2015-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643462摘要：癌症細胞即使在有氧的情況下，也會將葡萄糖水解產生乳酸，這種與正常細胞不同的代謝方式，稱有氧醣解 (aerobic glycolysis)，這種代謝改變亦稱為“Warburg Effect”。有愈來愈多證據顯示這些代謝相關改變的產物在許多腫瘤有其臨床病理的重要性，而最近一些關於癌細胞過度運用“有氧醣解”機制的研究發現其能量代謝來源有轉向非醣類碳來源 (特別是脂肪酸或麩醯胺酸)，以維持粒腺體完整及功能。這種代謝改變亦稱“mitochondrial uncoupling”，且現已有證據顯示其與血液腫瘤抗藥性有關。癌症幹細胞已被發現與腫瘤復發，轉移及抗藥性有關。最近已有研究指出癌症幹細胞的代謝與正常細胞有許多差異，例如“過度醣解”表現，而這些代謝改變也被發現與化療抗藥性有關。而最近一篇用 proteomics 來研究乳癌的報告也發現乳癌腫瘤幹細胞某些代謝相關的基因亦增高表達，這些研究資料統合而言顯示腫瘤細胞，特別是癌症幹細胞，其代謝途徑與正常細胞有明顯差異，而且這種差異可能有其臨床意義，包括腫瘤再發、轉移及抗藥性。上皮間質轉化 (epithelial mesenchymal transition, 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。雖然上皮間質轉化病理病因機制已被廣泛研究，但這些研究絕大部分多集中在探討轉錄因子，特別是 Snail，Slug，Twist，Zeb-1及 Zeb-2 的機制，目前並無針對上皮間質轉化之代謝相關變異的全面性研究。此外 ，有愈來愈多證據顯示癌症幹細胞與進行 EMT 的細胞在細胞及分子層級有高度類似，這些資料暗示癌症幹細胞與進行 EMT 的細胞可能在代謝變異上有相似性。在目前的計畫中，我們將上個計畫所採用的 Snail-乳癌 EMT 模式擴展到一個已被廣為接受的 TGF-誘導乳房細胞模式來研究細胞進行 EMT 時的代謝基因產物及代謝物的變化，及其意義。初步我們發現代謝相關的基因 hyaluronan synthase 2 (HAS2)在乳腺細胞 EMT 時高度表達 且發現 HAS2 在 metaplastic carcinoma， 一種與 EMT 相關的乳癌亞型，會高度表達，且進一步發現其表達與 triple-negative and basal-like phenotypes 相關且與 poorer overall survival 高度相關(preliminary result)。這些初步結果高度支持 EMT 過程中的某些代謝相關基因有病理臨床意義。除了 HAS2，我們也初步發現兩個代謝相關基因4-aminobutyrate aminotransferase (ABAT) and heparan sulfate (glucosamine) 3-o-sulfotransferase 2 (HS3ST2) 也在乳腺細胞 EMT 時高度表達(preliminary result)，我們將進一步分析其意義。 另外我們也收集乳腺細胞有 EMT 與沒有 EMT 時的培養液(preliminary result)，也將接著分析其 metabolite, 找出 potential metabolism-related genes, 分析其意義。因此在此計劃的延續計劃中, 我們將結合微短陣的方式和 2-D 電泳及質譜儀的方式(透過與 NTU Genomic center Microarray、Protein 及 Metabolomics Core 的合作)，用全方面(global approach)的方式，來找出 mRNA 層級及蛋白質/代謝物層級在乳房細胞進行 EMT 時所發生的變化，進一步我們會先從此兩種層級所找到的共同或類似代謝途徑著手，例如經 Metabolomics Core 如發現某代謝物增加，則我們會比對 microarray或 protein core 的結果，尋找參與該 metabolite 代謝相關基因(如酵素之 RNA、蛋白)是否亦高表現。進一步我們會著重在這些經 validate 的標的基因/代謝物，依我們實驗室過去的背景及技術 (background 及 preliminary result)，進行功能性的分析及進一步以免疫組織化學來看該基因的蛋白質/metabolite 產物表現與病理及臨床之相關性。我們希望能在此計畫中，藉由 core lab 的合作及實驗室的背景經驗，尋找出 EMT 相關的代謝變異及其意義，相信從 metabolism 的角度切入，我們將可對 EMT 及癌症幹細胞的病理病因機制及可能的抗藥性機制有更進一步的了解。 <br> Abstract: Cancer cells produce energy via the conversion of glucose into lactate, despite the presence of oxygen, a process known as aerobic glycolysis. The metabolic shift toward glycolysis was referred as the “Warburg Effect”. The expression of metabolism-related products has been shown to be associated with clinicopathologic parameters in various types of neoplasm. Recent studies in the mechanism underlying the increased dependence of cancer cells on aerobic glycolysis for energy generation have suggested that a shift to the oxidation of nonglucose carbon sources (such as fatty acids or glutamine) to maintain mitochondrial integrity and function, a process referred as mitochondrial uncoupling, may occur in cancer cells and may link to chemoresistance in hematological malignancy. Cancer stem cells have been linked with resistance to conventional chemotherapy, tumor recurrence and metastatic potential. Recent studies have provided evidences suggesting that cancer stem cells may have altered metabolism, such as enhanced glycolytic phenotype, and these alterations may be related to chemoresistance. In line with this notion, a recent study investigating the proteomic profiling of cancer stem cells have revealed metabolism-related proteins differentially upregulated in cancer stem cells. These data taken together suggests that there are potential difference in metabolism pathways between normal cells and cancer cells and even cancer stem cells, and these alterations may have clinical implication, including tumor recurrence, metastasis and chemoresistance. Epithelial-mesenchymal