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Vol. 18, Issue 11, 4615-4624, November 2007

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Direct Repression of Cyclin D1 by SIP1 Attenuates Cell Cycle Progression in Cells Undergoing an Epithelial Mesenchymal Transition

Jakob Mejlvang^*, Marina Kriajevska^*, Cindy Vandewalle, Tatyana Chernova, A. Emre Sayan^*, Geert Berx, J. Kilian Mellon^*, and Eugene Tulchinsky^*

*Department of Cancer Studies and Molecular Medicine and Medical Research Council Toxicology Unit, University of Leicester, Leicester LE1 9HN, United Kingdom; and Unit of Molecular and Cellular Oncology, Department for Molecular Biomedical Research, Flanders Institute for Biotechnology-Ghent University, BE-9052 Gent, Belgium

Submitted May 3, 2007; Revised August 27, 2007; Accepted September 4, 2007
Monitoring Editor: Ben Margolis

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Zinc finger transcription factors of the Snail/Slug and ZEB-1/SIP1 families control epithelial-mesenchymal transitions in development in cancer. Here, we studied SIP1-regulated mesenchymal conversion of epidermoid A431 cells. We found that concomitant with inducing invasive phenotype, SIP1 inhibited expression of cyclin D1 and induced hypophosphorylation of the Rb tumor suppressor protein. Repression of cyclin D1 was caused by direct binding of SIP1 to three sequence elements in the cyclin D1 gene promoter. By expressing exogenous cyclin D1 in A431/SIP1 cells and using RNA interference, we demonstrated that the repression of cyclin D1 gene by SIP1 was necessary and sufficient for Rb hypophosphorylation and accumulation of cells in G1 phase. A431 cells expressing SIP1 along with exogenous cyclin D1 were highly invasive, indicating that SIP1-regulated invasion is independent of attenuation of G1/S progression. However, in another epithelial-mesenchymal transition model, gradual mesenchymal conversion of A431 cells induced by a dominant negative mutant of E-cadherin produced no effect on the cell cycle. We suggest that impaired G1/S phase progression is a general feature of cells that have undergone EMT induced by transcription factors of the Snail/Slug and ZEB-1/SIP1 families.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
An important event in the development of malignant epithelial tumors is epithelial-mesenchymal transition (EMT), a process of generation of motile and invasive mesenchymal cells from polarized epithelia. Because EMT plays a fundamental role at certain stages of normal development (gastrulation, neural crest migration, somitogenesis), it has been suggested that some elements of embryonic transdifferentiation programs are exploited by cells of growing carcinoma (Thiery, 2003). Cells undergoing EMT are characterized by massive alterations in gene expression patterns. They acquire expression of mesenchymal but loose epithelial markers. A central event in EMT is loss of epithelial cadherin (E-cadherin), a surface receptor that plays an essential role in the formation of adherens junctions and that is often mutated or lost in cancer cells (Thiery and Chopin, 1999, Thiery, 2003).

In recent years, several direct transcriptional repressors of E-cadherin (Snail, Slug, ZEB-1, SIP1, and E47) have been identified (Batlle et al., 2000; Cano et al., 2000; Comijn et al., 2001; Perez-Moreno et al., 2001; Bolos et al., 2003; Eger et al., 2005). These proteins act downstream in EMT-inducing signal transduction pathways activated by TGF, FGF, and EGF growth factors, integrin engagement, and hypoxia (Imai et al., 2003; De Craene et al., 2005a; Krishnamachary et al., 2006; Imamichi et al., 2007). ZEB-1/SIP1 and Snail/Slug family members directly interact with the response elements in the proximal e-cadherin gene promoter and actively repress transcription recruiting transcriptional corepressors such as CtBP or mSinA (Furusawa et al., 1999; Shy et al., 2003; Peinado et al., 2004). More recently, direct repression of other epithelial genes by Snail and SIP1 has been reported (De Craene et al., 2005b; Vandewalle et al., 2005; Moreno-Bueno et al., 2006). In addition, Snail/Slug and ZEB-1/SIP1 proteins mediate up-regulation of genes implicated in cell invasion and motility (e.g., vimentin, members of the matrix metalloproteinase (MMP) family of proteases, fibronectin). The mechanisms of transcriptional activation is less clear; in some cases, indirect activation of genes implicated in EMT by Snail and SIP1 takes place (Jorda et al., 2005; Taki et al., 2006). In contrast to Snail and Slug, ZEB-1, and SIP1 proteins interact with transcriptional coactivators pCAF and p300 (Postigo et al., 2003; van Grunsven et al., 2006). This biochemical difference may indicate that Snail and SIP1 family members activate expression of mesenchymal markers via fundamentally different mechanisms. In vivo studies demonstrated that Snail/Slug and ZEB-1/SIP1 proteins have different functions in embryonic development and are involved in the control of distinct EMT programs. Snail regulates gastrulation, and snai1^–/– mutant embryos exhibit severe defects in EMT required for generation of the mesoderm cell layer (Carver et al., 2001). On the other hand, experiments with snai2-deficient mice (Jiang et al., 1998) and generation of conditional snai1^–/– knockout embryos demonstrated that neither Snail nor Slug is required for the delamination and migration of neural crest cells (Murray and Gridley, 2006). In contrast, homozygous mutant embryos lacking zfhx1b, the gene encoding SIP1, display early arrest in cranial neural crest migration (van de Putte et al., 2003).

