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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cancer Res. Author manuscript; available in PMC Jun 15, 2009.
Published in final edited form as:
PMCID: PMC2631428
NIHMSID: NIHMS59581

Snail up-regulates pro-inflammatory mediators and inhibits differentiation in oral keratinocytes

Abstract

The transcriptional repressor, Snail2, is over-expressed in head and neck squamous cell carcinomas (HNSCCs) relative to non-malignant head and neck mucosal epithelium, and in locally recurrent relative to non-recurrent HNSCCs. We investigated the mechanisms by which Snails might contribute to the pathogenesis of HNSCCs using cell biological and molecular analyses. Oral keratinocytes that expressed Snails acquired an enhanced ability to attract monocytes and to invade a dense interstitial collagen matrix. They were also found to up-regulate production of pro-inflammatory cytokines and cyclooxygenase-2 (COX2), which have previously been shown to correlate with malignancy. Induction of nuclear factor-kappa B transcriptional activity by Snails was weak and not sufficient to account for the elevated levels of COX2, interleukin-6, interleukin-8 or CXCL1. In addition, expression of Snails in oral keratinocytes impaired desquamation in vitro and strongly repressed expression of both ELF3 and matriptase-1, which play important roles in the terminal differentiation of keratinocytes. Re-expression of matriptase-1 in Snail-expressing cells partially rescued desquamation. This implicates Snails as contributing to malignancy both at the early stages, by impeding terminal differentiation, and at later stages, when invasion and inflammation are important.

Keywords: Snail, keratinocyte, head and neck squamous cell carcinoma, inflammation, differentiation

Introduction

Head and neck cancer is the sixth most common cancer and has seen little improvement in mortality over several decades (1). The keratinocytes that give rise to head and neck squamous cell carcinoma (HNSCC) are characterized by impaired terminal differentiation, dysplasia and invasion of extracellular matrices. HNSCCs are frequently associated with inflammation and pro-inflammatory cytokines (2). The transcription factor nuclear factor-kappa B (NFκB) has been implicated in driving their expression, but the mechanisms underlying increased NFκB activity are not clear, and are complicated by the fact that some cytokines can themselves increase NFκB activity in target cells.

The Snail family of transcriptional repressors has been associated with several forms of carcinoma as well as having important roles in development and injury responses (3). In head and neck tissues, both malignancy and local recurrence following treatment have been associated with a gene expression signature that includes Snail2 (4). Snails can cause an epithelial-mesenchymal transition and invasion (3). In the present study, we investigated the biological effects of Snail expression in human oral keratinocytes to determine how increased levels might contribute to HNSCC malignancy. We found that Snails promote changes in keratinocyte behavior and gene expression that would facilitate progression to malignancy by altering their abilities to terminally differentiate, invade extracellular matrices and recruit pro-inflammatory leukocytes.

Methods

Cell culture

Materials were obtained from Sigma-Aldrich (St Louis, MO), unless otherwise indicated. WSU-HN13 cells (5) were transfected using Polyfect (Qiagen, Hilden, Germany) with a plasmid (pCEFL2) driving expression of TVA, the receptor for RCAS viruses, from the EF1alpha promoter and selected for resistance to 0.4mg/ml G418. Clones were screened for TVA by fluorescence after immunostaining with antibodies and transduction with enhanced green fluorescent protein (EGFP)-Renilla luciferase (RLh) cloned into RCASYGWB, an RCAS proviral vector (6). One clone, HN13(TVA), was chosen for further studies. Genes were introduced into HN13(TVA) by transduction using RCASYGWB. Using this procedure, >90% cells were transduced. Open reading frames of human Snail1, Snail2, RelA and matriptase-1 were amplified by polymerase chain reaction (PCR) and cloned as C-terminal myc (Snails), Glu-Glu (RelA) or HA (matriptase-1) epitope tag fusions into pCEFL2 and RCASYGWB. Snail1(delSNAG) lacks amino acids 2–7 of the SNAG domain, which is essential for repressor activity. Snail1-ERT2/pCEFL2 was constructed by cloning the Snail1 open reading frame 5’ to the ERT2 tamoxifen-inducible cassette (7) and transfected into MDCK and HN13 cells. Clones (MDCK/Snail1-ERT2 and HN13/Snail1-ERT2) were selected on their response to 4-hydroxytamoxifen (4OHT) morphologically and by repression of a mouse E-cadherin promoter luciferase reporter gene in transient transfections. Control cells (HN13/EGFP-ERT2) were stably transfected with a plasmid in which the enhanced green fluorescent protein (EGFP) open reading frame substituted for Snail1. HN13/Snail-ERT2 cells were induced with 4OHT continuously for 7 days prior to RNA isolation.

