2012-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643526摘要：上皮-間質轉化 (EMT)是一種由極化的上皮細胞群轉變為鬆散而能移動的纖維細胞樣的細胞之過程。而這種過程後來發現在腫瘤之進展，包括侵犯和轉移，亦扮演重要角色。因此若能透過全基因方式了解上皮-間質轉化過程之詳細基因表現變化，將有助我們了解胚胎發育及腫瘤發展的複雜機制與變化。我們於上一個計劃中,與M.D AndersonCancer center 的洪明奇院士的合作中,運用Snail 突變穩定轉殖細胞株模式,透過微矩陣的方法找到潛在可能於Snail 引發的上皮間質轉移過程中會過度表達或減少表達的基因,初步於這些基因中,我們進一步探索其臨床病理與預後影響及生物學意義.當中,我們發現過度表達基因中的moesin ,UCHL1 ,SPARC,其蛋白(免疫染色)與RNA (微矩陣)的量都特別高表達於轉化型乳癌(metaplastic carcinoma),而此種乳癌亞型正是目前已被了解於病理病因上與上皮間質轉化過程高度相關的乳癌亞型.更甚者,我們發現在一般最常見的腺管型乳癌 (ductal carcinoma)中,這3 種經由此Snail 轉殖模式找到的EMT高表達基因的表現是與basal phenotype 高度相關,且與不良存活高度相關.我們進一步在乳癌細胞株上證實 Snail 的確可以誘發 moesin 及 UCHL1 的表達,而且在38 個原發乳癌的微矩陣資料中發現 Snail mRNA 量的確與moesin 及 UCHL1 高度相關.這些資料強烈支持透過這個 Snail 轉質模式所找到的潛在EMT 高表達基因的確有其臨床病理及預後意義,也高度值得再於此計畫中進一步再探索(Arm 1).而於進一步尋找這些基因的生物學意義的過程中,我們透過一系列功能性研究(functional assays),包括 MTTassay, wound healing migration assay , transwell invasion assay ,3-D collagenassay ,soft agar colony-formation assay 及 MTT chemosensitivity ,發現於乳癌細胞株中減少 (knockdown ) UCHL1 會減少其侵略性 (invasiveness),而把 UCHL1 gene置入(knock in)一株沒 UCHL1 表達的乳癌細胞株M10,則會增加其侵略性,這些功能性研究的結果強烈支持為何乳癌腫瘤組織表達 UCHL1 會與 basal phenotype 及不良存活預後有關.因此,依據這些已有的資料結果,我們相信透過這種 Snail 轉職模式找到的EMT 相關基因,不只是高表達(uprgulation),而且包括低表達(downregulation)的基因,應該都有其重要的臨床病理與預後的意義,且可經由in vitro 證實其實際與Sanil 的引起相關 (casual effect),因此我們將在此計畫中在已有的被驗證 (validated )的方法基礎上(functional assays)進一步在探索這些EMT-related 基因的生物學意義(Arm 2).此外,在上個計劃中,我們亦探索 Snail-mediated EMT 的epigenetic 機制,我們發現在此 Snail 轉殖 model 中,許多與維持上皮型態有關的基因,包括 E-cadherin ,TACSTD1 ,claudin 3, 4, 及 7, CRB3 及PPl 的基因啟動子(promoter) 在Snail-mediatel EMT 中出現高度DNA 甲基化,進而被抑制表達,而當這些Snail-mediatelEMT 細胞給予 Aza ,一種 DNA methyltransferase 抑制劑,則此種甲基化會被移除,而細胞重新表現上述這些被抑制的基因. 更重要的是我們運用免疫沉降(immunoprecipitation)及染色質免疫沉降(chromatin immunoprecipitation)發現於EMT 的乳癌細胞中, Snail 會與 DNA methyltransferase 3a 結合於這些上皮相關基因的promoter 上,而造成DNA 甲基化.此一結果為 Snail-mediatel EMT 的epigenetic 機制提供了重要的解釋.除了 epigenetic 機制,有愈來愈多的資料顯示 genetic 機制在 EMT亦變演重要的角色,因此於此一新計畫中,我們將進一步探索 EMT 的genetic 機制(Arm3).我們計畫以轉化型乳癌為材料,因為此乳癌亞型被認為基病理病因與 EMT 高度相關.我們計畫運用 array CGH (comparative genomic hybrirdization )的方法,分析轉化型乳癌的上皮癌處 (carcinomatous component)與肉癌處 (sarcomatous component)的gemomic 不同處,而後者正是被認為由前者發生EMT 而來 因此透過此方法 我們預期能找到與EMT 有關的基因變異 (genetic profiles ),這將有助於我們更進一步了解 EMT的機制 .<br> Abstract: Epithelial-mesenchymal transition (EMT), originally discovered from studies of embryonicdevelopment, is the process of disaggregating structured polarized epithelial units into singlemotile fibroblastoid cells to enable cell movement and morphogenesis. The process of EMThas later gained wide recognition as a potential mechanism for the progression of malignancy,for example invasion and metastasis. Accordingly, a better understanding of the EMT-relatedgenes identified through a genome-wide approach would contribute to the knowledge of thecomplex pathophysiology of EMT underlying development and cancer progression. In ourprevious project (98-2320-B-002-052-MY3), by using the Snail variant MCF7-6SA cell linesystem as a model (in collaboration with Dr. Hung Mien-Chie in MD Anderson CancerCenter), we have identified genes upregulated and downregulated in Snail-induced EMT bymicroarray. We have in part explored the clinicopathologic and biologic role of some of thegenes and in part investigated the mechanism of Snail-mediated EMT. Specifically, among thegenes upregulated in Snail-mediated EMT, we have shown that the protein level (byimmunohistochemistry) and RNA level (by oligonucleotide microarray) of moesin, ubiquitincarboxyl-terminal esterase L1 (UCHL1) and SPARC are significantly highly correlated withmetaplastic carcinoma (MCB) which has been shown to be pathogenetically closely