In a number of clinical studies, transcription of genes encoding Snail/Slug and ZEB-1/SIP1 proteins has been detected in breast (Blanco et al., 2002; Elloul et al., 2005), ovarian (Elloul et al., 2005), gastric (Rosivatz et al., 2002), and hepatocellular (Sugimachi et al., 2003) carcinoma cells, and Snail immunoreactivity significantly correlated with breast cancer metastasis (Zhou et al., 2004). Activation of Snail, Slug, E47, ZEB1, and SIP1 is an important, but not the only instrument that is utilized by cancer cells to acquire motile characteristics. Inactivation of e-cadherin by gene mutations (Berx et al., 1998; Guilford et al., 1998) or consistent cleavage of the E-cadherin extracellular domain chronically exposed to matrix metalloproteinases secreted by stromal cells may be sufficient to trigger a process ultimately leading to EMT in tumor cells (Lochter et al., 1997). Recently, we explored a model of functional inhibition of E-cadherin in squamous carcinoma cells A431 by a dominant negative E-cadherin mutant (Andersen et al., 2005). Expression of this mutant triggered a program of gradual EMT, which eventually resulted in activation of vimentin and increased cell motility.

In nonpathological conditions, EMT represents the profound de-differentiation program that must be incompatible with cell proliferation (Burstyn-Cohen and Kalcheim, 2002). Indeed, in 8.5 dpc mouse embryos, cells expressing Snail are characterized by decreased incorporation of bromodeoxyuridine (BrdU; Vega et al., 2004). In Madin-Darby canine kidney (MDCK) cells and in primary keratinocytes, Snail family members induce cell cycle arrest in G1 phase and hypophosphorylation of the retinoblastoma (Rb) protein (Vega et al., 2004; Turner et al., 2006). Complex cell cycle–regulating networks are dependent on cell–cell adhesion, integrin signaling, cell spreading, and actomyosin contractility (Walker et al., 2005). Therefore, there are many potential molecular schemes by which EMT may affect cell proliferation in embryonic development and cancer. However, in cancer cells, the interrelationship between cell growth and EMT can be circumvented by the defects in the molecular pathways controlling the cell cycle. In this study, we analyze cell cycle progression in two EMT models based on conditional expression of either SIP1 or a dominant negative E-cadherin mutant Ec1WVM in the same cell line. We show that SIP1, but not Ec1WVM, induces accumulation of cells in the G1 phase of cell cycle. This effect is largely mediated by the direct transcriptional repression of the cyclin D1 gene by SIP1.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Plasmids
Vectors expressing myc-tagged wild-type SIP1 (pUHDmyc-SIP1) and myc-tagged SIP1 with the mutated C-terminal Zn finger (pTREmyc-SIP1ZFmut) have been described (Comijn et al., 2001; van Grunsven et al., 2003). To generate a doxycycline (DOX)-regulated cyclin D1 expression vector (pBIcyclD1), the cyclin D1 coding sequence was amplified and cloned into pBI vector (BD Bioscience, Clontech, Palo Alto, CA). To analyze cyclin D1 promoter activity, we generated two luciferase reporter vectors, pwtCCND1LUC and pmutCCND1LUC. To generate pwtCCND1LUC, a fragment of the cyclin D1 5'-flanking sequence (–1025 to +18) was cloned into pGL3 basic vector (Promega Biotech, Madison, WI). To create pmutCCND1LUC, three Z-boxes with coordinates –1014 to –1010; –857 to –853, and –300 to –290 were mutated by introducing a single nucleotide substitution (5'-AGGTG replaced by 5'-AGATG) using conventional PCR-based methods.

