shRNA SNAIL 导致的细胞表型改变

The effects of shRNA-mediated gene silencing of transcription factor SNAI1on the biological phenotypes of breast cancer cell line MCF-7Yan Lu •Lina Yu •Minlan Yang •Xiangshu Jin •Zhijing Liu •Xiaowei Zhang •Liping Wang •Dongjing Lin •Yuanyuan Liu •Min Wang •Chengshi QuanReceived:18August 2013/Accepted:15November 2013/Published online:29November 2013ÓSpringer Science+Business Media New York 2013Abstract To research the effects of silencing transcrip-tion factor SNAI1on the in vitro biological phenotypes of breast cancer cell line MCF-7,based on the gene sequence of SNAI1,we linked shRNA with the green fluorescent protein-expressing eukaryotic expression vector pGCsi-lencer TM U6/Neo/GFP,and transfected it into MCF-7cells.The SNAI1gene-silencing effect was authenticated by RT-PCR and immunofluorescence.We then examined the effect of gene silencing on the expression of epithelial and mesenchymal markers and on their biological phenotypes of the target cells.Finally,we explained that SNAI1was bound to E-cadherin in MCF-7cells by ChIP.Silencing SNAI1upregulated the expression of epithelial markers claudin-4,claudin-7,and E-cadherin,while expression of the mesenchymal marker matrix metalloproteinase-2was downregulated.The capacity for proliferation,migration,and invasion was diminished.SNAI1binds to the E-cad-herin gene promoter and inhibits its transcription.We can conclude that silencing gene SNAI1inhibits expression of properties that are associated with the malignant phenotype of MCF-7cells and reverses the epithelial–mesenchymaltransition process by regulating relevant target gene E-cadherin.Keywords SNAI1ÁGene silencing ÁBiological phenotypes ÁMCF-7IntroductionDuctal carcinoma of the breast is one of the most common malignant tumors in women,and its incidence is increasing year by year [1,2].Tumor metastasis is the main reason for mortality from this disease [3].In breast cancer,the expression level of SNAI1(snail homolog 1),a transcription factor,correlates with tumor grade and lymph node metas-tasis [4].SNAI1can regulate the expression of a target gene by E-box (CAGGTG\CACCTG)which can specifically bind upstream of the target promoter to inhibit the expression of the gene,resulting in initiation of epithelial–mesenchymal transition [5–11]which is also mainly characterized by downregulation of E-cadherin [12,13].SNAI1induces epithelial–mesenchymal transition which increases metas-tasis through loss of apical–basolateral polarity and gain of mesenchymal characteristics to enhance motility [14].Consequently,we have postulated that SNAI1plays a key role in the invasion and metastasis of breast cancer through binding to E-cadherin gene promoter.To test this hypothesis,we designed the plasmid pGCsi-lencer TM U6/Neo/GFP,which can silence the expression of SNAI1.The plasmid was transfected into MCF-7cells which have high endogenous SNAI1expression.We then examined the expression of epithelial and mesenchymal markers as well as the effect of SNAI1silencing on proliferation,migration,and invasion in vitro.SNAI1binding to E-cadherin gene promoter in MCF-7cells was authenticated by ChIP.Yan Lu and Lina Yu have contributed equally to this paper.Y.Lu ÁM.Yang ÁX.Jin ÁZ.Liu ÁX.Zhang ÁL.Wang ÁD.Lin ÁY.Liu ÁM.Wang ÁC.Quan (&)The Key Laboratory of Pathobiology,Ministry of Education,Department of Pathology,College of Basic Medical Sciences,Jilin University,126Xinmin Avenue,130021Changchun,China e-mail:quancs@L.YuThe First Hospital Cancer Center of Jilin University,Changchun,ChinaMol Cell Biochem (2014)388:113–121DOI 10.1007/s11010-013-1903-4Materials and methodsCell cultureHuman mammary epithelial cell line HBL-100,human breast cancer cell lines MCF-7and MDA-MB-231,and mousefibroblast cell line NIH3T3were cultured in high glucose DMEM medium(GIBCO,USA)supplemented with10%fetal bovine serum(FBS;Hyclone,USA)at 37°C,in the presence of5%CO2.Transient transfectionBased on Ding et al.