Key points are not available for this paper at this time.
Snail, a transcriptional repressor of E-cadherin expression, plays a role in the process of epithelial-mesenchymal transition. However, the molecular basis of the role of snail in epithelial-mesenchymal transition has not been fully clarified. Here we show that the expression of snail in epithelial Madin-Darby canine kidney (MDCK) and A431 cells enhances both cell detachment and attachment. Snail did not confer resistance to anoikis induced by loss of contact but instead enhanced cell attachment to extracellular matrices such as fibronectin. This attachment was inhibited by Arg-Gly-Asp (RGD) peptides. Up-regulation of the promoter activity of integrin αV was observed in snail-expressing MDCK (MDCK/snail) cells. Snail also enhanced MDCK cell migration toward osteopontin that is a ligand for integrin αVβ3. We confirmed the reduction of basement membrane proteins such as laminin (LN) α3, β3, and γ2 (laminin-5/LN-5) and of receptors for LN-5 such as integrins α3, α6, or β4 in MDCK/snail or in snail-expressing A431 (A431/snail) cells. Nevertheless, suppression of LN-α3 chain by transient transfection of small interference RNAs resulted in no enhancement of cell detachment. We also found an induction of matrix metalloproteinase-3 in MDCK/snail and A431/snail cells. However, the inhibition of matrix metalloproteinase-3 showed no significant effect on the detachment of MDCK/snail cells. These results suggest that snail enhances cell detachment by multiple mechanism and leads to cell migration and reattachment at a second site, at least in part, by changing the expression of integrins in the cells. Snail, a transcriptional repressor of E-cadherin expression, plays a role in the process of epithelial-mesenchymal transition. However, the molecular basis of the role of snail in epithelial-mesenchymal transition has not been fully clarified. Here we show that the expression of snail in epithelial Madin-Darby canine kidney (MDCK) and A431 cells enhances both cell detachment and attachment. Snail did not confer resistance to anoikis induced by loss of contact but instead enhanced cell attachment to extracellular matrices such as fibronectin. This attachment was inhibited by Arg-Gly-Asp (RGD) peptides. Up-regulation of the promoter activity of integrin αV was observed in snail-expressing MDCK (MDCK/snail) cells. Snail also enhanced MDCK cell migration toward osteopontin that is a ligand for integrin αVβ3. We confirmed the reduction of basement membrane proteins such as laminin (LN) α3, β3, and γ2 (laminin-5/LN-5) and of receptors for LN-5 such as integrins α3, α6, or β4 in MDCK/snail or in snail-expressing A431 (A431/snail) cells. Nevertheless, suppression of LN-α3 chain by transient transfection of small interference RNAs resulted in no enhancement of cell detachment. We also found an induction of matrix metalloproteinase-3 in MDCK/snail and A431/snail cells. However, the inhibition of matrix metalloproteinase-3 showed no significant effect on the detachment of MDCK/snail cells. These results suggest that snail enhances cell detachment by multiple mechanism and leads to cell migration and reattachment at a second site, at least in part, by changing the expression of integrins in the cells. The molecular basis of the EMT 2The abbreviations used are:EMTepithelial-mesenchymal transitionECMextracellular matrixLNlamininCol IVcollagen IVFNfibronectinBMbasement membraneMDCKMadin-Darby canine kidneyDMEMDulbecco's modified Eagle's mediumFCSfetal calf serumPBSphosphate-buffered salineBSAbovine serum albuminMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideRTreverse transcriptionsiRNAsmall interference RNApoly-HEMApoly-2-hydroxyethyl methacrylateLDHlactate dehydrogenaseCMVcytomegalovirusFLfirefly luciferaseRLRenilla luciferasePIpropidium iodideCMconditioned mediumDCdeoxycholic acid. process involves changes in the expression, distribution, and function of a number of proteins that play a role in extracellular matrix remodeling or in cell-cell adhesion, such as MMPs, and E-cadherins (1Kiemer A.K. Takebuchi K. Quinlan M.P. Oncogene. 