related toEMT. Importantly, we have further shown in breast cancer cohort that the expression of thesethree proteins is significantly correlated with basal phenotype and poor overall survival.Furthermore, we have further confirmed in vitro in breast cancer cell line that moesin andUCHL1 can be upregulated by Snail. The in vitro data validate the in vitro finding of moesinand UCHL1 as upregulated genes in Snail-mediated EMT (by oligonucleotide microarray)and the in vivo finding of the differentially high expression (by IHC and microarray) ofmoesin and UCHL1 in MCB. In line with the in vitro causal relation between Snail andmoesin and UCHL1, we observed statistically significant correlation between the mRNAlevels of Snail and moesin and UCHL1 in a microarray data base based on 38 invasive breastcarcinoma. To validate the biological significance of EMT-related genes identified throughsuch an approach, we have shown, through a series of functional assays, including MTT assay,wound healing migration assay, Transwell invasion assay, 3-D collagen assay, soft agarcolony-formation assay and chemosensitivity MTT assay, that knockdown of UCHL1decreases tumor invasiveness and increases chemosensitivity and knock in of this geneincreases invasiveness. These in vitro data together supports the in vivo finding that UCHL1expression was significantly associated with MCB and basal-type invasive carcinoma whichare thought to be more aggressive breast cancer subtype. Taken together, these preliminarydata strongly supports that genes identified through such a model may have importantclinicopathological and prognostic impacts in breast cancer and the causal relationshipbetween Snail and these genes can be clearly validated in vitro. Therefore, in one major armof the coming project, we will continue on searching the clinicopathologic and mechanisticroles of these EMT-related genes (both upregulated and downregulated), based on our wellestablished and validated models and methodology. In the other arm of the preceeding projectin the exploring the epigenetic mechanism of Snail-mediated EMT, we have shown thatseveral of the downregulated genes, especially genes related to the maintenance of epithelialphenotypes, including E-cad, TACSTD1, claudin 3, 4 and 7, PPL and CRB3, have promoterhypermethylation and the downregulation phenotype could be reversed by DNA methylaseinhibitor, Aza. In exploring the pathogenetic role of Snail in the promoter DNAhypermethylation and dowuregulation of these genes, we have critically shown byimmunoprecipitation of the binding of Snail with DNMT3a and have further shown bychromatin immunoprecipitation of the recruitment of DNA methyltransferase 3a by Snail tothe promoter and contribute to the promoter methylation of selected target genes, E-cadherin,TACSTD1 and ER. These data together provide a critical insight into the epigeneticmechanism of Snail mediated EMT in breast cancer model. Since accumulating evidenceshave shown that genetic modification may play a role in EMT, we will investigate the geneticmechanism of EMT in the other major arm of the coming project. We will compare thegenetic profiles, by using array CGH (comparative genomic hybridization), betweencarcinomatous component and different sarcomatous components in each case of MCB. Wehypothesize that the sarcomatous components may in some way represent EMT of thecarcinomatous components in each case of MCB. Therefore, by comparing the geneticprofiles between carcinomatous and sarcomatous components, we expect to find potentialgenetic alterations underlying EMT.上皮-間質轉化epithelial-mesenchymal transitionmetaplastic carcinomaSnail transfectant