Cell Lines and Transfections
To generate A431 clones with the inducible expression of wild-type or mutant SIP1 (Tet-On system), we used a clone of A431 cells expressing Tet-responsive transcriptional activator rtTA (Andersen et al., 2005). Cells were transfected either with the pUHDmyc-SIP1 or pTREmyc-SIP1ZFmut along with the pTK-Hyg vector (BD Bioscience Clontech). Selection of stable clones was carried out in the presence of 60 µg/ml hygromycin B. Clones with concurrent DOX-regulated expression of SIP1 and cyclin D1 were obtained by cotransfecting A431/SIP1 cells with pBIcyclD1 and pPuro (BD Bioscience Clontech; conveys resistance to puromycin), followed by the selection of puromycin-resistant cells in the presence of 0.5 µg/ml puromycin. Transfections of plasmid DNA were performed by electroporation with a single pulse of 250 V and 250 µFd by using the Gene Pulser Xcell electroporation system (Bio-Rad Laboratories, Hercules, CA). Established cell lines were cultured in DMEM supplemented with 10% fetal bovine serum with or without DOX (2 µg/ml).

Immunofluorescence
For immunofluorescent staining, cells were grown for 2 d in 10-well glass microscope slides (VWR International, Fontenay-sous-Bois, France). Cells were washed and fixed in acetone/methanol (1:1) solution for 3 min on ice. After rinsing, the slides were incubated with primary antibodies for 1 h at room temperature, rinsed, and incubated with Alexa 488–conjugated rabbit anti-mouse IgG (Pierce, Rockford, IL) for 1 h. The anti-vinculin antibody was from BD Biosciences, Transduction Laboratories. Cells were examined and photographed using a confocal inverted microscope (Axiovert 200M; Zeiss, Oberkochen, Germany). To monitor BrdU incorporation, cells were pulse-labeled with BrdU for 40 min and stained with DAPI and an anti-BrdU antibody (Roche, Mannheim, Germany) according to the protocols supplied with the Detection kit I (Roche). Proportion of BrdU-positive cells was quantified in several microscopic fields and are presented as mean ± SD.

Western Blotting
Proteins (10 or 20 µg) were denatured, separated on 6% or precast 4–20% gradient SDS-polyacrylamide gels (Invitrogen, Carlsbad, CA), and then transferred to Immobilon-P membranes (Millipore, Bedford, MA) by the standard procedure. After protein transfer, blots were incubated in blocking solution with primary antibody at a dilution of 1:1000 (for anti-myc tag antibody, clone 9E10; Santa Cruz Biotechnology, Santa Cruz, CA) and 1:500 (for anti-cyclin D1, anti-p21, anti-p16, anti-p27, and anti-Rb antibodies; Santa Cruz Biotechnology). Immunoreactive proteins were detected using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ).

cDNA Microarray Analysis
Construction of 20K microarrays, probe labeling, hybridization, and scanning were carried out at the MicroArray Facility of the Flanders Interuniversity Institute for Biotechnology. Changes in spot intensities >1.8 or <0.55 were regarded as significant in this system.

RNA Interference
Purified and annealed synthetic oligonucleotides specific for cyclin D1 or control small interfering RNA (siRNA) were purchased from Ambion (Austin, TX). Target sequence for cyclin D1 was validated previously by the company. Cells (n = 2 x 10⁶) were transfected with 0.2 nmol of siRNA by nucleofection technique in buffer V (nucleofection protocol T-20). The nucleofector device and a nucleofection kit were obtained from Amaxa (Köln, Germany) and used in accordance with the manufacturer's recommendations. At 48 h after transfection, cells were harvested, counted, and processed for fluorescence-activated cell sorting (FACS) analysis or Western blotting.

Determination of Cyclin D1 mRNA Stability
Cells were maintained in the presence or absence of DOX for 48 h. Then, actinomycin D (ActD) was added at the concentration of 5 µg/ml for various time periods. Total RNA was isolated, and transcription of cyclin D1, 28S gene, and fosl1 was analyzed by RT-PCR or quantitative real time PCR.

FACS Analysis
A431/SIP1 and A431/SIP1/cyclD1 cells or cells nucleofected with siRNA were grown in the presence or absence of DOX for 48 h, harvested, fixed in 70% ethanol, treated with RNase (1 mg/ml), and stained with propidium iodide (PI; 50 µg/ml). The cellular DNA content was evaluated using FACS flow cytometer.