[15],shRNA(containing sense and antisense sequences linked by a hairpin loop:TTCAAGA-GA)was designed and constructed into the vector pGCsi-lencer TM U6/Neo/GFP(GeneChem,China).The MCF-7cells were then transfected with SuperFectÒTransfection Reagent(QIAGEN,USA).The negative control cell line was generated by infecting cells with the vector pGCsi-lencer TM U6/Neo/GFP constructed with oligonucleotides, which has no homology to the human gene.The control group was the MCF-7cell line that has not been treated. Reverse transcription-polymerase chain reaction(RT-PCR)Total RNA was isolated with TRIZOL(Invitrogen,USA) and reverse transcribed with M-MLV reverse transcriptase (TaKaRa,Japan).The primers(Sangon,China)used for RT-PCR were devised by software Prime premier5.0and are listed in Table1.Western blot analysisCells were harvested and lysed in300l l ice-cold Radio Immunoprecipitation Assay Lysis Buffer supplemented with 1%phenylmethylsulfonylfluoride,then centrifuged at 12,0009g,at4°C for30min to remove cell debris.Their concentration was determined by a BCA Protein Assay Kit (Pierce Chemical Co.,Rockford,IL,USA).Cell lysates were separated in10%SDS-PAGE and then transferred onto a nitrocellulose membrane(Millipore,Temecular,CA,USA). The membrane was then incubated with5%nonfat milk (Applichem)at37°C for1h,and incubated with1:1,000 diluted primary antibody SNAI1(ENOGENE,CHINA)at 4°C overnight and1:1,000diluted secondary antibody for 1h at room temperature thereafter.The secondary antibody was appropriate IgG,conjugated to horseradish peroxidase. The blots werefinally stained using an ECL Western blotting system(GE,Fairfield,CT,USA).ImmunofluorescenceThe assay was performed to evaluate the expression of SNAI1as previously described[16].In our study,cells were incubated with primary SNAI1antibody(1:300),at4°C overnight.The secondary antibody was Alexafluor488anti-rabbit IgG(1:250dilution;CST,USA).Cells were visualized with a laser scanning confocal microscope(Olympus,Japan).Cell proliferation analysisIn order to measure cell proliferation,cells were seeded in 24-well plates at a low density(29104cells/well)and cul-tured as described above.The number of cells was countedTable1Primers and information for RT-PCR MMP-2matrix metalloproteinase-2Gene Primers Length(bp)Annealingtemperature(°C)CyclesSNAI150-GCCTAGCGAGTGGTTCTTCTG-301685430 50-TAGGGCTGCTGGAAGGTAAA-30Claudin-450-TGCACTCTGCGAACGTTAAG-301415635 50-GCGATGCCCATTACCTGTAG-30Claudin-650-TTCATCGGCAACAGCATCGT-303455635 50-GGTTATAGAAGTCCCGGATGA-30Claudin-750-GGAGATCCCAGGTCACACAT-301405030 50-CAGGGTCTGCCCTAGTCATC-30E-cadherin50-CGGTGGTCAAAGAGCCCTTACT-301715832 50-TGAGGGTTGGTGCAACGTCGTTA-30MMP-250-CTTCCAAGTCTGGAGCGATGT-301185230 50-TACCGTCAAAGGGGTATCCAT-30GAPDH50-TGTTGCCATCAATGACCCCTT-301875525 50-CTCCACGACGTACTCAGCG-30every24h over the next7days.Three replicate counts were performed in each of three independent experiments.Wound healing assay19105cells were seeded per well into a24-well plate and cultured as described above.When cells had grown to confluence,a straight wound was created in each mono-layer by dragging a10-l l pipette tip through the mono-layer.Images were taken under a microscope(Olympus, Japan)at0and24h after wounding to determine the width of the wounded area.The relative migration distance(%of recovery)was calculated as(W0-W24)/W09100%.In each experiment,three replicate wells were measured.Invasion assayWe cultured NIH3T3cells as described above and replaced the medium with serum-free DMEM when the cells were at logarithmic growth phase.The supernatant was collected after 24h and passed through a0.22-l mfilter to remove the cell debris.Cells of three groups were harvested from monolayer culture by trypsinization,centrifuged to collect them into a 1.5-ml eppendorf tube,and stained with0.5ml Rhodamine (1mg/ml)for30min.We planted59104stained cells on a Matrigel membrane which was in the upper compartment of the Millipore chamber.The supernatant was placed into the lower compartment as a chemoattractant.The chambers were then incubated for5h at37°C in the presence of5%CO2. The Matrigel