2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643527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。轉化型乳癌(metaplastic carcinoma of breast, MCB)典型是由上皮癌部份(ductal carcinoma)及類肉癌(sarcomatous)部份混雜而成，這當中類肉癌部份因具備鬆散及移動性類纖維細胞的特性，因此被視為是由上皮癌經上皮-間質轉化過程而轉變形成的。這種型態上之特點，使這種腫瘤成為研究上皮-間質轉化過程的很好材料。我們推測經由比較這兩群腫瘤基因表現差異，應可找出潛在與上皮-間質轉化過程有關之基因群。在上一個計畫中，我們運用微矩陣的方法分析MCB 基因表現並比較MCB 和DCB(ductal carcinoma of breast)腫瘤基因表現。的確，我們發現這些差異基因群中有許多有已知或推測的EMT 角色、功能，這當中我們亦發現如SPARC 及vimentin都顯著與乳癌不良預後有關，這印證EMT 相關基因與腫瘤不良預後的相關性，也進一步印證這些基因群在研究EMT 的可靠性與正確性。除此之外，我們亦注意到這些差異基因群中有許多有基因群表現與我們建立的Snail 轉殖細胞(Snail-6SA vs. wild type)基因群表現趨勢類似，這亦印證MCF7-Snail transfectants in vitro EMT model 的可行性。因此，在此計畫的第一部份，我們將延續EMT 相關基因與人類乳癌組織腫瘤臨床病理預後的探索。為兼顧檢體純度，同時考量生物學意義及避免culture artifact，我們會先著重於檢視重疊於MCB/IDC 及MCF7-Snail transfectants 的差異基因群。鑒於之前的經驗，我們發現有許多基因並無適用的抗體，因此我們將採quantitative RT-PCR或對一些特別有意義的基因自製抗體來探索EMT 相關基因與人類乳癌組織腫瘤臨床病理預後相關性。延續計畫的第一部份，我們發現有些Snail-高度表達的基因亦同時高度表達於MCB/IDC 的差異基因群，強烈暗示Snail 在這些基因的過度表達扮演重要的角色。因此，在此計畫的第二部份，我們將著重於研究Snail 與這些EMT 相關基因的關係。具體而言，初步我們將研究Snail 與GPX/GSTP1, moesin/podoplanin/vimentin andIMP-1/IMP-3 過度表現之關係(請詳見計畫的目的部分).此外，我們將於計畫的第三部份延續探討Snail 引發EMT 的機制，特別注重於轉譯的抑制調控。於前一計畫中，我們初步發現EZH2 (key component of Polycomb Repressive Complex 2) 在Snail 引發EMT 的機制的角色，我們將繼續探索EZH2 與PRC2 其他成員的角色，包括不同Histone lysinemethylation 的情形。總和而言，我們期望能於此計畫中經由綜合in vivo tumor 與invitro Snail-transfectants model，運用微矩陣的方法分析，而找到與人類乳癌組織腫瘤臨床病理預後有關的基因。而在機制方面，我們希望能瞭解Snail 與這些EMT 相關基因的關係，同時瞭解Snail 引發EMT 的機制，特別是EZH2 與PRC2 其他成員於轉譯的抑制調控的角色。這些訊息將有助於我們進一步了解關於上皮-間質轉化過程及腫瘤的發展。<br> Abstract: Epithelial-mesenchymal transition (EMT) is the process of disaggregating structuredpolarized epithelial units into single motile fibroblastoid cells to enable cell movement andmorphogenesis. The process of EMT has later gained wide recognition as a potentialmechanism for the progression of malignancy, for example invasion and metastasis, whichwere attributed to the loosening of epithelial characteristics and the acquisition of migratoryand highly matrix invasive phenotypes. Accordingly, a genome-wide approach to theidentification of genes related to EMT would be critical to understand the complexpathophysiology of EMT underlying cancer progression. Metaplastic carcinoma of breast(MCB) is classically composed of carcinomatous and sarcomatous components, with the latercomponent morphologically characterized by the less cohesive and more fibroblastoid growthpatterns reminiscent of EMT. These morphological features would make MCB an ideal in vivomodel to study the genetic programming involved in EMT. During my previous project, wehave investigated the molecular characteristics of MCBs and compare them with those ofDCBs (ductal carcinoma of breast) using microarray analysis of samples from four MCBs and34 stage-matched DCBs. By using this genome-wide approach, we have identified potentialgene signatures discriminating MCBs and DCBs, of which several are of special interest andhave known or inferred roles related to EMT. Furthermore, we have preliminarily surveyedthree of these discriminator genes and found two of them (vimentine and SPARC) to havesignificant clinicopathological and prognostic impacts, reinforcing the critical roles ofEMT-related genes both pathologically and clinically. Besides, several of those discriminatorgenes from primary breast cancer were also cross-validated in our MCF7-Snail transfectantsin vitro EMT model, and this in turn justifies the use of the mutant-Snail transfectant as avalid model system for the study of EMT. Therefore, in the Arm 1 of this proposal, we willcontinue our efforts to explore the clinicopathological and prognostic roles of thoseEMT-related genes identified in our previous project. We will firstly focus on thoseEMT-related genes which are common to discriminatory gene lists obtained from MCF7-Snailtransfectants and primary human breast cancer tumor tissues. In continuous with Arm 1, wenote that some of those genes up-regulated in Snail-transfectant are also highly up-regulatedin the discriminator gene list in MCB/IDC primary tumors. This strongly implicates apathogenetic role of Snail in some of these EMT-related genes. Therefore, in Arm 2, we willinvestigate the pathogenesis, particularly the potential roles of Snail, in the regulation of theseEMT-related genes. Specifically, we will start with the following genes of great interest:GPX/GSTP1, moesin/podoplanin/vimentin and IPM-1/IMP3. We have also focused onelucidating the potential mechanism of Snail-mediated EMT, specifically on thetranscriptional regulation, by using this in vitro model. Preliminarily, we have identified thepossible role of EZH2 (key component of Polycomb Repressive Complex 2) in theSnail-mediated EMT and we will continue, in Arm 3, to work on this critical issue, especiallythe roles of EZH2 and other members of PRC2 and the methylation status of different lysineresidues of histones. Through the survey of the clinicopathological and prognostic impacts ofthese genes, we will be able to identify certain EMT-related genes or gene lists to haveclinical and pathologic impacts. Mechanistically, we will be able to gain an insight in thepotential role of Snail in the regulation of some critical EMT-related genes and mechanistic ofSnail-mediated downregulation during EMT, especially the potential role of PRC2.Information from such approach may contribute to the knowledge of EMT, a key events intumorigenesis and cancer progression.上皮-間質轉化epithelial-mesenchymal transitionmetaplastic carcinomaSnail transfectant