Three-dimensional Matrigel Invasion Assay
Invasion was analyzed in inverse invasion assay as previously described (McGarry et al., 2004) with minor modifications. A431/SIP1 and A431/SIP1/cyclD1 cells were maintained with or without DOX for 48 h. Cells (n = 6 x 10⁴) were seeded on the underside of the polycarbonate filter of a Transwell chamber containing 100 µl of matrigel basement membrane matrix (Becton Dickinson, Oxford, United Kingdom) diluted 1:1. Cells were allowed to adhere for 3 h and washed by DMEM. Transwell chambers were placed in wells filled with 1 ml of DMEM with or without DOX. In 3 d, cells were fixed in methanol and stained for 1 h in PI solution (10 µg/ml). Optical sections were scanned at 10-µm intervals using the confocal microscope Zeiss Axiovert 200M. To perform statistical analysis of the invasive potential of A431/SIP1 and A431/SIP1/cyclD1 cells, the amount of cells entering matrigel and remaining at the filter was calculated in 12 optical fields. The values were expressed as a percentage of cells that penetrated matrigel.

Cell Adhesion and Transwell Migration Assays
Cell adhesion assay was carried out essentially as previously described (Mejlvang et al., 2007). Ninety-six–well tissue culture plates were coated with 50 µg/ml human fibronectin or 50 µg/ml rat collagen type I (all from BD Biosciences). Cells were allowed to adhere for 15 min. In some experiments, a blocking antibody AIIB2 known to prevent adhesion to both substrates (Brockbank et al., 2005) has been mixed with the cells for 10 min before the assay.

A directed transwell migration assay was performed using 24-well transwell plates containing 8-µm pore-size polycarbonate filters (Corning Costar, Cambridge, MA). Cells (n = 10⁵) were cultured with or without DOX for 48 h, seeded in culture inserts, and maintained overnight. Adhered cells were allowed to migrate toward gradient of serum used as a chemoattractant in the lower chamber for 2 h. Cells that migrated to the underside of transwell filters were fixed, stained with a Gurr rapid staining kit (BDH, Dagenham, United Kingdom), and counted by bright-field microscopy at a magnification of x200 in four random fields using the ImageJ program.

Nuclear Run-On Assay
Nuclear run-on assay was based on the incorporation of biotin-16-uridine-5'-triphosphate (biotin-16-UTP) in nascent transcripts according to Patrone et al. (2000). Briefly, cells were maintained with or without DOX for 48 h. Cells were harvested and consequently resuspended in buffer I (10 mM Tris-Cl, pH 7.4, 3 mM MgCl₂, 10 mM NaCl, 150 mM sucrose, 0.5% NP40) or buffer II (10 mM Tris-Cl, pH 7.4, 3 mM MgCl₂, 10 mM NaCl, 150 mM sucrose) and centrifuged at 500 x g for 10 min. Nuclei were then resuspended in buffer III (40% glycerol, 50 mM Tris-HCl, pH 8.5, 5 mM MgCl₂, 0.1 mM EDTA) and quickly frozen.

To perform nuclear run-on reactions, 2 x 10⁶ nuclei were incubated in a reaction buffer (4 mM of each NTP, 200 mM KCl, 20 mM Tris-Cl, pH 8.0, 5 mM MgCl_2, 4 mM dithiothreitol, 200 mM sucrose) for 30 min at 29°C and stopped by adding RNase-free DNase I. In some reactions (negative controls), 0.5 mM UTP instead of biotin-16-UTP was used. Total RNA was isolated by TRIzol (Invitrogen) extraction, and biotinylated RNA was purified using agarose-conjugated streptavidin beads. Beads were washed two times with 15% formamide and five times with 2x SSC. Isolated biotinylated RNA was used for RT-PCR.

Real-time Quantitative PCR
RNA was isolated using TRIazol reagent. cDNA synthesis was carried out using random hexamers and Superscript II (Invitrogen). PCR was performed using SYBR Green PCR Master Mix in the PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Primers were designed to cross exon–exon boundaries and used at the concentration 900 nM. Each sample was run in triplicate. The C_T (threshold cycle when fluorescence intensity exceeds 10 times the SD of the baseline fluorescence) values for the target amplicon and endogenous control (28S) were determined for each sample. Quantification was performed using the comparative C_T method (C_T).

