Immediate and delayed effects of E-cadherin inhibition on gene regulation and cell motility in human epidermoid carcinoma cells

Abstract

The invasion suppressor protein, E-cadherin, plays a central role in epithelial cell-cell adhesion. Loss of E-cadherin expression or function in various tumors of epithelial origin is associated with a more invasive phenotype. In this study, by expressing a dominant-negative mutant of E-cadherin (Ec1WVM) in A431 cells, we demonstrated that specific inhibition of E-cadherin-dependent cell-cell adhesion led to the genetic reprogramming of tumor cells. In particular, prolonged inhibition of cell-cell adhesion activated expression of vimentin and repressed cytokeratins, suggesting that the effects of Ec1WVM can be classified as epithelial-mesenchymal transition. Both short-term and prolonged expression of Ec1WVM resulted in morphological transformation and increased cell migration though to different extents. Short-term expression of Ec1WVM up-regulated two AP-1 family members, c-jun and fra-1, but was insufficient to induce complete mesenchymal transition. AP-1 activity induced by the short-term expression of Ec1WVM was required for transcriptional up-regulation of AP-1 family members and down-regulation of two other Ec1WVM-responsive genes, S100A4 and igfbp-3. Using a dominant-negative mutant of c-Jun (TAM67) and RNA interference-mediated silencing of c-Jun and Fra-1, we demonstrated that AP-1 was required for cell motility stimulated by the expression of Ec1WVM. In contrast, Ec1WVM-mediated changes in cell morphology were AP-1-independent. Our data suggest that mesenchymal transition induced by prolonged functional inhibition of E-cadherin is a slow and gradual process. At the initial step of this process, Ec1WVM triggers a positive autoregulatory mechanism that increases AP-1 activity. Activated AP-1 in turn contributes to Ec1WVM-mediated effects on gene expression and tumor cell motility. These data provide novel insight into the tumor suppressor function of E-cadherin.

FIG. 1.

Characterization of stable A431 clones expressing Ec1WVM. (A) Phase-contrast images of NT-2 and W3 clones. (B) Detection of wild-type E-cadherin and Ec1WVM in clones with altered (W1 to W6) and epithelial morphology (NT-1 and NT-2). A total of 20 μg of proteins was analyzed by Western blotting with antibodies as indicated. (C) 2D gel (isoelectrofocusing) autoradiographs of [³⁵S]methionine-labeled proteins from NT-2 and W3 cells. Only fractions of 2D gel autoradiographs are shown. The positions of keratin 13, keratin 13 variant (NT-2 panel), and vimentin (W3 panel) are indicated by arrows.

FIG. 2.

Validation of Atlas cDNA microarray data. Transcription of genes identified in W1 to W6, NT-1, and NT-2 clones was analyzed by Northern blotting. Equal loading was verified by hybridization with the labeled polyU probe.

FIG. 3.

Ec1WVM mutant induces rapid response in A431 cells. (A) Characterization of the 31D6 clone with DOX-regulated expression of Ec1WVM. Induction of Ec1WVM by DOX treatment for 48 h results in cell dissociation and morphological alterations. Immunoblot analysis of Ec1WVM and endogenous E-cadherin expression is shown in the upper part of the panel. 31D6 cells were maintained in the presence or absence of DOX for 48 h and analyzed with anti-myc and anti-E-cadherin antibodies. (B) Ec1WVM affects transcription of fra-1, c-jun, S100A4, and igfbp3 in 31D6 cells. Total RNA was extracted from 31D6 cells maintained without DOX or with DOX for the indicated periods of time. Gene expression was examined by Northern blotting using ³²P-labeled probes as indicated. The membrane was probed with labeled polyU probe to demonstrate equal loading. (C) Ec1WVM activates AP-1-driven transcription in 31D6 cells. 31D6 cells were transfected with the AP-1-dependent reporter pTREx5Luc or with pRSVLuc along with the control β-galactosidase-expressing vector pCMVβ-gal and maintained in the presence (+) or absence (−) of DOX. At 48 h posttransfection, luciferase activity was measured and normalized to the β-galactosidase activity. The results (average and standard deviations) are expressed as the relative activation of luciferase in DOX-treated cells (gray bars) compared to that in untreated cells (white bars). (D) Ec1WVM does not influence TCF/LEF transcriptional activity. 31D6 cells were transfected with pTOPFLASH or pFOPFLASH reporters along with pCMVβ-gal and maintained in the absence or presence of DOX for 48 h. Relative TCF/LEF transcriptional activity was defined as ratio of pTOPFLASH/pFOPFLASH luciferase activities normalized to the β-galactosidase level detected in each transfection. The results (means ± standard deviations) of three independent experiments are shown.

