Expression of E-Cadherin in Epithelial Cancer Cells Increases Cell Motility and Directionality through the Localization of ZO-1 during Collective Cell Migration

Abstract

Collective cell migration of epithelial tumor cells is one of the important factors for elucidating cancer metastasis and developing novel drugs for cancer treatment. Especially, new roles of E-cadherin in cancer migration and metastasis, beyond the epithelial-mesenchymal transition, have recently been unveiled. Here, we quantitatively examined cell motility using micropatterned free edge migration model with E-cadherin re-expressing EC96 cells derived from adenocarcinoma gastric (AGS) cell line. EC96 cells showed increased migration features such as the expansion of cell islands and straightforward movement compared to AGS cells. The function of tight junction proteins known to E-cadherin expression were evaluated for cell migration by knockdown using sh-RNA. Cell migration and straight movement of EC96 cells were reduced by knockdown of ZO-1 and claudin-7, to a lesser degree. Analysis of the migratory activity of boundary cells and inner cells shows that EC96 cell migration was primarily conducted by boundary cells, similar to leader cells in collective migration. Immunofluorescence analysis showed that tight junctions (TJs) of EC96 cells might play important roles in intracellular communication among boundary cells. ZO-1 is localized to the base of protruding lamellipodia and cell contact sites at the rear of cells, indicating that ZO-1 might be important for the interaction between traction and tensile forces. Overall, dynamic regulation of E-cadherin expression and localization by interaction with ZO-1 protein is one of the targets for elucidating the mechanism of collective migration of cancer metastasis.

Keywords: E-cadherin; ZO-1; cancer metastasis; cell patterning; directionality; motility.

Figure 3

Tight junction proteins are related to cell migration. (a) Cell lysates were subjected to immunoblot analysis for ZO-1, E-cadherin, β-catenin, CLND-1, -2, -3, -4, -6, -7, -9, and β-actin. (b) Total RNA extracted from AGS and EC96 gastric cancer cells were subjected to RT-PCR with ZO-1, CLDN1, and CLDN7-specific primers. Averages of three independent experiments with error bars are presented. ** p < 0.01; *** p < 0.001. (c) EC96 cells were transfected with 100 nM of ZO-1, CLDN1, CLDN7 siRNA (“siZO-1, siCLDN1, siCLDN7) or scramble control siRNA (“siControl”) or 1 μg of the CLDN1 expression vector. In vitro cell migration was tested by the wound healing assay. Averages of five independent experiments with error bars are presented. *** p < 0.001. (d) AGS, EC96, and E-cad. KD (E-cadherin-knockdown EC96) cell lysates were subjected to immunoblot analysis for E-cadherin, ZO-1, CLDN7, and β-actin. (e) AGS, EC96, ZO-1 KD (ZO-1-knockdown EC96), and CLDN7 KD (CLDN7-knockdown EC96) cell lysates were subjected to immunoblot analysis for E-cadherin, ZO-1, CLDN7, and β-actin.

Figure 1

Experimental set-up of the isotropic free-edge expansion model and expansion of AGS and EC96 cell islands. (a) Schematic of circular-shaped cell island patterning for the isotropic free-edge expansion model; (i) placing a PDMS stencil in a 35 mm cell culture dish; (ii) adding cell suspension media over the stencil in the dish; (iii) incubating the dish for the cells to settle down within the holes of the stencil; (iv) removing the stencil to release the cell island to begin the expansion assay. (b) Schematic of the experimental model for isotropic free-edge expansion. (c) Bright-field images of AGS and EC96 cell islands at 0 h and 9 h after release (scale bar = 200 μm). (d) The trajectory of the cells within the AGS and EC96 cell islands. The stronger red color of the arrows indicates more time passed. (e) Relative increase rate of the AGS and EC96 cell islands. The error bars represent the standard errors calculated from separate assays on each group (n = 5).

Figure 2

Comparison of motility and directionality of AGS and EC96 cell islands. (a) The trajectory from the initial locations of the inner and boundary cells within the AGS and EC96 cell islands. The stronger red color of the dots indicates more time passed. (b) Color-code map of the directional persistence of the cells within the AGS and EC96 cell islands. The brighter dots indicate higher directional persistence. (c) Average path length and directional persistence along with the same distance from center to edge of the AGS and EC96 cell islands. The error bars represent the standard deviations calculated from separate assays on each group (n = 5). (d) Average directional persistence of inner and boundary cells within the AGS and EC96 cell islands. The error bars represent the standard errors calculated from separate assays on each group (n.s. not significant and *** p < 0.001; n = 5). (e) Color-code map of the directional persistence of the lowest 25% (gray edge circles) and the highest 25% (black edge circles) of motile cells within the AGS and EC96 cell islands. Gray circles represent mid-quartile (25–75%) motile cells within each group population.

Figure 4

Analysis of motility and directionality of cell islands composed of tight junction protein knockdown cell lines and comparison with AGS and EC96. (a) Bright-field images of CLDN7 and ZO-1 KD cell islands at 0 h and 9 h after release (scale bar = 200 μm). (b) Relative area increase of cell islands composed of the AGS (blue circle), EC96 (red square), CLDN7 KD (cyan triangle), and ZO-1 KD (green diamond) cells over time. The error bars represent the standard deviations calculated from separate assays on each group (n = 5). (c) The trajectory of the cells within the CLDN7 KD and ZO-1 KD cell islands. The stronger red color of the arrows indicates more time passed. (d) Average path length along the same distance from center to edge of the AGS (blue circle), EC96 (red square), CLDN7 KD (cyan triangle), and ZO-1 KD (green diamond) cell islands. The error bars represent the standard deviations calculated from separate assays on each group (n = 5). (e) Color-code map of the directional persistence of the cells within the CLDN7 KD and ZO-1 KD cell islands. The brighter dots indicate higher directional persistence. (f) Average directional persistence along the same distance from center to edge of the AGS (blue circle), EC96 (red square), CLDN7 KD (cyan triangle), and ZO-1 KD (green diamond) cell islands. The error bars represent the standard deviations calculated from separate assays on each group (n = 5). (g) Color-code map of the directional persistence of the low 25% (gray edge circles) and high 25% (black edge circles) of motile cells within the CLDN7 KD and ZO-1 KD cell islands. Gray circles represent mid-quartile (25–75%) motile cells within each group population. (h) Average directional persistence of low and high motility cells within the AGS (blue), EC96 (red), CLDN7 KD (cyan), and ZO-1 KD (green) cell islands. The error bars represent the standard errors calculated from separate assays on each group (n.s. not significant, * p < 0.05 and *** p < 0.001; n = 5).

Figure 5

Analysis of differential expression of ZO-1 protein in migratory cells. (a) Double-immunofluorescence labeling for β-catenin (green) and ZO-1 (red) in inner cells (left panel) and boundary cells (right panel) of AGS, EC96, and ZO-1-knockdown EC96 cells. Scale bar: 100 µm. (b) AGS, EC96, and ZO-1-knockdown EC96 cells were subjected to immunofluorescence staining for ZO-1 (green) and Actin (red) at the leading edge. Scale bar: 100 μm. (c) EC96 cells were subjected to double-immunofluorescence labeling for ZO-1/Actin (c-i) and (c-ii) or ZO-1/Paxillin (c-iii) at the leading edge. Scale bar: 100 μm.