ARD1 Inhibits Lung Adenocarcinama Metastasis through Reducing Cdc42/Rac1 Activity and Reorganizing F-actin Cytoskeleton Structure.

肺癌為世界十大癌症死因之首，導致肺癌死亡率居高的原因，主要為癌細胞之遠端轉移率高而造成治療失敗。因此研究抑制癌細胞轉移之機轉，在癌症治療領域佔有重要的角色。癌細胞轉移的病理機制包括細胞移動、浸襲、血管新生、及細胞增生。不同型態的細胞轉移會受到不同的機轉調控。找出抑制腫瘤浸襲基因並釐清其機制，可做為作為病理診斷上的新方法，及未來治療癌症的方向。 F-肌動蛋白結構的改變是導致細胞移動的主要機轉。F-肌動蛋白的重組主要受到小型G蛋白質所調控，如Rho、Rac1及Cdc42。小型G蛋白活性可傳遞細胞外的化學訊息至下游的接受者，而使細胞骨架結構重組，因此抑制小型G蛋白質酵素活性可減少癌細胞轉移及浸襲的能力。 ARD1 在1985年即被發現在酵母菌中扮演著轉換細胞有絲分裂及發育機轉之角色，ARD1和其共同作用者NATH可形成一個穩定的結構，此結構具有N基乙醯轉移酵素之活性，並參與許多生物性功能如細胞分化，熱敏感性，細胞凋亡等。本篇研究結果顯示，臨床上分析ARD1蛋白表現量較低的病人，與癌病惡性度較高，有淋巴轉移，較早癌病復發率及較短存活時間有正相關。ARD1在非肺癌腫瘤部位的蛋白表現量較腫瘤組織表現量高，在統計上也有顯著的意義（p<0.05）。在體外細胞株實驗當中，ARD1的核糖核酸及蛋白表現量和轉移浸襲能力有高度負相關性。在ARD1過度表現之轉殖細胞株中發現顯著降低細胞的轉移及浸襲能力；相反的在ARD1剔除轉殖株中則促進癌細胞轉移能力。進一步探討ARD1所調控之轉移能力之機轉則發現，ARD1可降低小型G蛋白質的活性，而導致F-肌動蛋白骨架重整，降低filopodia的形成即使細胞轉變為非轉移性型態而有效抑制細胞轉移能力。我們也證明了ARD1本身之N基乙醯轉移酵素活性並不影響其調控細胞轉移及浸襲之能力。最後藉由動物實驗，再次印證ARD1在活體實驗當中也有效抑制癌細胞轉移能力。因此，ARD1為一個新發現之抑癌基因並可作為將來治療癌症轉移的標的因子。Lung cancer is by far the most common cause of cancer death in the world, and metastasis is the major cause of treatment failure and death in cancer patients. The pathogenesis of cancer metastasis involves cell migration, invasion, angiogenesis, and cell proliferation. Different types of cell migration are regulated by different mechanisms. Identification of novel tumor migration-associated genes and elucidation of their mechanism of action may provide new insights into the pathogenesis and management of cancer metastasis. Reorganization of the actin cytoskeleton is the primary mechanism of cell motility and is essential for most types of cell migration. Actin reorganization is regulated by Rho family small GTPases such as Rho, Rac, and Cdc42. These small GTPases transmit extracellular chemotactic signals to downstream effectors. Inhibition of Rho family small GTPase signaling suppresses the migration and invasion of cancer cells. Thus, control of cell migration via the actin cytoskeleton provides the possibility of regulating cancer cell migration and metastasis. Arrest defective protein 1 (ARD1) is first described in Saccharomyces cerevisiae. It is characterized as an N-acetyltransferase and forms heterodimer with human N-acetyltransferase (NATH). The ARD1-NATH complex shows N-acetyltransferase activity and involves in many biological processes such as cell cycle regulation, viability, temperature sensitivity, and differentiation. We recently identified ARD1 as a novel migration suppressor and a prognosis marker of metastasist in lung adenocarcinoma patients. In the present study, we found that ARD1 was expressed higher in non-tumor part than matched tumor part of lung adenocarcinoma patients and reversely correlated with the migration and invasion abilities in lung adenocarcinoma cell lines. We demonstrated that ARD1 inhibited migratory and invasive abilities, and depolymerized F-actin structure through reducing Rho family Cdc42 and Rac1 GTPases activities. Moreover, in vivo animal model showed that ARD1 inhibited distal organ metastasis and primary tumorigenesis. In conclusion, we considered ARD1 as a tumor suppressor gene and it inhibited cancer cell migration and metastasis through reducing Cdc42 and Rac1 activity together with F-actin structure reorganization. Therefore, ARD1 may be a potential target for cancer treatment.Content 中文摘要………………………………………………………………………………2 Abstract………………………………………………………………………………..3 Preface………………………………………………………………………………...5 Ⅰ. Human Lung adenocarcinoma…………………………………………5 Ⅱ. Tumor metastasis……………………………………………………..…7 Ⅲ. Arrest defective protein 1 (ARD1)………………………………….….9 Ⅳ. Rho family small G proteins……………………………………….…..11 Chapter：ARD1 Inhibits Lung Adenocarcinoma Metastasis through reducing Cdc42/Rac1 Activity and Reorganizing F-actin Cytoskeleton Structure. Introduction………………………………………………………………..13 Materials and Methods……………………………………………………17 Results……………………………………………………………………...26 Discussion……………………………………………………………….…38 Figures and Figure Legends……………………………………………...42 Tables………………………………………………………………….…...67 Reference………………………………………………………………..…6