的全長、N端、C端以及sFRP3全長和C端蛋白都會促進β-catenin的穩定性，使β-catenin能在細胞質累積並進入細胞核中。而Sphere-forming assay的結果顯示，sFRP1全長蛋白會抑制sphere的形成，相反地，sFRP3全長蛋白則是會促進sphere-forming的能力。綜合以上的實驗結果得知，sFRP蛋白能夠影響Wnt訊息傳導路徑的活化，而這樣的影響是經由不同的機轉來調控Wnt訊息傳導路徑的活性。我們在此提供了一些嶄新的方法來檢視其生物學上的影響和作用方式，並且了解sFRP1和sFRP3在調控Wnt訊息傳導路徑中所扮演的角色以及對於癌細胞的幹細胞特性和腫瘤新生能力的影響。
並列摘要
Based on the results of our study, sFRP proteins have an association with Wnt3a through cysteine-rich domain (CRD) which is located at the N terminus; while the C terminus [netrin-related (NTR) domain] associates with β-catenin. The result derived from Luciferase reporter assay proves that overexpression full-length sFRP1 can apparently inhibit the level of TopFlash reporter activities to be almost as low as the basal level. In addition, the deletion mutants, the N and C terminus of sFRP1, also inhibit activities of TopFlash reporter. In the sFRP3-overexpressed stable clone, the transcriptional activity of TCF/β-catenin is enhanced, whereas the deletion mutants, the N and C terminus of sFRP3, highly inhibit activities of TopFlash reporter. Furthermore, our data showed that sFRP1 and sFRP3 antagonize Wnt3a-elicited LRP6 co-receptor activation, which also leads to the fall in its phosphorylation level. Interestingly, we observed that the deletion mutants of sFRPs would modulate Wnt3a-elicited LRP6 co-receptor activation. The N terminus of sFRP3 inhibits the phosphorylation level of LRP6 co-receptor, whereas overexpression the C terminus of sFRP1 enhances the phosphorylation level of LRP6 co-receptor. The activity of glycogen synthase kinase-3β (GSK-3β) which is in the downstream of LRP6 is also modulated by sFRP proteins. Moreover, it showed that overexpression sFRP1 could inhibit GSK-3β phosphorylation, whereas overexpression sFRP3 enhances the phosphorylation level of GSK-3β. Also, we observed that the full length, N, and C terminus of sFRP1 and the full length and C terminus of sFRP3 would stabilize β-catenin, which contributes to β-catenin accumulation in the cytosol and further entering into the cell nucleus. As the result showed in the sphere-forming assay, full-length sFRP1 would inhibit the formation of sphere. On the contrary, full-length sFRP3 facilitates the capability of sphere-forming. From what has been mentioned above, we can come to the conclusion that sFRP proteins are capable of modulating the Wnt signaling pathway. They control the activity of Wnt signaling pathway through different mechanisms. In this study, we provide several brand-new approaches to survey the biological effects of sFRPs. Furthermore, these findings characterize the roles of sFRP1 and sFRP3 involved in regulating Wnt activities and provide a new perspective regarding their biological effects and mode of action, distinguished by capable of modulating stemness and tumor-initiating abilities.
並列關鍵字
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