membranes were scanned by a laser scanning confocal microscope(Olympus,Japan)to see how many cells had migrated through the Matrigel.Two replicates were performed in each of three independent experiments. Chromatin immunoprecipitation assayThe experiment was performed as described by the EZ-ChIP kit protocol(MILLOPORE,USA).Briefly,the steps were as fol-lows:Cells were cultured to confluence in10-cm dishes (19107–59107cells per dish).Proteins were cross-linked to DNA,by adding formaldehyde drop-wise directly to the media for afinal concentration of1%,and then incubated at37°C for 10min.Incubated with2.5M glycine for5min at room temperature to terminate formaldehyde cross-linking.The cells were removed mechanically in cold PBS and transferred into 15-ml tubes.They were then harvested by centrifugation and resuspended in lysis buffer(contains protease inhibitors).The lysate was sonicated to shear the DNA to an average fragment size of200–1,000bp.The fragment size was fol-lowed on a1.5%agarose gel.The reaction mixture was cen-trifuged,the supernatant was transferred to a new tube,and 100l l of each sonicated sample was removed.900l l ChIP dilution buffer and20l l509Protease Inhibitor Compounds were added to the each of sonicated samples.The antibody was diluted to a concentration of1:500.RNA polymerase II anti-body was added as a positive control and rabbit IgG antibody was used as a negative control.60l l of protein A/G beads and salmon protease were added to all samples which were then immunoprecipitated overnight with rotation at4°C.The pro-tein A/G beads were harvested by centrifugation(2,0009g)and washed with buffer.The DNA was then eluted and PCR ana-lysis was performed to detect the promoter regions of E-cad-herin(promoter,5’-ACTCCAGGCTAGAGGGTCAC-3’and 5’-CCGCAAGCTCACAGGTGCTTTGCAGTTCC-3’). Statistical analysisStatistical analysis was performed using the statistical software SPSS12.0for t test and one-way ANOVA.The criterion for statistical significance was P\0.05. ResultsSNAI1expression in mammary cell linesRT-PCR and western blot analysis showed that there was lower SNAI1expression in human mammary epithelial cell line HBL-100and higher expression in the two human breast cancer cell lines.MCF-7cells had higher expression levels than MDA-MB-231(Fig.1a,b).Therefore,we chose MCF-7as the focus of our study.SNAI1gene-silencing effectRT-PCR demonstrated that after transfection with shRNA, SNAI1was significantly downregulated incomparison Fig.1Expression of SNAI1in different cell lines.We evaluated the expression of SNAI1in mammary epithelial cell line HBL-100and breast cancer cell lines MCF-7and MDA-MB-231(a,RT-PCR;b, Western blot).SNAI1expression was higher in MCF-7cells than in the other two cell lineswith the control and negative control group between which there was no significant difference in SNAI1expression (Fig.2a,b).Figure2c shows that following transfection with shRNA,SNAI1expression was downregulated,as the greenfluorescence was weaker in the nucleus compared with the other two groups,and that SNAI1was located in the nucleus.Expression of epithelial and mesenchymal markersRT-PCR analysis shows that,after silencing of SNAI1,the epithelial markers claudin-4,claudin-7,and E-cadherin were upregulated.At the same time,the mesenchymal marker matrix metalloproteinase-2was downregulated (Fig.3a,b).Thus,we postulate that SNAI1silencing can lead to epithelial–mesenchymal transition.Effect of SNAI1silencing on cell behaviorsAfter growing the cells for7days in24-well plates,we counted the cell number each day and found that cells in the shRNA group proliferated significantly more slowly than the other two groups(Fig.4a).After wounding the monolayer, we measured the relative migration distance of the cells in each group and calculated their relative mobility at0and 24h(Fig.4b,c).At24h,the in vitro migration rate of the shRNA group was significantly lower than the rate of the other two groups.After SNAI1silencing,the migration distance(Fig.4d lower panel)and the