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標題: 探討PAK2的上游調控機制及其對於肺癌進程的影響
To investigate the upstream mechanism of PAK2 and it's function in lung cancer progression
作者: 林德珊
Te-Shan Lin
關鍵字: 肺癌;Lung cancer;PAK2
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Mutat Res 752, 10-24
肺癌,是近年來最具高發生率和極高死亡率之惡性腫瘤,癌症轉移則是導致病患死亡的主因。過往YWHAZ (14-3-3ζ) 蛋白已被證實在肺腺癌中藉由透過結合β-連鎖蛋白 (β-catenin) 與轉錄因子T-cell factor 4 (TCF-4) 在核內結合並形成複合體,進而促進肺癌轉移與侵襲能力。先前實驗室透過即時定量聚合酶反應 (QPCR) 與微陣列分析 (Microarray) 交叉比對後,找出YWHAZ-β-catenin-TCF4複合體的可能下游調控基因p21-蛋白活化激酶2 (p21 protein-activated kinase 2, PAK2)。PAK2屬絲氨酸/蘇氨酸蛋白激酶,可被多種上游信號如G蛋白Rho家族的Rac和Cdc42啟動,在進化上高度保守。PAK2已被證實在多種腫瘤中均有異常表達,並參與多種調控腫瘤發生的細胞過程包括細胞增殖、細胞凋亡以及細胞骨架的重組,然而目前對PAK2在肺癌轉移上機制仍然不清楚。因此,為了探討YWHAZ-β-catenin-TCF4複合體對PAK2在腫瘤中的調控機制,首先利用生物資訊學方法分析PAK2轉錄起始位置及預測啟動子上的TCF-4結合區域,以聚合酶連鎖反應合成PAK2啟動子-2077/+59片段。接著利用5'片段缺失構築及共同表現β-catenin基因或YWHAZ基因以進行冷光報導基因分析,結果發現在PAK2啟動子-2077/-1547為重要調控轉錄活性區域,而-478/+59區域具一定活性。利用TRANSFEC軟體及JASPAR網站對這兩區域分析,發現具兩個TCF-4轉錄因子結合位-1985/-1977 與-1467/-1459,點突變結果顯示可能透過TCF-4位點以調控PAK2。接著於CL1-0中建立大量表現PAK2之穩定細胞株P1、P2、P3、P4用來進行細胞功能試驗。免疫螢光染色顯示PAK2表現於細胞質和細胞核內。在非依賴性與依賴性細胞聚落形成試驗結果皆顯示大量表現PAK2具顯著的聚落形成。細胞遷移能力試驗不論transwell或傷口癒合試驗,PAK2大量表現時會增加細胞移動的能力,確認PAK2在肺癌中會促進腫瘤的生長並促進細胞的移動能力。

Lung cancer is the leading deceases of the cancer-related death worldwide. However, the prognosis for lung cancer patients is rather poor by lack of early marker detected. To investigate the mechanisms involved in tumorigenesis and metastasis would be necessary for patients' therapy. In our group's previous studies, it has been found that YWHAZ associates with TCF-4 and β-catenin in the nucleus, and YWHAZ-β-catenin-TCF-4 complex further promote EMT, cell invasion and migration in lung cancer. Also, previous studies reveal that YWHAZ-β-catenin-TCF-4 complex can regulate four candidate genes: ATF3, DMTF1, DUSP1 and PAK2.
PAK2, also known as p21-activated kinases 2, belongs to the PAKs family of serine/threonine kinases which can be activated by small G proteins: Rac and Cdc42. PAK2 is believed to promote tumorigenesis through regulatin cell proliferation, apoptosis and cytoskeleton remodeling. In order to investigate the upstream mechanism of PAK2, luciferase reporter and site-directed mutagenesis assay were used to check promoter activity. At first, PAK2 promoter region from -2077 to +59 was cloned. Then, three 5' terminal deleted fragment promoters were constructed. Luciferase assay results demonstrated that there is an important regulation region in -2077/-1547 fragment, and core promoter region of PAK2 is located at -478 bp to +59 bp. Furthermore, two TCF-4 binding motif sites -1467 and -1985 were predicted by TRANSFAC analysis. Both mutation vectors of TCF-4 binding site showed significantly lower luciferase activity compared with the wild type, indicating PAK2 may be activated by TCF-4. While PAK2 promoter region co-expressed with β-catenin or YWHAZ were analyzed, it is interesting to note the results show PAK2 may not be regulated by β-catenin but by YWHAZ. To investigate the function of PAK2 in lung cancer progression, stable cell lines were established. Overexpressing PAK2 in CL1-0 cell enhances cell colony formation and migration. In conclusion, PAK2 promoted cell motility by regulated of YWHAZ-TCF-4 interaction in lung cancer.
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