Luciferase Reporter Assay
To determine transcriptional activity of cyclin D1 reporters, A431/SIP1 cells were transfected with 1 µg reporter constructs. The efficiency of each transfection was monitored using 400 ng cotransfected -galactosidase expression vector, pCMV-gal (BD Biosciences). Cells were maintained with DOX for 48 h and lysed, and the luciferase activity was measured with a Lumat LB9501 tube luminometer (Berthold Detection Systems, Pforzheim, Germany). The luciferase activity was normalized to the activity of -galactosidase determined using o-nitrophenyl--D-galactopyranoside (Sigma, Poole, Dorset, United Kingdom) as a chromogenic substrate.

Chromatin Immunoprecipitation Assay
A431/SIP1 cells were cultured for 24 h in the presence or absence of DOX. Cross-linking, immunoprecipitation, and DNA purification were performed using chromatin immunoprecipitation (ChIP)-IT kit (Active Motif, Carlsbad, CA) according to the manufacturer's protocol. Immunoprecipitated DNA was analyzed by real-time quantitative PCR.

Statistics
Results are expressed as the mean ± SD. Student's t test was used to evaluate the differences between groups.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
SIP1-mediated EMT in A431 Cells: Switch from a Proliferative to an Invasive State
To study the physiological effects of SIP1 in carcinoma cells, we generated clones of the squamous carcinoma cell line A431 with the DOX-regulated expression of 6xMyc-tagged SIP1 (clone A431/SIP1). Treatment with DOX resulted in rapid accumulation of SIP1 in 95–98% of nuclei of A431/SIP1 cells, leading to cell scattering and dramatic morphological conversion from an epithelial cell state to a fibroblast-like phenotype. On the other hand, A431 cells expressing 6xMyc-tagged SIP1 with the mutated C-terminal Zn-finger retained entirely polarized epithelial morphology (Figure 1A). Expression of wild-type SIP1 promoted cytoplasmic redistribution of the adherens junctions and tight junction proteins (data not shown). The staining of DOX-treated cells with phalloidin or an anti-vinculin antibody indicated that SIP1-inhibited cell–cell adhesion, but promoted formation of focal adhesions and disappearance of F-actin from intercellular borders (Supplementary Figure S1A). In addition, SIP1-activated cell adhesion to collagen and fibronectin (Supplementary Figure S1B). We used a cDNA microarray technique to obtain a global view on the number of genes regulated by SIP1 in A431 cells. Forty-eight hours of SIP1 induction led to prominent changes in gene expression profiles. We found that out of 20,000 genes analyzed, SIP1 repressed 281 genes by a factor of 0.55 or less and activated 204 genes by a factor of 1.8 or more (corresponds to the 2.4% of human genome; Supplementary Tables SIA and SIB).

View larger version (22K): [in this window] [in a new window]   Figure 1. SIP1 induces cell invasion and inhibits cell growth. (A) Characterization of DOX-regulated A431 clones expressing SIP1 or SIP1ZFmut. Phase-contrast images of DOX-inducible A431 cell clones expressing myc-tagged SIP1 or SIP1ZFmut. Western blots show the expression of wild-type or mutant SIP1 in cells maintained with or without DOX for 48 h. (B) Inverse invasion assay of DOX-treated or untreated A431/SIP1 and A431/SIPZFmut cells. Confocal microscope sections of PI-stained cells were used to analyze the invasion assay. The row corresponding to 0 µm shows cells on the underside of the filter. Other sections show cells invaded into matrigel at different distances as indicated. The experiment was repeated four times, and results of a typical experiment are shown. (C) SIP1 inhibits cell growth. A431/SIP1 or A431/SIP1ZFmut cells were cultured with and without DOX, and cell number was counted at different time points as indicated. Experiments were repeated three times with similar results. Data shown are mean ± SDs of triplicate experiments.

To analyze the effects of SIP1 on cell growth, we seeded equal amounts of cells on six-well culture plates, maintained them with and without DOX, and counted them in 24, 48, 72, and 96 h. Already after 24 h of DOX-treatment, SIP1 significantly decreased the doubling time of A431 cells (p < 0.05; Figure 1C). Consistent with this observation, A431/SIP1 cells incubated with DOX for 48 h incorporated 3.2-fold less BrdU than cells maintained in the absence of DOX (see Figure 3C). As expected, expression of SIP1 with the mutated C-terminal Zinc-finger domain produced no effect on cell proliferation or matrigel invasion (Figure 1, B and C). Taken together, these data demonstrated that SIP1-induced EMT program encompasses a global genetic reprogramming and switch from a proliferative to an invasive type of cell behavior.