number(Fig.4d upper panel)of cells that went through Matrigel to the polycar-bonate membrane level were less than the other two groups. There was no significant difference between the control and the negative control group cells(Fig.4).Therefore,we concluded that SNAI1can promote the proliferation, migration,and invasion of MCF-7cells.SNAI1is bound to the E-cadherin geneFigure2c shows that SNAI1was located in the nucleus and may have some function related to epithelial–mesenchymal transition in the nucleus.Chromatin immunoprecipitation demonstrated that SNAI1was bound to the E-cadherin gene promoter in MCF-7cells(Fig.5b).Furthermore, SNAI1silencing was associated with upregulated E-cad-herin(Fig.3a,b).Therefore,we can concluded that SNAI1 is a transcriptional factor,which can bind to the E-cadherin gene and inhibits its transcription.DiscussionTranscription factor SNAI1is a member of the snail tran-scription inhibitor super-family,with more than50family members which have been characterized in metazoans.In humans,it is distributed in the placenta,heart,lung,brain, liver,and skeletal muscles[17].At the carboxy terminus of the SNAI1gene,there are four highly conserved zincfingers which specifically bind to the E-box sequence(CAGGTG\CAGGTG)of the upstream target gene promoter region and have the capacity of initi-ating the epithelial–mesenchymal transition process by inhibiting the activity of the target gene promoter[18]. Epithelial–mesenchymal transition is characterized by the loss of apical–basolateral polarity,gain ofmesenchymal Fig.2Expression of SNAI1in different groups.RT-PCR(a,b)andimmunocytofluorescent analysis(c)show that SNAI1expression waslower in the shRNA group than the other two groups.The data areexpressed as mean±SD,N=3;*P\0.01versus control andnegative group(b).After silencing,SNAI1expression wasdownregulated,as the greenfluorescence was weaker in the nucleuscompared with the other two groups(c).Three independent exper-iments were done(M marker,C control,N negative control,S SNAI1-shRNA)characteristics,extracellular matrix degradation,weakened intercellular adhesion,and enhanced cell motility [8,19,20].The epithelial–mesenchymal transition process is critical for embryonic development,wound healing,cell differentia-tion,cell migration,and tumor metastasis [21,22].On the contrary,mesenchymal cells can acquire characteristics of epithelial cells through transformation,namely,mesenchy-mal–epithelial transition.Therefore,we have postulated that reversing the epi-thelial–mesenchymal transition phenotype might reduce the invasive and metastatic capabilities of early cancers and prolong the survival of tumor-bearing animals.In this study,we used RNAi technology to silence SNAI1gene expression and studied the resulting biological phenotypes of MCF-7cells.After silencing SNAI1,the epithelial markers E-cad-herin,claudin-4,and claudin-7were upregulated and the mesenchymal cell marker matrix metalloproteinase-2was downregulated.The results show that silencing SNAI1activated the epithelial cell-associated genes.The cells then regained their epithelial polarity and capacities for cell–cell adhesion,thus demonstrating the unique morphology of epithelial cells.Silencing SNAI1induced downregulation of matrix metalloproteinase-2and reduced the ability of the cells to degrade extracellular matrix and collagen.In other words,the cells lost the ability of migration and invasion,in concert with the loss of mesenchymal to epithelial transformation.Other in vitro experiments have obtained results that are consistent with our data.After antisense SNAI1was transfected into colon cancer cell line HT-29,the cell pseudopodia became shortened and the cells became round,suggesting mesenchymal cell-like phenotype transition to an epithelioid phenotype [23].In the present study,we also examined the proliferative capacity of the test cells in vitro.After silencing SNAI1,the proliferation rate of the MCF-7cells was diminished.In a similar experiment,Olmeda et al.