Transcription analysis

The human and mouse E-cadherin promoters were amplified by PCR from genomic DNA and cloned into pGL3r2.1basic (Promega, Madison, WI). NFκB activity was measured using NFêB-Luc (Stratagene, La Jolla, CA). Luciferase activities were measured using Dual-Glo reagents (Promega, Madison, WI). Microarrays used Cy-5 and Cy-3 labelled cDNAs hybridized to a human GEM2 cDNA array as described previously (8). Specific mRNAs were quantitated by reverse transcription and real-time PCR with Superscript III and Platinum Sybr Green Supermix (Invitrogen, Carlsbad, CA) in a Rotorgene 6000 thermocycler/analyzer (Corbett Research, Sydney, Australia), normalized using glyceraldehyde 3-phosphate dehydrogenase mRNA and corrected for the amplification efficiencies of glyceraldehyde 3-phosphate dehydrogenase and the gene of interest. Primer sequences are listed in Supplementary Table SI. Cytokines in media were quantitated by ELISA (ThermoScientific, Woburn MA).

Migration assays

Keratinocyte migration was assayed by using a blue pipette tip to “scratch” corridors of cells from a confluent monolayer, incubating for 16h at 37C, fixing and staining with Evans blue. The widths of 16 random places along the original scratch lines were measured using Canvas SE software (ACD Systems, Victoria, BC, Canada) and the “wound” closure was compared with the EGFP/RCASYGWB-transduced control cells. Human monocyte migration was measured by plating 105 THP-1 cells in fresh medium into the interior of an 8µm porous polyethylene 24-well plate cell culture insert (BD, Bedford, MA) and filling the outer chamber with medium conditioned by HN13(TVA) cells or their Snail-expressing derivatives. After 20h at 37C, cells that had traversed the membrane were quantitated by incubation for 1–4h in 1mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, lysing the cell pellet in DMSO and reading the absorbance at 550nm. Interleukin inhibitors were from R&D Systems (Minneapolis, MN).

Invasion assays

Invasion of collagen I matrix was measured by plating cells at a confluent density into 14mm glass chamber dishes (MatTek, Ashland, MA) previously prepared by drying down fluorescein isothiocyanate-labelled type I collagen that had been gelled at neutral pH (9). The resulting density of collagen is approximately 20mg/ml, similar to that in a real tumour tissue. After 48h, the cultures were fixed in 4% paraformaldehyde in PBS, counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and imaged by confocal microscopy. Invasion was calculated from the positions of the nuclei of the leading cells relative to the top of the collagen matrix, as measured from Z-stacks.

Results

Activation of Snail1-ERT2 with 4-hydroxytamoxifen recapitulates Snail1 up-regulation in epithelial cells

Ginos et al. identified Snail2 as part of a gene expression signature associated with malignancy and local recurrence of head and neck tumours (4). Further analysis shows a significant association of malignancy and local recurrence with Snail2 expression, independently of other genes (Supplementary Figure S1). To identify Snail-regulated genes, Snail1 was expressed as a 4OHT-inducible C-terminal fusion protein (Supplementary Figure S2A), enabling target genes to be identified by comparing 4OHT-induced and un-induced cells. To ensure that 4OHT induction of cells expressing Snail1-ERT2 faithfully replicated Snail1 up-regulation, it was tested in the well-characterized MDCK cell line. Dependent upon treatment with 4OHT, MDCK cells stably expressing Snail1-ERT2 repressed E-cadherin promoter activity, decreased the amount of endogenous E-cadherin made by those cells (Supplementary Figure S2), redistributed E-cadherin from the plasma membrane, acquired an EMT-like morphology and invaded a collagen matrix from preformed cysts (Supplementary Figure S3). Thus, the activation of Snail1-ERT2 by 4OHT had the same effects as the ectopic expression of Snail genes in MDCK on E-cadherin, cell morphology and invasion (3, 10).

Identification of Snail1 regulated genes in oral keratinocytes

To identify Snail1 regulated genes in oral keratinocytes, mRNAs in 4OHT- and solvent-treated HN13/Snail1-ERT2 cells were compared on cDNA microarrays and genes of interest were validated as Snail1 targets by real-time PCR analysis of cDNAs (Supplementary Table S2). Several genes regulated by Snail1 fell into two groups: epithelial-specific genes that were down-regulated, and inflammation-associated genes that were up-regulated. Snail1 down-regulated the epithelial cell-cell adhesion proteins of adherens and tight junctions, E-cadherin and claudin-1, respectively, as has been reported elsewhere (3). Another adherens junction molecule, EpCAM (TACSTD1), was also markedly down-regulated. Matriptase-1 (ST14), a cell surface serine proteinase required for complete terminal differentiation of keratinocytes (11), was down-regulated, as was ELF3, an epithelial-specific transcription factor required for the expression of genes such as small proline-rich proteins, transglutaminase-3 and profilaggrin during terminal differentiation of keratinocytes (12, 13). Pro-inflammatory genes whose expression was up-regulated by Snail1 activity included interleukin-1 alpha (IL1a), interleukin-1 beta (IL1b), interleukin-6 (IL6), interleukin-8 (IL8), CXCL1 and the prostaglandin synthetic enzyme COX2 (PTGS2). Control cells expressing EGFP-ERT2 showed that the altered expression of all genes was due to Snail1, and not to 4OHT or the ERT2 moiety.

