探討Rhotekin基因於癌症病變過程中所扮演的角色

Abstract

胃癌全球罹患率與致死率皆高居癌症的第二位，但我們對胃癌之致病機轉了解仍有限。實驗室以差異性表現分析，發現Rhotekin (RTKN) 蛋白在胃癌組織中表現量高於週邊正常組織。目前對於 RTKN 的功能研究知道其會和 Rho、 Tax interacting protein-1 一起促進 serum response elememt 的活化，而我們實驗室之前則發現有RTKN 大量表現的細胞具有抗凋亡的能力可能是因 RTKN 影響NF-κB的活性，另外也發現 RTKN 降低了 RhoA 下游 ROCK 的活性並造成Rac 活性上升而影響細胞骨架的重整，減少 stress fiber 生成並增加細胞 surface protrusion 和移動的能力。在本論文中，我們進一步探討 RTKN 在細胞轉型(cell transformation) 及遷移(migration)過程中所扮演的角色。我們根據細胞株中所擁有RTKN 的表現量挑選出合適的細胞株，分別在低表現量的細胞株(H1299, AZ-521)內大量表現RTKN以及在高表現量的細胞株(NUGC-3)內利用sh-RNA 專一性抑制內生性的RTKN 之表現。初步發現，過度表現RTKN 使得細胞喪失接觸性抑制(contact inhibition of growth)而造成細胞飽和度增加，促使細胞骨架重整造成細胞型態改變，增加細胞 遷移(Migration)及細胞入侵(Invasion)之能力，並且發現細胞過度表現RTKN 促進其非固著性依賴生長(anchorage-independent growth)之能力。這些現象說明了RTKN 參與了細胞轉型(transformation)及遷移(migration)，因此可能與胃癌生成以 及轉移惡化有關。我們藉由本研究之分析RTKN 所給予細胞轉型之特性，建議RTKN 於胃癌生成或惡化中扮演一重要的角色及可能之機制。

Gastric cancer is ranked the fourth common cancer and the second leading cause of cancer death worldwide (almost 1 million deaths/year).Using ordered differential display, we have identified rhotekin, the gene encoding Rho effector rhotekin (RTKN) as one of the genes overexpressed in human gastric cancer. To further understand the role of Rho/RTKN involved in the pathogenic development of GC, in this study, we propose to dissect Rho signaling pathways that are mediated by RTKN, and special efforts will be focused on the ones contribute to malignant transformation and tumor metastasis. To assess the function of RTKN in cellular transformation, we ectopically expressed RTKN in cultured cell, and monitored the biological readouts of RTKN expression. The data shown, RTKN expression suppressed contact inhibition of movement in both H1299 as well as HEK293 cells. RTKN In consistence, overexpression of RTKN conferred an increased saturation density in H1299 cells. Overexpression induced extensive cell surface protrusions with a concomitant decrease of stress fibers and focal adhesions. We have further generated stable clones : H1299 and AZ-521, RTKN low expresser that stably expressed RTKN, and NUGC-3 , RTKN high expresser that stably knock downed RTKN, and tested for transforming activity. The stable clone displayed increased migration and invasion ability when compared to the parental cells. In addition, the stable clone developed anchorage-independent growth in soft agar whereas the parental cells showed reduced growth in soft agar. Taken together, we conclude that overexpression of RTKN may promote cell transformation and cell migration, and thus and thus contribute to tumor metastasis.