[24]silenced the SNAI1gene in MDA-MB-231cells which are typically poorly differentiated and easily transplanted.60days after inoculating the silenced cells and control cells into mice,both the number of metastases and the tumor size in the SNAI1-silenced group were significantly less than the control group,indicating that SNAI1inhibited cell prolif-eration in vivo.This observation is also consistent with our experimental data.Olmeda et al.postulated that SNAI1might be a downstream molecule of TGF-b ,PI3K,or a signal pathway and suggested that the reduction of SNAI1might diminish cell proliferation capacity through a signal transduction pathway.In our study,silencing SNAI1inhibited cell proliferation in association with upregulation of E-cadherin,claudin-4,and claudin-7,suggesting that the connections between cells and their anchorage in local position may inhibit mitosis.This concept is supported by the work of St Croix et al.[25]who found that E-cadherin is not only a mediator of epithelial adhesion but also an important contact cell growth inhibitor.By upregulating cell cycle inhibitor p27,E-cadherin acted on the cell cycle to inhibit cell prolifer-ation.Thus,it may be that SNAI1affects cell proliferation indirectly through E-cadherin expression.Silencing SNAI1appears to inhibit MCF-7cell migra-tion in several ways.First,in our study,SNAI1directly lowered the expression of E-cadherin by binding to its promoter and inhibiting its transcription and translation.E-cadherin is the main component for epithelial adhesion connection [26].It can be connected to b -catenin to form an E-cadherin/b -catenin complex,causing the cells to be connected to each other and restricting their migration or invasion [27].In the process of tumorigenesis and pro-gression,the decline in expression of E-cadherin can weaken cell adhesion,thus causing tumor cells to exitfromFig.3Expressions of epithelial and mesenchymal markers.mRNA level of the expression of epithelial markers claudin-4,claudin-6,claudin-7,and E-cadherin and mesenchymal marker MMP-2(a ,b ).Except claudin-6,expression of epithelial markers in the experimental group was upregulated in comparison with the control and negative control groups.Expression of the mesenchymal marker MMP-2was downregulated.Data represent one of at least three independent experiments.Each value represents the mean ±SD.*P \0.01versus the parent control cells (M marker,C control,N negative control,S SNAI1-shRNA)the primary tumor,invade the surrounding tissue,and subsequently diffuse through blood vessels [28].Second,we found that silencing SNAI1upregulated the expression of key components of the tight junction—claudin-4and claudin-7—which play a decisive role in the constitution of epithelial and endothelial tissue [29].Tight junction com-ponents may strengthen the connections between cells in order to maintain organizational structure and the local steady state.Therefore,we postulate that the diminished capacity of cell migration in vitro after silencing SNAI1may be related to the upregulation of E-cadherin,claudin-4,and claudin-7.Similar to our results,Fabre-Guillevin [30]also found that in the breast cancer cell line MDA-MB-231,the ability of cells to migrate in vitro was significantly weakened when SNAI1was inhibited.Moreover,in human colorectal cancer cell lines HCT-116and HT29[31]and hepatoma cell line HepG2[32],the results were also consistentwithFig.4Effect of SNAI1silencing on cell behaviors in MCF-7cells.a Cell proliferation curve.There was a significant difference between the numbers of cells in each group from the second day,but no significant difference in cell proliferation between the control group and the negative control group.SNAI1transfection of MCF-7cells significantly inhibited their proliferative ability (The data are expressed as mean ±SD,N =6,*P \0.01versus control and negative group).b Wound healing assay to detect cell migration ability.After wounding the monolayer,we measured the relative migration distance of the cells in each group and calculated