以磷酸化蛋白質體學剖析參與肺癌幹細胞特性之訊息傳遞

Abstract

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.