Arf Tumor Suppressor and miR-205 Regulate Cell Adhesion and Formation of Extraembryonic Endoderm from Pluripotent Stem Cells

Induction of the Arf tumor suppressor in response to hyperproliferative stress following oncogene activation activates a p53-dependent transcriptional program that limits the expansion of incipient cancer cells. Although Arf is not expressed in most tissues of fetal or young adult mice, it is physiologically expressed in the fetal yolk sac, a tissue derived from the extraembryonic endoderm. We demonstrate that expression of the mouse p19Arf protein marks late stages of extraembryonic endoderm differentiation in cultured embryoid bodies derived from either embryonic stem cells or induced pluripotent stem cells, and that Arf inactivation specifically delays the differentiation of the extraembryonic endoderm lineage, but not the formation of other germ cell lineages from pluripotent progenitors. Arf is required for the timely induction of extraembryonic endodermal cells in response to Ras/Erk signaling and, in turn, acts through p53 to ensure extraembryonic endoderm lineage development, but not maintenance. Remarkably, a significant temporal delay in extraembryonic endoderm differentiation detected during the maturation of Arf-null embryoid bodies is rescued by enforced expression of miR-205, a micro-RNA up-regulated by p19Arf and p53. Introduction of miR-205 into Arf-null embryonic stem cells rescues defective ExEn formation and elicits a program of gene expression that controls the migration and adhesion of embryonic endodermal cells. This occurs, at least in part, through atypical regulation of genes that control the epithelial-to-mesenchymal transition in cancer cells. Our findings suggest that noncanonical and canonical roles of Arf in extraembryonic endoderm development and tumor suppression, respectively, may be conceptually linked through mechanisms that govern cell-to-cell attachment and migration. 4 samples total two each at two time points in ESC development At each time point one sample was treted with miR-205 and the other with a GFP control vector

癌基因激活后，肿瘤抑制因子Arf（Arf tumor suppressor）可响应过度增殖应激（hyperproliferative stress）而被诱导，进而激活依赖于p53的转录程序，限制潜在癌细胞（incipient cancer cells）的扩增。尽管在大多数胎儿或年轻成年小鼠的组织中均未检测到Arf的表达，但该蛋白在胎儿卵黄囊（fetal yolk sac）——一种源自胚外内胚层（extraembryonic endoderm）的组织——中存在生理性表达。本研究证实，小鼠p19Arf蛋白（p19Arf protein）可标记由胚胎干细胞（embryonic stem cells, ESC）或诱导多能干细胞（induced pluripotent stem cells）衍生的培养拟胚体（embryoid bodies）中胚外内胚层分化的晚期阶段；且Arf失活可特异性延缓胚外内胚层谱系的分化，却不会影响多能祖细胞（pluripotent progenitors）向其他生殖细胞谱系（germ cell lineages）的形成。Arf是响应Ras/Erk信号通路（Ras/Erk signaling）及时诱导胚外内胚层细胞所必需的，且其可通过p53通路保障胚外内胚层谱系的发育（而非维持）。值得注意的是，Arf基因敲除（Arf-null）拟胚体成熟过程中出现的胚外内胚层分化显著时序延迟，可通过强制过表达（enforced expression）miR-205得以挽救——该微小RNA（micro-RNA）可被p19Arf与p53上调。将miR-205导入Arf基因敲除的胚胎干细胞，可挽救缺陷型胚外内胚层形成，并激活调控胚胎内胚层细胞迁移与黏附的基因表达程序。这一过程至少部分通过异常调控癌细胞中调控上皮间质转化（epithelial-to-mesenchymal transition）的基因实现。本研究结果表明，Arf在胚外内胚层发育中的非经典功能，与其在肿瘤抑制中的经典功能，或可通过调控细胞间附着与迁移（cell-to-cell attachment and migration）的机制在概念上建立关联。本数据集共包含4个样本，涵盖胚胎干细胞发育过程中的两个时间点，每个时间点设置两个样本；每个时间点下，一组样本经miR-205处理，另一组则以GFP对照载体进行转染。