the relative mobility at 0and 24h.In the shRNA group,the migration rate at 24h was significantly lower than the other two groups (N =6,*P \0.01).Mobility of control group and negative group was notsignificantly different (c ).d Invasion assay to detect cell invasion ability.Cells were planted on a Matrigel surface in the upper well of a Boyden chamber,and then incubated at 37°C for 5h.Conditioned medium was used as a chemoattractant.After incubation of the chamber,we used confocal microscopy to scan the cells in each group layer by layer.Cells that had migrated through the Matrigel layer to the polycarbonate membrane level were counted (d ,upper panels ).Cell migration distance is shown in (d lower panels ).Invasive ability of the shRNA group was significantly less than the other two groups;the shRNA group had no detectable cells that had passed through the Matrigel layer (e ,N =6,**P \0.001);M polycarbonate membrane,C control,N negative control,S SNAI1-shRNAthe present experiment that cell migration was significantly weakened.Tumor invasion influence factors include reduction of intercellular adhesion,increased mobility of the tumor cells,and enzymatic degradation of the basement membrane and extracellular matrix by enzymes such as matrix metallo-proteinases,cathepsins,and plasminogen activators [33].In our experiments,silencing SNAI1significantly downregu-lated the expression of matrix metalloproteinase-2and inhibited the cellular invasive ability in vitro.Silencing SNAI1also downregulated the expression of matrix metalloproteinase-9.Matrix metalloproteinase-2and matrix metalloproteinase-9are gelatinases [34].Their substrates are degenerated collagen and the main constituent of base-ment membrane,type-IV collagen.Matrix metalloprotein-ase-2can also degrade fibronectin and laminin,which contribute to the process of tumor invasion [35].We postulate the existence of a unique mechanism by which these genes are regulated through silencing SNAI1.In our experiments,after gene silencing,SNAI1was mainly expressed in cytoplasm,instead of in both nucleus and cytoplasm,and it upregulated E-cadherin.Further-more,SNAI1was bound to E-cadherin in MCF-7cells;we concluded that SNAI1,as a transcriptional factor,works in nucleus to bind to E-cadherin gene promoter and inhibitsits transcription,resulting in epithelial–mesenchymal transition.In the promoter region upstream of E-cadherin [36],claudin-4,and claudin-7[37],there are three,eight,and eight E-box structures,respectively,to which SNAI1could directly bind,thus playing a transcriptional repressor role.Thus,silencing SNAI1could reduce its normal inhibitory role,causing upregulation of E-cadherin,claudin-4,and claudin-7.Although claudin-6has three E-boxes as well,its expression did not change significantly.This shows that though the gene claudin-6also consists of E-boxes,SNAI1did not directly bind to them.This is similar to the regulation of claudin-1.Ohkubo and Ozawa [38]found that there are two E-boxes in the claudin-1promoter region and RT-PCR results showed that the transferred exogenous SNAI1gene did not cause alteration of claudin-1expression.Luciferase reporter experiments also indicated that claudin-1gene promoter activity was not changed.These showed that E-box was not the unique gene region for SNAI1to bind to the gene promoter region,which can inhibit transcription.Furthermore,SNAI1gene silencing also led to downregu-lation of MMP-2,while the promoter region of the matrix metalloproteinase gene family does not have an E-box [39,40].Therefore,SNAI1did not directly act on the tran-scription phase of matrix metalloproteinases and further research is needed.Since current research has shown that the expression level of SNAI1also influences occludin,claudin-3,and ZO-1[41],we will include analysis of these mole-cules in our next experiments.In summary,we have found that silencing SNAI1,which is a transcriptional inhibitor that can bind to E-cadherin gene promoter and inhibits its transcription,leads to changes in