2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38608To discover novel metastasis suppressor genes that are clinically relevant in human oral cancer, we used human oral cancer cells and employed DNA microarray technology to identify genes whose expression diminishes as a function of metastasis. We then evaluated the expression profile and identified one of these diminished genes, RhoGDIβ, whose expression was down-regulated in oral cancer cells. The greatest RhoGDIβ loss in invasive and metastatic cancer tissue might link the RhoGDIβ gene to cancer metastasis. I propose that cells with the restore the expression of RhoGDIβ might lose their ability to metastasize. In this regard, I will set out to study the biochemical features and functional roles of RhoGDIβ, which might be involved in the development of the metastasis of oral cancer. Furthermore, in another set of experiment we found RhoGDIβ might associate with ZAK through yeast two-hybrid experiment. ZAK is a novel mixed lineage kinase-like protein, containing a leucine-zipper (LZ) and a sterile-alpha motif (SAM), was cloned in the lab. ZAK gene encodes a protein of 800 amino acids that contains a kinase catalytic domain, a leucine-zipper and a SAM. ZAK is proposed to function as mitogen-activated protein kinase kinase kinase in pathways leading to MAPK cascade. The expression of ZAK in mammalian cells specifically leads to the activation of the JNK/SAPK pathway as well as the activation of transcription factor, NF-κB. ZAK activates JNK/SAPK mediated by downstream target, MKK7. Moreover, ZAK activity may be involved in actin organization regulation. In this regard, the primary goal of this project is to study on the regulation of ZAK on this ZAK associated protein (RhoGDIβ) that might play its role on the development of metastasis and I will conduct comprehensive studies for the next three years to fulfill this proposal. For the first year, I will focus on the investigation of the expression of RhoGDIβ in oral cancer cells compared with normal counter parts and study the mechanisms of RhoGDIβ on the regulation of migration, invasion and metastasis of oral cancer cells and in nude mice. For the second year, I will focus on the determination on the physical binding site in ZAK for RhoGDβ. Characterize the function of Rac in the development of metastasis stimulated by RhoGDIβ. Moreover, analyze the gene expression profile in RhoGDIβ-expressing cell. The studies on the mechanisms of RhoGDIβ to induce Rac mRNA expression will be addressed. The final year, employ siRNA to silence RhoGDIβ and ZAK expression in oral cancer cells. To determine if silencing of these genes might affect the development of metastasis. Moreover, to map the region on ZAK required for binding to RhoGDIβ and determine the putative phosphrylation sites in RhoGDIβ. RhoGDIβ associated proteins will be determined by using yeast-two hybrid techenology for further study RhoGDIβ signal transduction pathways.為了探討在人類口腔癌中，新的轉移抑制基因有臨床的重大意義，我們使用人類口腔癌 細胞、利用DNA 微陣列技術(microarray technology)去確認使轉移功能表現降低的基 因。接著我們鑑定此表現的圖形數據，確認這些減弱的基因其中之一，RhoGDI　，在 口腔癌細胞中的表現是down-regulated 的。在侵略性及轉移的腫瘤組織裡，此 RhoGDI　的減少可能與腫瘤轉移有關。RhoGDI　重新表現的細胞可能失去轉移的能 力。我將開始研究RhoGDI　與肺癌轉移發展相關的生化特徵及其所扮演的功能性角 色。除此之外，本實驗室透過yeast two-hybrid 實驗證明RhoGDIβ 能與某一蛋白質激 酶, ZAK,結合。 ZAK 基因是一個含有800 個胺基酸蛋白質激脢，其中包含了一個蛋白質激脢區、一個 Leucine zipper、及一個SAM 區。ZAK 是屬於MAPKKK 家族，能夠活化MAPK 訊息 傳導途徑。在本實驗室發現，當細胞表現ZAK 時，能夠同時活化JNK/SAPK 訊息傳導 途徑及活化NF-κB 轉錄因子。當ZAK 活化JNK/SAPK 途徑時需要利用MKK7。另外 ZAK 亦能調節細胞骨架actin 的結構。 本計劃的主軸為研究ZAK 與腫瘤轉移抑制蛋白(RhoGDIβ)之間的交互關係及其在調控 腫瘤轉移之研究。我們將利用三年的時間來完成此研究；第一年，我們將比較正常細胞 及轉移口腔癌細胞中RhoGDIβ 之表現，以期了解RhoGDIβ 的表現與轉移之關係，並 利用細胞培養及裸鼠系統來研究RhoGDIβ 調控癌細胞爬行、侵襲及轉移之機制。第二 年，我們將探討ZAK 與RhoGDIβ 之間的關係。首先研究ZAK 與RhoGDIβ 之間的結合 區域，並且嘗試研究Rac 蛋白在RhoGDIβ 中造成癌細胞轉移的機制。同時利用微矩陣 研究RhoGDIβ 所調控的基因組，並研究Rac 的表現是否由RhoGDIβ 所調控。爾後我 們將利用yeast two-hybrid 實驗找出在細胞中與RhoGDIβ 結合的蛋白質，以期更加了 解RhoGDIβ 在細胞中訊息傳導的機制。第三年，將利用RNAi 技術來減低RhoGDIβ 或 ZAK 的表現，以期了解ZAK 與RhoGDIβ 表現降低時對癌細胞轉移的影響，並研究 RhoGDIβ 被磷酸化的位置，以期了解ZAK 對RhoGDIβ 的調控機制。Project