transition (EMT), originally discovered from studies of embryonic development, is the process of disaggregating structured polarized epithelial units into single motile fibroblastoid cells to enable cell movement and morphogenesis. The process of EMT has later gained wide recognition as a potential mechanism for the progression of malignancy, for example invasion and metastasis. Although EMT has been pathogenetically extensively studied, the great majority of studies have been focused on transcription factors, such as Snail, Slug, Twist, Zeb-1 and Zeb-2. There is currently no reports, if any, investigating the metabolism-related alterations in a global approach. Moreover, it has been shown that cancer stem cells may have cellular and molecular features similar to cancer cells undergoing EMT and this may suggest potential association/similarity between metabolism alterations occurring in EMT and cancer stem cells. In the present project, we have extended from our previous Snail mutant-induced EMT breast cancer cell model to the well-established TGF- induced EMT of breast cells system model to study the potential metabolic alterations and significance during EMT. We have preliminarily found a metabolism-related gene, hyaluronan synthase 2 (HAS2), that is upregulated during TGF-b induced EMT. We have further found clinicopathological correlation between HAS2 and triple-negative and basal-like phenotypes (Preliminary result 2) as well as a poorer overall survival in primary breast carcinoma (Preliminary result 3). Furthermore, we have found very high expression of HAS2 in metaplastic carcinoma (Preliminary result 4), a breast carcinoma subtype pathogenically highly related to EMT. Moreover, we have observed upregulation of HAS2, at RNA and protein level, in TGF-b induced EMT in human breast cells MCF10A (Preliminary result 5). This in vitro result supports our in vivo finding. Together, these preliminary results validate the hypothesis that metabolism-related genes during EMT may have clinicopathologic significance in breast cancer. In addition, among other metabolism-related genes altered during EMT, we have also preliminarily found two candidate genes, 4-aminobutyrate aminotransferase (ABAT) and heparan sulfate (glucosamine) 3-o-sulfotransferase 2 (HS3ST2), to be upregulated in the MCF10A cell system induced to undergo EMT (Preliminary result 6). We are currently investigating the function of these candidate metabolism-related genes altered during EMT. With the support of these preliminary result and the laboratory techniques we have established, we will continue, in the coming continuous project, to further identify and explore the significance of metabolism-associated genes up-regulated, as well as downregulated, during EMT. We will identify potentially metabolism-assicated gene products (from RNA gene product to protein to metabolite) through two major approaches, namely microarray-based transcriptome analysis, and 2-D electrophoresis or LC/MS/ mess spectrometry-based proteome and metabolome approach (supported by Microarray Core, Protein Core and Metabolomics Core Laboratory at NTU research center). We believe that based on the support from Core Lab and our previous background and experience (see background and unpublished result 1-9), we will be able to carry out such studies in the coming proposal. We hope to identify and validate potential metabolism-related molecules occuring during EMT which may contribute to knowledge of the complex pathogenesis of EMT.上皮-間質轉化代謝物epithelial-mesenchymal transitionbreast cellsmetabolism