Research Article The Transcription Factor Snail Induces Tumor Cell Invasion through Modulation of the Epithelial Cell Differentiation Program Bram De Craene,1 Barbara Gilbert,1 Christophe Stove,1,2 Erik Bruyneel,3 Frans van Roy,2 and Geert Berx1 1Unit of Molecular and Cellular Oncology and 2Molecular Cell Biology Unit, Department for Molecular Biomedical Research, VIB-Ghent University; and 3Laboratory of Experimental Cancerology, University Hospital of Ghent, Ghent, Belgium Abstract studies have focused on the global effects of Snail transcriptional activity, and information on the early response genes in cells Abberant activation of the process of epithelial-mesenchymal transition in cancer cells is a late event in tumor progression. expressing Snail is limited. Here, we show that conditional A key inducer of this transition is the transcription factor expression of human Snail (hSnail) in human colon cancer cells Snail, which represses E-cadherin. We report that conditional promotes an epithelial-mesenchymal transition–like process in expression of the human transcriptional repressor Snail in which loss of intercellular adhesion coincides with induction of colorectal cancer cells induces an epithelial dedifferentiation invasiveness. To identify transcriptional changes that are specific program that coincides with a drastic change in cell responses toward hSnail expression, a comparative differential morphology. Snail target genes control the establishment of gene expression analysis using cDNA microarrays was done. This several junctional complexes, intermediate filament networks, molecular functional analysis revealed that Snail induction leads to and the actin cytoskeleton. Modulation of the expression of a general (de)regulation of epithelial differentiation, metabolism, and signal transduction. Using chromatin immunoprecipitation, we these genes is associated with loss of cell aggregation and induction of invasion. Chromatin immunoprecipitation identified several new direct cellular targets of Snail. These experiments showed that repression of selected target genes molecular data provide a better understanding of the functional is associated with increased binding of Snail to their consequences of Snail expression during tumor progression. promoters, which contain consensus Snail-binding sites. Thus, Snail constitutes a master switch that directly represses the Materials and Methods epithelial phenotype, resulting in malignant carcinoma cells. Cell culture and generation of stable cell lines. DLD-1TR21 cells were (Cancer Res 2005; 65(14): 6237-44) cultured in RPMI with 10% FCS, 100 units/mL penicillin, and 100 Ag/mL streptomycin. Linearized pcDNA4/TO-hSnailMyc/His plasmid was stably Introduction transfected by electroporation. Clones were isolated after 2 weeks of Snail is a transcriptional repressor that plays a central role in selection on 500 Ag/mL zeocin and 10 Ag/mL blasticidin (Invitrogen, A epithelial-mesenchymal transition, a process by which epithelial Carlsbad, CA). Expression was induced using doxycycline (1 g/mL, Sigma, St. Louis, MO). cells lose their polarity and are converted to a mesenchymal DLD-1TR21-hSnailMyc/His cells were retrovirally transduced with pFB- phenotype (1). Epithelial-mesenchymal transition is important in Neo-hEcad. Cells were selected on 300 Ag/mL neomycin for 2 weeks. many developmental processes, such as gastrulation and neural DLD-1TR21 cells were retrovirally transduced with either the pFB-Neo- crest migration, but its deregulation in cancer cells can lead to enhanced green fluorescent protein (EGFP) or the pFB-Neo-EGFP-hSnail tumor progression. Multiple signaling pathways seem to converge vector. After selection for 2 weeks, cells underwent two cycles of sorting to Snail expression during different normal developmental using the FACSVantage (Becton Dickinson, San Jose, CA). steps but also during tumor progression. Besides their involvement All constructs used were obtained with standard cloning techniques. in epithelial-mesenchymal transition, Snail family members have Sequence verification was carried out with sequence-specific primers. been implicated in a variety of other processes, such as apoptosis Quantitative real-time PCR. Design of primers and probes, cDNA and left-right asymmetry (2, 3). Up to now, the mechanism by which synthesis, and PCR amplification were described previously (9). In addition, we used TaqMan Gene Expression Assays (Applied Biosystems, Foster City, Snail influences these different cellular processes remains largely CA). Sequences of primers and probes are listed in Supplementary Table S1. unresolved. Snail can repress E-cadherin through binding to The average