FIG. 4.

Effect of Ec1WVM on tumor cell motility. Wounds were created in confluent cultures of NT-2, W3, and 31D6 cells cultured with (+) or without (−) DOX for 48 h prior the experiment. Wounds were marked and photographed after 0, 8, or 17 h. Experiments were repeated three times, and results of a typical experiment are shown. Wound closure at various time intervals was measured in arbitrary units and represented in graphs.

FIG. 5.

AP-1 is involved in transcriptional effects of Ec1WVM. (A) Characterization of G10 and B4 clones. DOX induces morphological transformation of G10 but not B4 cells. Phase-contrast and fluorescence microscopy of cells cultured with or without DOX for 48 h is presented. Nuclear localization of TAM67-GFP in DOX-treated G10 cells is demonstrated. Expression of TAM67-GFP fusion protein, Ec1WVM, and c-Fos was examined in DOX-treated or untreated G10 and B4 cells by Western blotting with anti-c-Fos, anti-GFP, and anti-myc antibodies. The effect of DOX treatment on AP-1-dependent transcription in B4 and G10 cells is shown. Cells were transiently transfected with the AP-1-regulated reporter pTREx5Luc, and the activity was determined as described in the legend to Fig. 3. (B) Northern blot analysis of S100A4, igfbp-3, c-jun, and fra-1 gene expression in G10 and B4 clones. G10 cells were cultured without DOX or with DOX treatment for indicated time periods (left). RNA was isolated, blotted, and hybridized to S100A4 and igfbp-3 probes. A Northern blot hybridization of RNA from G10 and B4 cells untreated or treated with DOX for 48 h is shown (right). RNA was hybridized to labeled probes as indicated. Hybridization to polyU confirms equal loading.

FIG. 6.

AP-1 controls motility of B4 and G10 cells. G10 and B4 cells were cultured with or without DOX. Where indicated, DOX was added 48 h prior to the experiment. Wounds were made in confluent cell cultures, marked, and photographed after indicated time periods. Wound closure at various time intervals was measured in arbitrary units and represented in graphs. Ectopic expression of c-Fos, Ec1WVM, and TAM67-GFP is indicated in brackets.

FIG. 7.

AP-1 family members c-Jun and Fra-1 are essential for increased motility of W3 cells. (A) RNA interference-mediated inhibition of c-Jun and Fra-1 expression in W3 cells. Cells were transfected with siRNAs targeting c-Jun and Fra-1. Scrambled siRNA was used as a control. The extent of silencing was determined by Western blotting as indicated. (B) Knockdown of c-Jun or Fra-1 retards wound closure. W3 cells were transfected with scrambled siRNA or specific siRNA inhibiting c-Jun or Fra-1 expression. Cell migration was analyzed in wound-healing assays after indicated time intervals. (C) Cell migration was analyzed in transwell motility assay. Expression of c-Jun, Fra-1, or c-Jun and Fra-1 in combination was silenced by RNAi in W3 cells. A total of 10⁵ cells were seeded onto 8 μM polycarbonate transwell filters and allowed to migrate toward fetal calf serum gradient. Cells that migrated to the lower surface of the filter were stained and counted microscopically. Migration was normalized to that of W3 cells transfected with the control siRNA. Data are means ± the standard deviations of triplicate experiments. The experiments were repeated three times with similar results.

FIG. 8.

Expression of Ec1WVM does not alter phosphorylation of EGFR. NT-2, W3, or 31D6 cells were serum depleted for 24 h and treated with indicated concentrations of EGF for 5 min. Expression of Ec1WVM in 31D6 cells was induced by adding DOX for 48 h. EGFR phosphorylation was detected in Western blotting using a phospho-specific antibody.