Transition into S Phase of the Cell Cycle Is Inhibited by SIP1
Having demonstrated inhibition of cell growth by SIP1, we analyzed the effect of SIP1 on cell cycle distribution. FACS analysis of A431/SIP1 cell cultures maintained with or without DOX for 48 h showed that SIP1 increased proportion of cells in G1 phase (Figure 2A). Percent of cells passing through S phase, G2, and mitosis was two times lower in cells undergoing EMT (24 ± 4 vs. 49 ± 3%). Because G1/S transition in mammalian cell cycle is regulated by Rb pathway and phosphorylation of the Rb protein is critical for G1/S progression, we examined the effect of SIP1 on the Rb phosphorylation. We found that in our system, accumulation of cells in G1 phase of the cell cycle was concomitant with the hypophosphorylation of Rb (Figure 2B). Microarray analysis revealed strong (6.7-fold) down-regulation of the CCND1 gene, which encodes cyclin D1, a critical regulator of Rb phosphorylation (Supplementary Table SIB). We confirmed SIP1-mediated repression of cyclin D1 on both mRNA and protein levels. Next, we analyzed expression of other key proteins regulating Rb phosphorylation and cell cycle progression through G1 phase (Figure 2B). Whereas the mRNA levels of cyclin D3, p21(Cip1), and p27(Kip1) remained not altered upon SIP1 induction, transcription of cyclin D2 was not detectable independently on the presence of DOX. Western blot analysis demonstrated similar levels of p27(Kip1) and lack of the expression of p16 protein in SIP1-expressing and nonexpressing cells. Unexpectedly, in the presence of SIP1, the expression of p21(Cip1) was reduced on protein level, although mRNA level was not affected (Figure 2B).

View larger version (23K): [in this window] [in a new window]   Figure 2. SIP1 negatively regulates G1/S transition. (A) FACS analysis of A431/SIP1 cells maintained in the absence or presence of DOX for 48 h. The diagram shows mean ± SDs of triplicate experiments. p < 0.0001 (t test). (B) Expression of molecules regulating G1/S transition in A431/SIP1 cells. Cells were cultured with or without DOX for 48 h, and the expression of indicated genes was analyzed by Western blotting or RT-PCR. Note the appearance of the hypophosphorylated form of Rb in DOX-treated cells.

View larger version (38K): [in this window] [in a new window]   Figure 3. SIP-1 attenuates cell cycle progression via cyclin D1 down-regulation. (A) RNAi-mediated repression of cyclin D1 leads to accumulation of A431/SIP1 cells in G1 phase of the cell cycle. FACS analysis of cell cycle distribution of A431/SIP1 cells, in which cyclin D1 expression was reduced either by SIP1 or by RNAi. Control cells were cultured with or without DOX for 48 h. Results are mean ± SD of three experiments. Right panel, Western blot analysis of cyclin D1 and Rb expression in A431/SIP1 cells transfected with the negative control siRNA or cyclin D1-specific siRNA. 20 µg of total proteins was loaded in each lane. (B) Exogenous cyclin D1 counteracts the effect of SIP1 on Rb phosphorylation and cell cycle distribution. Clones with simultaneous DOX-regulated expression of SIP1 and cyclin D1 were generated and the phosphorylation of Rb was analyzed by Western blotting. Bottom, cells were cultured in the presence or absence of DOX for 48 h, and cell cycle distribution was monitored by FACS. (C) DOX-induced A431/SIP1 and A431/SIP1/cyclinD1-2 cells differently incorporate BrdU after 40-min pulse labeling. After the labeling, BrdU incorporation was detected by fluorescence microscopy with the monoclonal anti-BrdU antibody. Total cell number was identified by blue fluorescence (DAPI DNA staining). Proportion of BrdU-positive cells was quantified in six microscopic fields and presented as mean ± SD. The experiment was repeated twice with similar results.

View larger version (27K): [in this window] [in a new window]   Figure 4. Ectopic expression of cyclin D1 does not influence SIP1-mediated cell invasion and motility. (A) Exogenous cyclin D1 does not compromise SIP1-activated invasion. DOX-treated or untreated A431/SIP1 and A431/SIP1/cyclinD1-2 cells were allowed to invade into matrigel. Cell number on the underside of the filter and at the distance of 20 µm was quantified after staining with PI. The percentage of invaded cells was quantified in twelve microscopic fields. The diagram represents mean ± SD of triplicate experiments. (B) Enforced expression of cyclin D1 does not influence SIP1-activated cell motility. A431/SIP1 and A431/SIP1/cyclinD1-2 cells were maintained with or without DOX for 48 h. Migration was analyzed in transwell motility assay. Bar graphs summarize the results of three separate experiments (mean ± SD).

