A431癌細胞之纖維連接蛋白素及轉麩胺酵素2表現量 與細胞轉移潛力之關係探討

Abstract

癌細胞的入侵與轉移，始終是治癒癌症最大的難題。之前文獻顯示，腫瘤的Fibronectin (FN) 表現量與癌細胞入侵能力及Matrix Mmetalloproteinases (MMPs) 的釋放量有關。為了癌症轉移的研究，實驗室已建立一套完整系統: 利用Boyden chamber assay從母代A431癌細胞 (A431P) 經三次循環篩選出更具入侵能力的細胞株 (A431III)。同時A431III也被發現會分泌更多的MMP-9以及具有較強的移動、貼附與延展能力。然而，這些特性增強的原因仍處於未知的階段。 此篇研究，我們觀察到A431III內生性FN的表現量與細胞外分泌量有大量提高的趨勢。利用siRNA技術來抑制FN表現量的情況下，顯示A431III的高入侵能力有完全被抑制的現象，其傷口癒合能力也有明顯減弱的趨勢。 Tissue transglutaminase (TG2) 傳統被認為是催化蛋白與蛋白間連接的酵素，最近被發現與腫瘤生成有相關性。2000年一篇報告顯示，TG2被發現具有另一項新的功能: 在細胞外扮演FN與Integrin相互連結的一個重要酵素。因此我們想進一步去研究A431P與A431III中TG2的表現量以及TG2與FN、Integrin的作用能力是否有所差異。實驗顯示，與大部分報告指出TG2隨著癌症轉移惡化而表現量減少的結果相反，TG2在較強入侵力A431III的表現量約是A431P的四倍。此外，TG2、FN及Integrin三者間的相互作用程度，在A431III中都比A431P要高出許多。利用siRNA來抑制TG2表現量的情況下，我們發現A431III原本較高的MMP-9分泌量、FN-Integrin作用力、FAK phosphorylation以及入侵、貼附、延展、移動能力都有降低的趨勢，甚至降到A431P的程度。 綜合以上結果，我們推測: A431III中TG2上升所增強的FN-Integrin交互作用，會活化FAK等相關的訊息傳遞，促使細胞入侵能力的上升。此外也顯示，本實驗室利用Boyden chamber assay所篩選出更具入侵力的癌細胞株，將會是一個研究癌症轉移相關機制的有利工具與系統。

The development of invasion and metastasis is a major impediment to the successful treatment of cancer. An increase in fibronectin (FN) expression in human tumor has been shown to be correlated with a high potential of tumor cell invasion and elevated matrix metalloproteinases (MMPs) secretion. We have previously shown that human A431 III sub-line isolated from the parental A431 cell (A431P) using Boyden chamber secreted higher levels of MMP-9 than A431P. The A431III cells exhibit elevated migratory, adherent, and spreading characteristics. However, the molecular mechanisms of highly metastatic features in A431III cells have not been fully understood. In this study, we observed that the amounts of endogenous FN in A431III sub-line were significantly higher than those in A431P cells. Moreover, we found that knock-down of endogenous FN by siRNA resulted in totally inhibition of the invasive potential and decreased the ability of wound healing in A431III cells. Since tissue transglutaminase (TG2) has been found to play an important role in tumor progression and could also act as a coreceptor for integrin-mediated binding of cells to FN, we further planed to investigate the roles of TG2 and its relationship with integrins in the potentials of cancer cell invasion and metastasis in highly invasive A431 cancer cell models. On contrary to other previous studies that showed that a decrease of TG2 expression and activity was accompanied by the increasing metastatic potential. Interestingly, our study showed that TG2 levels were significantly increased in A431III cells compared to A431P cells. Furthermore, the interactions of FN, TG2, and integrin β1/β3 were markedly higher in A431III than in A431P cells. After the knockdown of TG2, the invasive ability, MMP-9 secretion, the interaction between FN and integrin β1, FAK phosphorylation, and highly metastasis-related appearances of A431III were decreased to the levels as that of A431P cells. Thus, our results indicate that increased expression of TG2 could enhance association of FN with integrins and trigger integrin-mediated outside-in signaling, at least in part leading to enhancement of the metastatic potentials in A431III cells. Our data suggest that the highly invasive A431III sub-line would be an excellent model for investigating the mechanism of tumor metastasis through FN and TG2-mediated signaling.