臺灣博碩士論文加值系統

癌幹細胞（CSCs）是可能具有幹性特徵並引發腫瘤發生的癌細胞亞群。近期研究顯示，上皮間質轉化（epithelial-mesenchymal transition, EMT）不僅是腫瘤轉移中的關鍵步驟，且負責在各種癌細胞中產生幹性。骨髓間質幹細胞（bone marrow-derived mesenchymal stem cells, BM-MSCs）在腫瘤微環境中透過分泌旁分泌因子助長腫瘤進展，然而，參與響應於MSCs分泌因子的信號傳導途徑了解甚少。在本研究中，我們首先展現了間質幹細胞條件培養基(conditioned medium of Mesenchymal stem cells, MSC-CM）改變了上皮型肺癌細胞（LM cells）的一些EMT 標記蛋白表現量和球狀形成能力。另外，我們進行細胞因子陣列，發現MSC-CM包含了幾種細胞因子。為了進一步闡明參與肺部CSCs形成的MSC-CM調節信號傳導途徑，我們利用磷酸化蛋白質體學方法，並確定了在700個磷酸化蛋白上共1926個磷酸化位點。利用基因集富集分析，TGF-beta誘導的EMT和胚胎幹細胞基因集從磷酸化蛋白質體富集出來。此外，磷酸化蛋白和激酶的整合分析表明，MSC-CM通過LM細胞中的絲裂原活化蛋白激酶（MAPK）信號路徑增強了c-Fos的磷酸化。ERK抑制劑減少了經由MSC-CM所增強的c-Fos磷酸化與細胞移動力。我們的研究表明，MSC可以引發肺癌細胞的MAPK信號路徑，從而引發EMT的產生，這在研究肺癌幹細胞形成的領域中提供了一個有洞察力的了解。

Cancer stem cells (CSCs) is a subpopulation of cancer cells which might have stem-like characteristics and initiate tumorigenesis. Recent studies suggested that epithelial-mesenchymal transition (EMT) is not only as a crucial step in tumor metastasis, but also responsible for generating stemness properties in various cancer cells. Bone marrow-derived mesenchymal stem cells (BM-MSCs) contribute for tumor progression through paracrine factors secretion in tumor microenvironment, however, signaling pathways that involved in response to MSCs-secreting factors are poorly understood. Here, we first showed that conditioned medium of MSCs (MSC-CM) altered the expression of EMT markers and sphere-forming ability in epithelial-type lung cancer cells (LM cells). Furthermore, we performed cytokine array and found that MSC-CM contained several cytokines which might induce the signaling pathway. To further elucidate the MSC-CM-regulating signaling pathways that are involved in the formation of lung cancer stem cells, we performed phosphoproteomics and identified a total of 1926 phosphorylation sites on 700 phosphoproteins. By using gene set enrichment analysis, the gene sets of TGF-beta induced EMT and embryonic stem cell were enriched from pre-ranked phosphproteomic profiles. Moreover, integrative analysis of phosphoproteins and kinases suggested that MSC-CM enhanced phosphorylation of c-Fos through mitogen-activated protein kinase (MAPK) signaling pathway in LM cells. Phosphorylation of c-Fos on serine 374 and cell migration were decreased in LM cells treated with MSC-CM coupled with ERK1/2 inhibitor. Our studies implied that MSCs triggered the phosphorylation signal in MAPK pathway to generated EMT process in lung cancer cells which provide insight for formation of cancer stem cells.

中文摘要…………………………………………………………………………………i
ABSTRACT………………………………………………………………………….ii
LIST OF FIGURES…………………………...………………………………………vii
LIST OF TABLE….…………………………………………………………………viii
Chapter 1 Introduction……………………………….…………..…………………….1
1.1 Cancer stem cells in lung cancer………….……………………………………..1
1.2 Tumor microenvironment………………………………………………………2
1.3 Protein phosphorylation…………..…………………………………………….2
1.4 MAPK pathway………………………………………………………………3
1.5 Aims……………………………………………………………………………4
Chapter 2 Materials and methods……………………………………………………5
2.1 Cell culture……………………………………………….………………..…...5
2.2 Sphere-forming assay……………………………………………………….6
2.3 Protein extraction for MS analysis……………….……………………………6
2.4 Dimethyl labeling of peptides………………………………………………7
2.5 Phosphopeptide enrichment……………………………………………………8
2.6 NanoLC-MS/MS Analysis……………………………………………………9
2.7 Protein identification and quantification……………………………………...10
2.8 Gene set enrichment analysis………….………………………………………11
2.9 Analysis of predicted kinases……………………………………………….12
2.10 Immunoblotting…………………………………………………………....12
2.11 SCH772984 treatment of LM cells………………………………………….13
2.12 Wound healing assays……………………………………………………….14
Chapter 3 Results…………………………..………………………………………….15
3.1 EMT markers and stem cell properties of lung cancer cells were altered by MSC-CM……………………………………………………………………...…………15
3.2 Quantitative analysis of phosphoproteome profiles in epithelial-type lung cancer cells treated with MSC-CM……………………….........................………………16
3.3 Stem-like property and TGF-beta induced EMT were enriched by using phosphoproteomic profiles.……………………..……………...............................17
3.4 Motif analysis revealed that MAPK pathways were involved in response to MSC-CM in LM cells……………………….……………………………………17
3.5 MSC-CM enhanced phosphorylation of c-Fos on serine 374 through ERK1/2………………………………………………………….................……18
3.6 Cell motility were increased by MSC-CM through activation of ERK1/2…......20
Chapter 4 Discussion………………………………………………………………..…22
REFERENCE………………………………………………………………….…….26
FIGURES…………………………………………………………………………….37
TABLE………………………...………………………...……………………………..49
APPENDIX..……………………..………………………………………………….66

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