EGF-Mediated Transcriptional Coregulation of EpICD-LEF1 Alters Nanomechanical Properties of Endometrial Cancer Cells for Promoting Cell Migration. Homo sapiens

Epithelial cell adhesion molecule (EpCAM), a membrane protein known to modulate cell-cell adhesion, is also a signaling molecule internalized into the nucleus for transcriptional regulation. Here we demonstrate that activated EGF/EGFR is a signaling factor to drive the proteolysis of EpCAM. Cleavage of the extracellular fragment EpEX results in topographic fading of cell-surface EpCAM detected by antibody-conjugated cantilevers of atomic force microscope (AFM). As a result, internalization of the cytoplasmic domain EpICD forms a transcription factor complex with LEF1 that regulates gene transcription for enhanced cell-mobility functions. Comprehensive probing of cell surface using AFM tip (without antibody) reveals increased elasticity and non-stickiness of these cells, promoting epithelial to mesenchymal transition. While EpCAM cleavage may contribute to the loss of cell-surface adhesiveness, its internalized EpICD additionally regulates targets for promoting cell migration. Thus, this EGF/EGFR-modulated action on structural EpCAM and regulatory EpICD can enhance invasion potential of transformed cells. Overall design: RL95-2 were stimulated with EGF for 0,12,24,and 48 hr.Immunoprecipitation was carried out using antibodies against EpCAM and Lef-1, sequenced by Illumina HiSeq 2000

上皮细胞黏附分子（Epithelial cell adhesion molecule, EpCAM）是一类已知可调控细胞间黏附的膜蛋白，同时也是可内化进入细胞核以介导转录调控的信号分子。本研究证实，活化的表皮生长因子/表皮生长因子受体（EGF/EGFR）是驱动EpCAM发生蛋白水解的信号因子。细胞外片段EpEX的切割会导致原子力显微镜（atomic force microscope, AFM）抗体偶联悬臂梁所检测到的细胞表面EpCAM信号出现形貌衰减。在此过程中，胞质结构域EpICD发生内化，并与LEF1结合形成转录因子复合物，调控可增强细胞运动性的基因转录。采用无抗体的AFM探针对细胞表面进行全面探测后发现，此类细胞的弹性升高且黏附性降低，进而促进上皮间质转化（Epithelial-Mesenchymal Transition, EMT）。尽管EpCAM的切割可能导致细胞表面黏附性丧失，但其内化的EpICD还可额外调控促进细胞迁移的靶基因。综上，EGF/EGFR对结构性EpCAM及调控性EpICD的调控作用，可增强转化细胞的侵袭潜能。实验设计概述：将RL95-2细胞用EGF分别刺激0、12、24和48小时；采用针对EpCAM与Lef-1的抗体进行免疫沉淀（Immunoprecipitation, IP），并通过Illumina HiSeq 2000完成测序。