threshold cycle of triplicate reactions was used for all E-boxes in the E-cadherin promoter (4, 5). Other candidate subsequent calculations using the DCt method. repressors for E-cadherin are Slug, E12/E47, y-EF1 (ZEB1), and Immunocytochemistry. Immunocytochemistry was done using stan- SIP1 (ZEB2; reviewed in ref. 6). Furthermore, Snail suppresses the dard procedures (9). In addition, mouse monoclonal antibodies recognizing expression of claudins and occludins (7), and other epithelial genes E-cadherin (HECD-1, Takara, Kyoto, Japan), p120ctn (Transduction, San such as MUC1 and cytokeratin 18 (8). However, until now, few Jose, CA), plakophilin-2 (Progen, Heidelberg, Germany), and claudin-4 (Zymed, San Francisco, CA) were used. Collagen invasion and fast aggregation assay. The assays were done as described (9). Note: Supplementary data for this article are available at Cancer Research Online RNA preparation. Cells were grown to subconfluency, trypsinized, (http://cancerres.aacrjournals.org/). B.D. Craene is a research assistant, and C. Stove and G. Berx are postdoctoral washed, and resuspended in 4 mol/L guanidine thiocyanate buffer. Lysates fellows, with the Fund for Scientific Research, Flanders. were homogenized on ice with a syringe and needle. RNA was pelleted by Requests for reprints: Geert Berx, Department for Molecular Biomedical ultracentrifugation through CsCl buffer at 32,000 rpm. The pellet was Research, Unit of Molecular and Cellular Oncology, Technologiepark 927, B-9052 resuspended in 0.3 mol/L sodium acetate (pH 6.0) and precipitated with Ghent (Zwijnaarde), Belgium. Phone: 32-9-33-13-740; Fax: 32-9-33-13-609; E-mail: [email protected]. 100% ethanol. RNA was further purified by phenol/chloroform extraction I2005 American Association for Cancer Research. and ethanol precipitation before removal of genomic DNA by DNase www.aacrjournals.org 6237 Cancer Res 2005; 65: (14). July 15, 2005 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Cancer Research Figure 1. Induction of hSnail in the DLD-1TR21-hSnail cell line results in induction of invasion and loss of aggregation. A, quantitative real-time PCR analysis using hSnail-specific primers and probe shows the rapid and sustained induction of hSnail expression in the inducible cell line DLD-1TR21-hSnail. B, staining with an anti-cMyc antibody reveals nuclear staining for the Myc-tagged hSnail construct after adding doxycyclin to the medium. White bar, 25 Am. C, light microscopy images of treated and untreated cells. Cells start scattering after long-term hSnail induction. Untreated cells or mock cells always form tight clusters. The depicted cells were treated for 2 weeks. Black bar, 100 Am. D, invasion into type I collagen is induced by hSnail expression (+dox). As a positive control for invasion, we used the E-cadherin blocking antibody DECMA-1. Retroviral transduction of E-cadherin in the inducible cell line does not diminish the invasive capacity induced by hSnail. Assays were carried out 72 hours after induction. E, fast aggregation assay. No cell aggregates were detected in liquid cell suspensions at time 0 (0 minute). After 30 minutes, cell-to-cell aggregation was readily detected for noninduced cells (ÀDox-DECMA-1 at 30 minutes). hSnail induction resulted in loss of aggregation (+Dox-DECMA-1 at 30 minutes). DECMA-1 was used as a control for inhibition of aggregation. Assays were carried out 72 hours after induction. Cancer Res 2005; 65: (14). July 15, 2005 6238 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Snail Modulates the Epithelial Differentiation Program treatment in the presence of RNase inhibitor (Promega, Madison, WI). Table 1. Classification of repressed genes using the Web- Treated samples were then purified again in the same way. based gene ontology tools FatiGO and AmiGO cDNA microarray analysis. Construction of the four human 5K microarrays, probe labeling, hybridization, washing, and scanning were carried out at the MicroArray Facility of the Flanders Interuniversity Genes down-regulated by hSnail Institute for Biotechnology (details in ref. 10). A gene was scored as down- Epithelial differentiation regulated if the [normratio 3]Av + 2.33r[normratio 3]Av < 0.75 and the Cytoskeleton [normratio 2]Av < 0.57; a gene was scored as up-regulated if the [normratio 3] À 2.33r > 1.25 and the [normratio 2] > 1.75. Genes were Actin cytoskeleton Av [normratio 3]Av Av h selected only if they complied with these selection criteria in the three EPLIN Epithelial protein lost in neoplasm 0.39 hybridization experiments representing RNA samples harvested at three CSRP1 Cysteine and glycine–rich protein 1 0.48 time points after Snail induction. SLC9A3R1 Solute carrier family