View larger version (22K): [in this window] [in a new window]   Figure 5. SIP1 regulates cyclin D1 promoter activity rather than the stability of cyclin D1 mRNA. (A) A431/SIP1 cells were maintained with or without DOX for 48 h. ActD was added for 4 or 8 h and expression of cyclin D1, SIP1, and fosl1 was analyzed by RT-PCR. 28S rRNA was used as an ActD-insensitive control. Right panel, DOX and ActD were added simultaneously, and transcription of cyclin D1, SIP1, fosl1, and 28S rRNA was analyzed by RT-PCR 8 h later. (B) Quantification of cyclin D1 mRNA levels in A431/SIP1 cells maintained with or without DOX using real time PCR. Cells were treated with ActD for 4 h, and amplification was performed in triplicate. Data are presented as the mean ± SD. (C) Nuclei were isolated from A431/SIP1 cells maintained with or without DOX for 48 h and subjected to nuclear run-on assay. Isolation of nuclei, conditions of the reaction, and purification of biotinylated uridine-containing transcripts are described in Materials and Methods. Isolated transcripts corresponding to GAPDH, prss11, and cyclin D1 genes were amplified by RT-PCR and analyzed in an agarose gel. The scheme depicts fragments of the cyclin D1 mRNA detected by RT-PCR. Results are representative of two independent experiments.

View larger version (31K): [in this window] [in a new window]   Figure 6. SIP1 directly regulates transcription of the cyclin D1 gene. (A) SIP1 interacts with three Z-box sequences in the cyclin D1 promoter in vivo. ChIP analysis of cyclin D1 promoter sequences in DOX-treated or untreated cells with the 9E10 antibody. As control an irrelevant antibody was used. Enrichment of SIP1-bound sequences was quantified by real-time PCR. Mean ± SD are shown. n = 9; *p < 0.01; **p < 0.001 (t test). (B) Luciferase reporter constructs pwtCCND1LUC and pmutCCND1LUC were transfected into A431/SIP1 cells. Cells were cultured in the presence of DOX for 48 h. Assays were carried out in triplicate, and four independent experiments were performed; a representative result is shown (mean ± SD, n = 3). p < 0.05 (t test).

View larger version (30K): [in this window] [in a new window]   Figure 7. E-cadherin dominant negative mutant (Ec1WVM) induces invasion without affecting cell cycle. (A) DOX-activated expression of myc-tagged Ec1WVM in A431 cells (clone 31D6) induces cell scattering and invasion. Phase-contrast images of 31D6 cells treated with DOX for 48 h or left untreated. Right panel, results of a typical matrigel invasion assay of 31D6 cells. (B) FACS analysis of 31D6 cells cultured in the absence or presence of DOX for 48 h. Results are means ± SD of three experiments. Right panel, Western blot analysis of cyclin D1 and Rb expression levels in DOX-treated (48 h) and untreated A431/Ec1WVM cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In addition to the activation of canonical well-described EMT-related processes (cell dissociation, cell motility and invasiveness, global changes in gene expression pattern), SIP1 significantly stimulated adhesion of A431 cells to fibronectin and collagen I (Supplementary Figure S1B). In contrast, Slug inhibited adhesion of human epidermal keratinocytes to fibronectin and laminin-5 as a result of transcriptional repression of genes coding for 3, 1, and 4 integrin subunits (Turner et al., 2006). In A431 cells, transcription of these genes was not affected by SIP1 (data not shown), and the mechanism by which SIP1 activated cell–matrix adhesion remains unclear. However, results reported by Turner et al. and our data represent a rare example of a cell feature oppositely regulated by different Snail/Slug and ZEB-1/SIP1 proteins in two cell lines of common (epidermal) origin.

Snail/Slug and ZEB-1/SIP1 family members control distinct EMT programs that are implicated in many aspects of embryonic development, gastrulation, somitogenesis, and neural crest migration. It is therefore plausible to speculate that cancer cells recapitulate some elements of concealed embryonic differentiation programs to acquire metastatic capabilities. Given that normal differentiating cells do not proliferate, the intriguing question arises as to whether the EMT programs affect cell proliferation in cancer as well. However, to our knowledge, this issue has not been scrupulously addressed. In an important study by Vega et al. (2004) the expression of Snail has been shown to induce accumulation of MDCK cells in the G1 phase of the cell cycle. In addition, Vega et al. demonstrated that Snail inhibited phosphorylation of Rb, lowered expression of cyclins D2 and D1 and increased expression of p21(Cip1). Cyclin D2 has been shown to be a direct Snail target. However, the role of particular cell cycle regulators has not been addressed in this study. Here, we analyzed how an EMT program initiated by the expression of SIP1 affects cell cycle progression. We found that although SIP1 belongs to a protein family only distantly related to Snail/Slug, its effect on cell cycle distribution in human epidermoid A431 cells is similar to the effects of Snail in MDCK cells and Slug in normal keratinocytes (Turner et al., 2006). Moreover, in the present study, we demonstrated the essential role of cyclin D1 whose direct transcriptional repression by SIP1 was necessary and sufficient to affect Rb phosphorylation status and to inhibit progression through G1 into S phase in A431 cells. Taken together, these observations indicate that targeting G1/S checkpoint is a common feature of different EMT-inducing transcription factors in different cell lines, although the actual mechanisms of this targeting might be different.

