E-cadherin loss in RMG-1 cells inhibits cell migration and its regulation by Rho GTPases

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.

Keywords: CRISPR/Cas9n; Cell migration; Dispase; E-cadherin; RhoGTPse; β-catenin.

Fig. 1

Generation of EcadKO RMG-1 cells. A, Schematic illustration of E-cadherin gene structure and sequences around the target loci. The yellow boxes indicate exons encoding the E-cadherin protein. The gRNA target sequences and protospacer adjacent motif (PAM) sequences are indicated by black and red underlining, respectively. The arrows indicate the location of PCR primers. B, The genomic sequences around the target sites of wild-type (WT) and E− EcadKO RMG-1 cells. C, Cell morphology (Phase), cytoskeletal organization (F-actin), and protein expression and localization are shown. Cell morphology were visualized using phase-contrast microscopy. Images of actin cytoskeletons stained with rhodamine X-conjugated phalloidine (F-actin) and images of immunofluorescence staining were visualized using confocal laser scanning microscope (LSM 700). D, Immunoblots analysis of indicated proteins are shown. E, Representative gel electrophoresis images of indicated genes after RT-PCR. F, Immunoblot analysis of indicated proteins. Cells were treated with 3 μM of BIO, 10 μM of MG132, and 100 nM of Bortezomib for 12 h.

Fig. 2

E-cadherin loss increased cellular dissociation, and increased cell–substrate adhesion. A, Representative image of cell dissociation. Images of cells after exposure to mechanical stress by pipetting after substrate detachment (upper). Images of cells exposed to mechanical stress by pipetting after EGTA treatment (lower). B, The extent of cell dissociation was represented by the Np/Nc index. Both Np and Nc were counted in minimum five different fields. C, D, E, The relative number of cells adhering to the plates coated with collagen I (C), laminin 5 (D), and vitronectin (E) are indicated. F, Ratios of undetached cells after dispase treatment. Values are expressed as the mean ± SEM of five images per sample. Experiments were repeated three times. Statistical significance is indicated with asterisk (*P < 0.05 and **P < 0.01; Student t-test).

Fig. 3

Loss of E-cadherin compromised cell migration and response to ROCK and Rac inhibitors. A, Representative images of migrating cells indicating the times after lifting of the culture insert. B, Determination of the ratios of the gap width represented by the gap width at indicated times divided by the gap width at 0 h. Gap widths are expressed as mean ± SEM of five images per sample. C, Cell growth curves. D and E, Representative images of migrating WT (D) and EcadKO RMG-1 (E) cells in the absence or presence of ROCK and Rac inhibitors. F and G, Determination of the ratios of gap width in WT (F) and EcadKO RMG-1 (G) cells. Gap widths are expressed as mean ± SEM of five images per sample. H and I, Immunofluorescence staining of WT (H) and EcadKO RMG-1 (I) cells with anti-E-cadherin and anti-RhoA antibodies were visualized using LSM 700.