proliferation,migration,and invasion,resulting in transformation of the malignant phenotype to a more benign state in breast cancer cell line MCF-7.Acknowledgments This study was supported by the National Nat-ural Science Foundation of China (Code:81172499)and Science and Technology Development Plan of the Office of Science and Tech-nology Project in Jilin Province (Code:201115113).We thank Dr.William Orr,Department of Pathology,University of Manitoba,Canada,for his help in the preparation of the manuscript.References1.Jung KW,Won YJ,Kong HJ,Oh CM,Seo HG,Lee JS (2013)Cancer statistics in Korea:incidence,mortality,survival and prevalence in 2010.Cancer Res Treat 45:1–14.doi:10.4143/crt.2013.45.1.12.Cowin P,Rowlands TM,Hatsell SJ (2005)Cadherins and cate-nins in breast cancer.Curr Opin Cell Biol 17:499–508.doi:10.1016/j.ceb.2005.08.0143.May CD,Sphyris N,Evans KW,Werden SJ,Guo W,Mani SA (2011)Epithelial-mesenchymal transition and cancer stemcells:Fig.5Association of SNAI1with E-cadherin promoter region.a Genomic DNA was sheared by sonication to an average fragment size of 100–2,000bp.b Cross-linked chromosomal DNA was immunoprecipitated from HBL-100cell and MCF-7cell nuclear extracts using a ChIP-grade SNAI1antibody.SNAI1was bound to E-cadherin promoter in MCF-7cells,but not in HBL-100cells (input positive control,IgG negative control)a dangerously dynamic duo in breast cancer progression.BreastCancer Res13:202.doi:10.1186/bcr27894.Blanco MJ,Moreno-Bueno G,Sarrio D,Locascio A,Cano A,Palacios J,Nieto MA(2002)Correlation of Snail expression with histological grade and lymph node status in breast carcinomas.Oncogene21:3241–3246.doi:10.1038/sj.onc.12054165.Geradts J,de Herreros AG,Su Z,Burchette J,Broadwater G,Bachelder RE(2011)Nuclear Snail1and nuclear ZEB1protein expression in invasive and intraductal human breast carcinomas.Hum Pathol42:1125–1131.doi:10.1016/j.humpath.2010.11.004 6.Peinado H,Ballestar E,Esteller M,Cano A(2003)Snail mediatese-cadherin repression by the recruitment of the Sin3A/histone deacetylase1(HDAC1)/HDAC2complex.Mol Cell Biol 24:306–319.doi:10.1128/mcb.24.1.306-319.20047.Foubert E,De Craene B,Berx G(2010)Key signalling nodes inmammary gland development and cancer.The Snail1-Twist1 conspiracy in malignant breast cancer progression.Breast Cancer Res12:206.doi:10.1186/bcr25858.van der Wurff AA,Vermeulen SJ,van der Linden EP,Mareel MM,Bosman FT,Arends JW(1997)Patterns of alpha-and beta-catenin and E-cadherin expression in colorectal adenomas and carcinomas.J Pathol182:325–330.doi:10.1002/(SICI)1096-9896(199707)182: 3\325:AID-PATH865[3.0.CO;2-Y9.Dang H,Ding W,Emerson D,Rountree CB(2011)Snail1induces epithelial-to-mesenchymal transition and tumor initiating stem cell characteristics.BMC Cancer11:396.doi:10.1186/1471-2407-11-39610.Schwartz B,Melnikova VO,Tellez C,Mourad-Zeidan A,BlehmK,Zhao YJ,McCarty M,Adam L,Bar-Eli M(2007)Loss of AP-2alpha results in deregulation of E-cadherin and MMP-9and an increase in tumorigenicity of colon cancer cells in vivo.Onco-gene26:4049–4058.doi:10.1038/sj.onc.121019311.Mauro L,Catalano S,Bossi G,Pellegrino M,Barone I,Morales S,Giordano C,Bartella V,Casaburi I,Ando S(2007)Evidences that leptin up-regulates E-cadherin expression in breast cancer:effects on tumor growth and progression.Cancer Res67:3412–3421.doi:10.1158/0008-5472.CAN-06-289012.Thiery JP,Sleeman JP(2006)Complex networks orchestrateepithelial-mesenchymal transitions.Nat Rev Mol Cell Biol 7:131–142.doi:10.1038/nrm183513.Peinado H,Olmeda D,Cano A(2007)Snail,Zeb and bHLHfactors in tumour progression:an alliance against the epithelial phenotype?Nat Rev Cancer7:415–428.doi:10.1038/nrc2131 14.Singh A,Settleman J(2010)EMT,cancer stem cells and drugresistance:an emerging axis of evil in the war on cancer.Oncogene29:4741–4751.doi:10.1038/onc.2010.21515.Ding JX,Feng YJ,Yao LQ,Yu M,Jin HY,Yin LH(2006)Thereinforcement of invasion in epithelial ovarian cancer cells by17 beta-Estradiol is associated with up-regulation of Snail.Gynecol Oncol103:623–630.doi:10.1016/j.ygyno.2006.04.02316.Peng