Abstract
E-cadherin is an adherens junction protein that forms intercellular contacts in epithelial cells. Downregulation of E-cadherin is frequently observed in epithelial tumors and it is a hallmark of epithelial–mesenchymal transition (EMT). However, recent findings suggest that E-cadherin plays a more complex role in certain types of cancers. Previous studies investigating the role of E-cadherin mainly used gene-knockdown systems; therefore, we used the CRISPR/Cas9n system to develop E-cadherin-knockout (EcadKO) ovarian cancer RMG-1 cell to clarify the role of E-cadherin in RMG-1 cells. EcadKO RMG-1 cells demonstrated a complete loss of the adherens junctions and failed to form cell clusters. Cell–extracellular matrix (ECM) interactions were increased in EcadKO RMG-1 cells. Upregulation of integrin beta1 and downregulation of collagen 4 were confirmed. EcadKO RMG-1 cells showed decreased β-catenin levels and decreased expression of its transcriptional target cyclin D1. Surprisingly, a marked decrease in the migratory ability of EcadKO RMG-1 cells was observed and the cellular response to Rho GTPase inhibitors was diminished. Thus, we demonstrated that E-cadherin in RMG-1 cells is indispensable for β-catenin expression and β-catenin mediated transcription and Rho GTPase-regulated directionally persistent cell migration.
Highlights
E-cadherin forms adherens junctions between epithelial cells and interacts with the intracellular cytoskeletal networks
To confirm the role of E-cadherin in RMG-1 cells, we prepared rescue cells that restored the expression of wild-type mouse E-cadherin in EcadKO cells and designated it as EcadKO + Ecad cells
Y27632 accelerated the migration of WT cells (Fig. 3D, F) but not that of EcadKO RMG-1 cells (Fig. 3E, G). These findings suggest that Rac and ROCK do not regulate cell migration in the absence of E-cadherin in RMG-1 cells
Summary
E-cadherin forms adherens junctions between epithelial cells and interacts with the intracellular cytoskeletal networks. Reduced cell–ECM adhesion has been reported in E-cadherin knockout MCF10A (MCF10A CDH–/–) cells (1), suggesting that the effect of Ecadherin loss on cell–ECM interactions is cell type dependent. Cadherins are considered to negatively regulate this pathway [13] by sequestering β-catenin [14] In this context, it has become of interest to examine whether loss of E-cadherin activates βcatenin-dependent transcription in RMG-1 cells. The role of E-cadherin in Rho signaling [17,18] and Rac-based direction-sensing mechanism [19] during collective cell migration have been elucidated. We generated EcadKO RMG-1 cells and elucidated the role of E-cadherin in cell morphology, cell–cell and cell–substrate adhesion, β-catenin expression, β-catenin mediated gene expression, and cell migration and its regulation by Rho GTPases
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