To ensure that the genes identified by 4OHT induction were genuine targets of Snail1, their regulation was validated independently by transducing HN13(TVA) cells with RCAS retroviral vectors (Supplementary Figure S4). In each case, Snail1 caused an increase or decrease in expression corresponding to the 4OHT induction results, as determined by real-time PCR (Figure 1). ELF3, matriptase-1 and EpCAM were strongly repressed in HN13 cells by both Snail1 and Snail2, by a greater extent than E-cadherin and claudin-1 (Figure 1A). This repression was dependent on the presence of the SNAG domain. RelA had no effect on E-cadherin mRNA levels in these cells. Up-regulation of pro-inflammatory genes was also confirmed using a virally transduced Snail1 gene (Figure 1B), with COX2, IL6, IL8 and CXCL1 being particularly strongly up-regulated (4–8-fold). Snail2 induced these same genes. Increases in secreted pro-inflammatory protein levels were confirmed by immunoassays of the culture supernatants (Supplementary Table S3). Expression of IL6, IL8 and CXCL1 were similarly strongly induced in A431 keratinocytes, derived from an epidermoid SCC, but not in HaCaT non-SCC keratinocytes (Supplementary Table S4).

Figure 1
Regulation of Snail target genes in oral keratinocytes by Snails and RelA

The role of NFκB activity in Snail induction of pro-inflammatory genes

The pro-inflammatory cytokine genes up-regulated by Snails are also induced in keratinocytes by NFκB (14)(Figure 1, Supplementary Table S4), prompting us to search for a relationship between the two factors. Expression of the NFκB inhibitor, IκB, inhibited NFκB activity (Figure 2B, Supplementary Figure S5) and expression of IL6, IL8 and CXCL1, having a greater effect in Snail-induced cells than in control cells (Figure 2A), whereas COX2 was not consistently affected. A reduced expression of Snail genes in IκB expressing cells may have contributed to this down-regulation of Snail-induced genes (Supplementary Table S5). In co-transfection experiments, expression of an NFκB reporter gene was induced weakly by Snail2 (less than 2-fold) and not at all by Snail1 under conditions in which RelA caused a strong response (Figure 2B). The basal reporter gene activity was dependent on endogenous NFκB in the cells, as shown by the ability of IκB to inhibit it. In parallel experiments, Snail1 and Snail2 were able to repress transcription from both human (Figure 2C) and mouse (not shown) E-cadherin promoters by 50–75%. Overall, the data in Figure 1 and Figure 2 suggest that NFκB activity might be necessary for strong Snail-induced expression of IL6, IL8 and CXCL1, but that the induction is not mediated by an increase in NFκB activity. NFκB is less important for COX2 induction.

Figure 2
Transcriptional regulation by Snail in oral keratinocytes

Snails impair terminal differentiation and induce a pro-invasive, pro-inflammatory phenotype

Terminal differentiation is impaired in neoplasms of stratified squamous epithelia, and their progression to malignancy is associated with invasion of the underlying extracellular matrix and an influx of inflammatory cells. Thus, it was of interest to determine the effects of Snail expression on these characteristics. Snail1 and Snail2, but not RelA, produced similar morphological changes in HN13(TVA) keratinocytes. At low cell density, scattering and elongated projections at the edges of colonies were common (Figure 3A). HN13(TVA) can terminally differentiate and shed squames into the supernatant 10–14 days after plating, a process that Snail1 and Snail2 inhibited by >80% (Figure 3B–D), instead causing cells to pile up (Figure 3C). The squames shed by control HN13(TVA) maintained their structure in 1% SDS (Figure 3C), indicating a mature, highly cross-linked cytoskeletal structure. Both nucleated and non-nucleated squames were represented, in a ratio of approximately 2:1, although many nuclei stained weakly with DAPI, suggesting that their DNA was undergoing degradation. Upon dissolution of both control and Snail transduced cultures with 1% SDS, squames were released, identifying the final process of keratinocyte terminal differentiation, desquamation, as being inhibited by Snails. Interestingly, re-expression of matriptase-1 (2–3-fold above control levels, as measured by reverse transcription/real-time PCR), disruption of whose gene inhibits desquamation in humans and mice (15), partially restored desquamation in Snail-expressing keratinocytes (Figure 3D).