X,Mehta R,Wang S,Chellappan S,Mehta RG(2006)Pro-hibitin is a novel target gene of vitamin D involved in its antipro-liferative action in breast cancer cells.Cancer Res66:7361–7369.doi:10.1158/0008-5472.CAN-06-100417.Moreno-Bueno G,Portillo F,Cano A(2008)Transcriptional regu-lation of cell polarity in EMT and cancer.Oncogene27:6958–6969.doi:10.1038/onc.2008.34618.Ferrari-Amorotti G,Fragliasso V,Esteki R,Prudente Z,Soliera AR,Cattelani S,Manzotti G,Grisendi G,Dominici M,Pieraccioli M, Raschella G,Chiodoni C,Colombo MP,Calabretta B(2013)Inhib-iting interactions of lysine demethylase LSD1with snail/slug blocks cancer cell invasion.Cancer Res73:235–245.doi:10.1158/0008-5472.CAN-12-173919.Peinado H,Ballestar E,Esteller M,Cano A(2004)Snail mediatesE-cadherin repression by the recruitment of the Sin3A/histonedeacetylase1(HDAC1)/HDAC2complex.Mol Cell Biol 24:306–31920.Shimokawa M,Haraguchi M,Kobayashi W,Higashi Y,Mats-ushita S,Kawai K,Kanekura T,Ozawa M(2013)The tran-scription factor Snail expressed in cutaneous squamous cell carcinoma induces epithelial-mesenchymal transition and down-regulates COX-2.Biochem Biophys Res Commun 430:1078–1082.doi:10.1016/j.bbrc.2012.12.03521.Wang H,Wang HS,Zhou BH,Li CL,Zhang F,Wang XF,ZhangG,Bu XZ,Cai SH,Du J(2013)Epithelial-mesenchymal transi-tion(EMT)induced by TNF-alpha requires AKT/GSK-3beta-mediated stabilization of snail in colorectal cancer.PLoS ONE 8:e56664.doi:10.1371/journal.pone.005666422.Micalizzi DS,Ford HL(2009)Epithelial-mesenchymal transitionin development and cancer.Future Oncol5:1129–1143.doi:10.2217/fon.09.9423.Batlle E,Sancho E,Franci C,Dominguez D,Monfar M,BaulidaJ,Garcia De Herreros A(2000)The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells.Nat Cell Biol2:84–89.doi:10.1038/3500003424.Olmeda D,Moreno-Bueno G,Flores JM,Fabra A,Portillo F,Cano A(2007)SNAI1is required for tumor growth and lymph node metastasis of human breast carcinoma MDA-MB-231cells.Cancer Res67:11721-11731.doi:10.1158/0008-5472.CAN-07-231825.St Croix B,Sheehan C,Rak JW,Florenes VA,Slingerland JM,Kerbel RS(1998)E-Cadherin-dependent growth suppression is mediated by the cyclin-dependent kinase inhibitor p27(KIP1).J Cell Biol142:557–57126.Wang Z,Gong Q,Fan Q(2012)Expression of E-cadherin inangiomyolipoma.Hum Pathol43:2348–2353.doi:10.1016/j.humpath.2012.04.01127.Kitase Y,Shuler CF(2013)Microtubule disassembly pre-vents palatal fusion and alters regulation of the E-cadherin/ catenin complex.Int J Dev Biol57:55–60.doi:10.1387/ijdb.120117yk28.Chen D,Wang Y,Zhang K,Jiao X,Yan B,Liang J(2012)Antisense oligonucleotide against clusterin regulates human hepatocellular carcinoma invasion through transcriptional regu-lation of matrix metalloproteinase-2and e-cadherin.Int J Mol Sci 13:10594–10607.doi:10.3390/ijms13081059429.Matter K,Aijaz S,Tsapara A,Balda MS(2005)Mammalian tightjunctions in the regulation of epithelial differentiation and prolifer-ation.Curr Opin Cell Biol17:453–458.doi:10.1016/j.ceb.2005.08.00330.Fabre-Guillevin E,Malo M,Cartier-Michaud A,Peinado H,Moreno-Bueno G,Vallee B,Lawrence DA,Palacios J,Cano A, Barlovatz-Meimon G,Charriere-Bertrand C(2008)PAI-1and functional blockade of SNAI1in breast cancer cell migration.Breast Cancer Res10:R100.doi:10.1186/bcr220331.Fan F,Samuel S,Evans KW,Lu J,Xia L,Zhou Y,Sceusi E,Tozzi F,Ye XC,Mani SA,Ellis LM(2012)Overexpression of Snail induces epithelial-mesenchymal transition and a cancer stem cell-like phenotype in human colorectal cancer cells.Cancer Med1:5–16.doi:10.1002/cam4.432.Hu CT,Wu JR,Chang TY,Cheng CC,Wu WS(2008)Thetranscriptional factor Snail simultaneously triggers cell cycle arrest and migration of human hepatoma HepG2.J Biomed Sci 15:343–355.doi:10.1007/s11373-007-9230-y33.Kevans D,Wang LM,Sheahan K,Hyland J,O’Donoghue D,Mulcahy H,O’Sullivan J(2011)Epithelial-mesenchymal transi-tion(EMT)protein expression in a cohort of stage II colorectal cancer patients with characterized tumor budding and mismatch repair protein status.Int J Surg Pathol19:751–760.doi:10.1177/ 1066896911414566。