2016-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643522摘要：TGFβ是已知的上皮間質轉化 (epithelial mesenchymal transition, EMT)誘導因子，且對癌症進展扮演重要角色。雖然目前對TGFβ在癌症發生的生物學已有廣泛的瞭解，但TGFβ促進癌症因子仍然陸續被發現，例如PRRX1 最近被發現為TGFβ誘發的EMT 誘導因子，在許多腫瘤會促進侵襲性，這些發現印證了TGFβ在促進癌症作用上的複雜性，且支持仍會有一些未知的TGFβ誘發的EMT 誘導因子尚未被發現。在過去幾年內，我們研究主要集中在乳癌的EMT，運用全基因微矩陣方式，我們提供了分子證據支持MCB 與一般IDC 是不同subtype，且這些discriminating genes 中有許多與EMT 有關，而且我們亦進一步與MD Anderson Cancer Center 洪明奇院士合作，運用MCF7-Snail variant S6A cell line model證明在in vitro 實驗下，這些基因的表現與EMT 有密切相關。我們進一步利用此model，運用高效能lentivirus infection system 來做一系列功能性分析以探討這些基因的in vitro 生物學功能，並運用免疫組織化學的方式研究其in vivo 臨床病理意義。如此我們證明了moesin 及UCHL1 在乳癌之角色，我們亦發現UCHL1 在乳房葉狀瘤的惡性度亦扮演重要角色。在我們上一個計畫中，我們開始運用TGFβ誘導乳腺細胞EMT 模式研究EMT 相關的基因，包括與代謝有關之基因。經由微矩陣及蛋白體的資料分析中，我們找到一些有趣的標的基因。例如我們發現hyaluronan synthase 2 (HAS2)在EMT 過程會增高，我們亦進一步發現HAS2 與EMT 高度相關的MCB 會高表達，且相關同時亦有文獻發現其in vitro 亦與EMT 相關。我們也初步發現PRRX2 為TGFβ誘發之轉錄因子，會增強移動性及侵襲性及腫瘤生成性。我們亦發現PRRX2 本身會誘發EMT，且機制上會促進PLAT 之表現，進而促進侵襲。除此之外，我們也發現基因如 FBN1、SPOCK1、IGFL2、ALPK2、ABAT、HS3ST2 於EMT 過程中會高表達，我們於初步免疫化學中亦發現如FBN1、SPOCK1 亦高度表達於MCB 中，當中我們更發現SPOCK1 可能是一種新穎的肌上皮marker。因此在此新的2 年計劃中，我們將計畫運用包括功能性分析、機制及臨床病理意義來分析經由microarray 及proteomic data 所得到的TGFβ誘發而表現的EMT 相關基因。這當中包括我們目前已初步發現的基因，如PRRX2、FBN1、SPOCK1、IGFL2 ，我們將繼續完成其功能性、機制及臨床病理意義之分析。此外，我們亦將繼續分析經由此模式所得的TGFβ誘發的EMT 的其他相關基因，透過文獻分析，找到可能與EMT 相關的新穎基因，並依上述三主軸(功能性、機制及臨床病理意義) 分析這些基因之意義。我們相信透過基因體核心實驗室 (microarray、proteomics and metaboliteLab)的技術支援，並根據我們實驗室目前擁有的技術 (功能性分析包括: MTT、Transwell、Woundhealing、3D collagen、Soft agar、chemosensitivity；機制分析包括: Luciferase、reporter assay、chromatin IP；臨床病理意義分析包括: IHC、IFC、public data base 分析)，我們應當有能力執行此計劃，且透過此計劃，找到一些TGFβ-EMT 未知因子，包括高表達 (potential oncogenic factor)與低表達 (potential tumorsuppression)。這些結果將有助於我們更進一步了解TGFβ促進癌症及EMT 在乳癌的病理機制。<br> Abstract: Epithelial mesenchymal transition (EMT) is the process of disaggregation of structured polarised epithelialunits into single motile fibroblastoid cells to enable cell movement and morphogenesis. EMT-like processeshave been proposed to occur during tumour progression in carcinomas, particularly at the invasion andintravasation stages, during which tumour cells disassemble and migrate to sites distant from the primarytumours. TGF-β is a known EMT inducer and has been shown to play critical role in cancer promotion.Despite the extensive knowledge of TGF-β biology in the development of cancer, several factors that mediatethe cancer-promoting role of TGF-β continue to be identified. For example, paired-related homeobox 1 hasrecently been reported to be induced by TGF-β and to increase the invasiveness of multiple cancer typesthrough EMT. These findings are consistent with the complex roles of TGF-β involving multiple aspectsrelating to cancer progression and may suggest the presence of additional uncharacterised factors related toTGF-β-related cancer biology. During the past few years, we have been focusing on the pathogenesis ofEMT in breast cancer. By using microarray-based genome-wide approach, we have provided the geneticbasis for the recognition of MCB (metaplastic breast carcinoma) as a discrete subtype of breast cancerdistinct from IDC, and have identified gene signatures discriminating MCBs and IDCs, from which severalof those genes have been cross-validated in MCF7-Snail variant 6SA cell line system exhibiting EMT. Wehave continued to explore the clinicopathologic and biologic roles of genes identified through Snail-mediatedEMT models, using high-efficient lentiviral infection system, a series of functional assays, andimmunohistochemistry in tumor cohort. Through such approaches, we have demonstrated association ofaggressive behavior and such EMT-related genes as moesin, and ubiquitin carboxyl-terminal esterase L1 inbreast cancer, as well as in phyllodes tumor. In our previous project, we have bagun to apply the TGF-βinduction system in breast cells as a model to study potential metabolic alterations related to EMT. We haveidentified several genes of interest through microarray and proteomic data bases in this model. For example,we have identified hyaluronan synthase 2 (HAS2) to be upregulated in the EMT model and have validated itsrole in EMT by showing enrichment of HAS2 expression in MCB. We have preliminarily identified PRRX2(paired-related homeobox 2 transcription factor) as a TGF--dependent upregulated transcription factor andenhanced migration, invasion and tumorigenesis. We have further demonstrated PRRX2 as an EMT inducerand that PLAT may mediate the invasiveness. We have also identified upregulation of FBN1, SPOCK1,IGFL2, NOV, ALPK2, ABAT and HS3ST2, to be upregulated in the MCF10A cell system induced to undergoEMT and have started to investigate their functions (please see preliminary results). In the coming 2-yearproposal, we will continue to further study the pathogenesis of the abovementioned genes as well as furthernovel genes related or not directly related to EMT through three major approaches, namely functional roles,mechanism and clinicopathological significance. We will identify novel EMT-related genes through twomajor approaches, namely microarray-based transcriptome analysis, and 2-D electrophoresis or LC/MS/ massspectrometry-based proteomic and metabolome approach. We believe that based on the support from CoreLab and our previous background and experience scuh as the Snail-induced EMT breast cancer cells model,the microarray analysis plateform, the very high efficiency lentiviral knockdown and knockin system forvarious difficult-to-transfect cells, the various functional assays, we will be able to carry out such studies inthe coming proposal. We hope, through such approaches, we will identify additional uncharacterised factorsrelated to TGF-β-related cancer biology, especially EMT-related gene, including upregulated genes (potentialoncogenic factors) and downregulated genes (potential tumor suppressor). Such results derived maycontribute to the knowledge of the complex pathogenesis of TGF-β-related EMT.