Figure 3
Snails inhibit terminal differentiation of oral keratinocytes

Expression of Snail1, Snail2 or RelA increased the migration of HN13(TVA) cells (Figure 4A). Additionally, THP-1 monocytes showed a 50–120% higher migration towards medium conditioned by Snail-expressing keratinocytes than that of control keratinocytes (Figure 4B). Blocking IL1, IL6 and IL8/CXCL1 activity with inhibitory concentrations of IL1 receptor antagonist, antibodies to IL6 and CXCR2 inhibitors (SB225002 or antibodies), respectively, did not inhibit the Snail-induced THP-1 migration, indicating that another molecule(s) is responsible. Moreover, Snail expression doubled the rate of invasion by keratinocytes into a high density fibrillar interstitial collagen matrix (Figure 4C).

Figure 4
Snails promote migration and invasion of keratinocytes and monocytes

Discussion

We have shown that the expression of Snail transcriptional repressors in keratinocytes impairs their differentiation into shed squames, an effect that can be rescued by forced re-expression of the Snail down-regulated gene, matriptase-1. Gene targeting of claudin-1 (16) and matriptase-1 (11) have demonstrated them to be required for complete terminal differentiation of keratinocytes, null mice dying from impaired epidermal barrier formation. Recently, a human genetic disease characterized by impaired desquamation was shown to be caused by a mutation in matriptase-1 (15). Another Snail target, ELF3, drives the expression of terminal differentiation proteins (12, 13). Thus, down-regulation of these genes is consistent with the failure to shed squames in Snail-expressing keratinocytes. It has been shown previously that expression of Snail2 in keratinocytes of the upper airway tract increases with malignancy and local recurrence following treatment (4). Hyperplasia of stratified squamous epithelia is accompanied by impaired terminal differentiation, increasing the accumulation of viable keratinocytes in the tissue, and so it is likely that Snail2 contributes to this process during HNSCC formation.

Inflammation is commonly associated with cancer and the up-regulation of IL1a, IL6 and IL8 (2) has been observed in HNSCCs, enhanced NFκB activity being implicated as mediating this effect (14). We have demonstrated here that Snails can also up-regulate these pro-inflammatory cytokines in oral keratinocytes. Snails are known to be repressors and have not been reported to act as transcriptional activators. There are no consensus Snail-binding E-boxes within 300 base pairs upstream of the transcription start site of the COX2, IL6, IL8 or CXCL1 genes, whereas the ELF3, EpCAM and matriptase-1 genes, like the E-cadherin gene, have at least 2 sites. Deletion of the SNAG domain reduced the up-regulation of the pro-inflammatory genes, suggesting that recruitment of co-repressors was important for the effect. Thus, the up-regulation of the pro-inflammatory genes is likely to be an indirect effect e.g. through down-regulation of a repressor of these genes or competition for co-repressors. The poor induction of NFκB reporter gene activity by Snail1 (no induction) and Snail2 (1.7-fold) suggests that the up-regulation of the strongly induced genes is not mediated by increasing NFκB activity, although NFκB might be necessary for cytokine induction by Snails. Thus, two pathways may have evolved to up-regulate these molecules in keratinocytes, presumably to respond to different stimuli. In contrast to the cytokines, COX2 expression was weakly stimulated by RelA, whereas it was strongly stimulated by Snails. MAPK pathways seem to be the dominant routes of up-regulating COX2 expression in human keratinocytes in response to cytokines, with NFêB playing a minor role (17), unlike mouse keratinocytes and other human cell types. COX2, like Snail2, predicts poor outcomes for HNSCC patients (18).

Recently, microarray analysis of gross epidermis was used to identify Snail2 targets in normal adult mouse epidermal cells (19). Of the Snail targets mentioned here, only EpCAM was modified in mouse epidermis by disruption of both Snail2 loci (1.5-fold higher in Snail2-null mice). The levels of Snails in normal keratinocytes might be too low to substantially up- or down-regulate the target genes identified in this study. However, in keratinocytes that have undergone malignant transformation or wounding (4, 20), Snail expression is raised and the regulation of targets identified in this study would take place, thereby facilitating migration, invasion and the recruitment of leukocytes.

Supplementary Material

Supp

Acknowledgements

We thank Drs William Taylor and Jeff Rubin, National Cancer Institute, for MDCK cells and laboratory space, Drs Christina Ochsenbauer-Jambor and Mary Ann Accavitti, University of Alabama at Birmingham, for the gift of anti-TVA monoclonal antibodies, and Dr Henning Birkedal-Hansen, NIDCR, for supporting this study. This study was supported in part by grants from the Sydney Cancer Centre Foundation and Cancer Council NSW.

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