Immunohistochemical data on the expression of Snail/Slug and especially ZEB-1/SIP1 family members in tumor tissue are limited. A proportion of ZEB1-positive tumors has been identified by immunohistochemical analysis of aggressive endometrial and non-small lung cancer specimens (Dohadwala et al., 2006; Spoelstra et al., 2006). In oral squamous cell carcinoma, SIP1 was detected in 27% of tumor specimens. SIP1 expression correlated with lack of E-cadherin immunoreactivity and low disease-specific survival (Maeda et al., 2005). Similarly, Zhou et al. (2004) described extended E-cadherin-negative and Snail-positive areas in breast cancer surgical specimens, and this pattern significantly correlated with cancer metastasis. In another study, only a limited number of single Snail-positive cells has been detected at the periphery of tumor tissue in cervical squamous carcinoma and colon adenocarcinoma (Franci et al., 2006). Studies on EMT of MDCK cells (Vega et al., 2004) and data presented here suggest that cells maintaining control over G1/S transition and undergoing a rapid EMT in response to Snail or SIP1 acquire a growth disadvantage. Therefore, the functional status of the Rb pathway may determine the configuration of EMT programs utilized by cells of growing tumors. In carcinoma cells maintaining partial control over G1/S restriction point, members of the SIP1 and Snail protein families may induce a transient EMT, which will contribute to metastatic dissemination without stable repression of epithelial markers (e.g., E-cadherin) in primary tumors. This hypothesis may explain why complete EMTs are relatively rarely observed in human cancers (Christofori, 2006). One of the events perturbing the Rb pathway is overexpression of cyclin D1 that is frequently associated with carcinomas in humans (in part, as a result of amplification of the cyclin D1 gene; Malumbres and Barbacid, 2001; Knudsen et al., 2006). Concurrent expression of cyclin D1 and SIP1 in A431 cell line generated cells, which were capable of proliferating and invading into matrigel at the same time (Figures 3 and 4). We suggest that accumulated defects in the Rb pathway in vivo would permit a stable EMT, resulting in the appearance of most aggressive tumor cell variants.

In contrast to the SIP1 model, functional inhibition of E-cadherin by a dominant negative E-cadherin mutant induces a gradual EMT in A431 cells without attenuating the cell cycle (Figure 7). Therefore, prolonged inactivation of epithelial adhesion by matrix metalloproteinases secreted by stroma cells or e-cadherin gene mutations may represent a mechanism of a stable EMT in tumor cells retaining partial control over G1/S transition.

In conclusion, we have demonstrated that cyclin D1 is a new direct transcriptional target of SIP1. Taken together with previously published results (Vega et al., 2004; Turner et al., 2006), our data suggest that attenuated G1/S phase cell cycle transition is a common feature of EMT programs induced by Snail/Slug and ZEB-1/SIP1 proteins.

	ACKNOWLEDGMENTS

 
We thank Jiri Lukas (Danish Cancer Society, Copenhagen) for his valuable comments on the manuscript. We thank Kristin Verschueren (University of Leuven, Belgium) for pTREmyc-SIP1ZFmut plasmid. We acknowledge the support from the Department of Cancer Studies and Molecular Medicine (University of Leicester, United Kingdom) and European Urology Fellowship Programme. C.V. was supported by the Fund for Scientific Research, Flanders (FWO) and the "Centrum voor Gewelziekten," Ghent. G.B. is a postdoctoral fellow with the FWO. The research by C.V. and G.B. has been supported by the Association for International Cancer Research (AICR-United Kingdom), FWO-Flanders and Interuniversitaire Atractiepolen (Belgian Government).

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-05-0406) on September 12, 2007.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Eugene Tulchinsky (et32{at}le.ac.uk)

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