2001; 20: 217-226Crossref Scopus (70) Google Scholar, 2Pulyaeva H. Bueno J. Polette M. Birembaut P. Sato H. Seiki M. Thompson E.W. Clin. Exp. Metastasis. 1997; 15: 111-120Crossref PubMed Scopus (101) Google Scholar). Down-regulation of E-cadherin expression frequently occurs during the progression of carcinomas (3Takeichi M. Curr. Opin. Cell Biol. 1995; 5: 806-811Crossref Scopus (840) Google Scholar, 4Christofori G. Semb H. Trends Biochem. Sci. 1999; 24: 73-76Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar). Over the last few years, several transcription factors have been characterized as repressors of E-cadherin. These factors repress E-cadherin transcription through binding to the proximal E-boxes of the human or murine promoters for E-cadherin (5Bolos V. Peinard H. Perez-Moreno M.A. Fraga M.F. Esreller M. Cano A. J. Cell Sci. 2003; 116: 499-511Crossref PubMed Scopus (961) Google Scholar, 6Yang J. Mani S.A. Donaher J.L. Ramaswamy S. Itzykson R.A. Come C. Cell. 2004; 117: 927-939Abstract Full Text Full Text PDF PubMed Scopus (3158) Google Scholar). Among these repressors of E-cadherin, the zinc-finger factor snail induces a full EMT when overexpressed in epithelial MDCK cells leading to the acquisition of a motile/invasive phenotype (7Cano A. Perez-Moreno M.A. Rodrigo I. Locascio A. Blanco M.J. del Barrio M.G. Nat. Cell Biol. 2000; 2: 76-83Crossref PubMed Scopus (2989) Google Scholar, 8Peinado H. Marine F. Cubillo E. Stark H.J. Fusening N.N. Niet M.A. J. Cell Sci. 2004; 117: 2827-2839Crossref PubMed Scopus (146) Google Scholar). In agreement with this role of EMT induction, snail has been found to down-regulate the expression of epithelial genes, such as occludin and claudin (9Ohkubo T. Ozawa M. J. Cell Sci. 2004; 117: 1675-1685Crossref PubMed Scopus (286) Google Scholar), and to induce the expression of mesenchymal and invasive genes, such as FN and MMP-9 (7Cano A. Perez-Moreno M.A. Rodrigo I. Locascio A. Blanco M.J. del Barrio M.G. Nat. Cell Biol. 2000; 2: 76-83Crossref PubMed Scopus (2989) Google Scholar, 10Jorda M. Olmeda D. Vinyals A. Valero E. Cubillo E. Llorens A. Cano A. Fabra A. J. Cell Sci. 2005; 118: 3371-3385Crossref PubMed Scopus (201) Google Scholar). The conversion of tumor cells from an epithelial to the mesenchymal phenotype is closely associated with the acquisition of metastatic potential (11Birchmeier C. Birchmeier W. Brand-Saberi B. Acta Anat. (Basel). 1996; 156: 17-26Crossref Scopus (232) Google Scholar). Snail expression has been detected in an increasing number of human carcinoma and melanoma cell lines (12Peinado H. Portillo F. Cano A. Int. J. Dev. Biol. 2004; 48: 365-375Crossref PubMed Scopus (489) Google Scholar). More importantly in terms of cell motility, snail is expressed at the invasive front of epidermoid carcinomas (8Peinado H. Marine F. Cubillo E. Stark H.J. Fusening N.N. Niet M.A. J. Cell Sci. 2004; 117: 2827-2839Crossref PubMed Scopus (146) Google Scholar) and is associated with the invasiveness of ductal breast carcinomas and hepatocarcinomas (13Blanco M.J. Moreno-Bueno G. Sarrio D. Locascio A. Cano A. Palacios J. Nieto M.A. Oncogene. 2002; 21: 3241-3246Crossref PubMed Scopus (493) Google Scholar, 14Sugimachi K. Tanaka S. Kameyama T. Taguchi K. Aishima S. Shimada M. Sugimachi K. Tsuneyoshi M. Clin. Cancer Res. 2003; 9: 2657-2664PubMed Google Scholar). However, the precise role of snail in tumor progression has not been clarified. epithelial-mesenchymal transition extracellular matrix laminin collagen IV fibronectin basement membrane Madin-Darby canine kidney Dulbecco's modified Eagle's medium fetal calf serum phosphate-buffered saline bovine serum albumin 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reverse transcription small interference RNA poly-2-hydroxyethyl methacrylate lactate dehydrogenase cytomegalovirus firefly luciferase Renilla luciferase propidium iodide conditioned medium deoxycholic acid. BMs are dynamic, thin, sheet-like structures that consist of ECM proteins, on which epithelial cells reside. Disruption of the BM itself or integrins, the receptors for BM components, contributes to the tumor process (15Havenith M.G. Arends J.W. Simon R. Volovics A. Wiggers T. Bosman F.T. Cancer. 1988; 62: 2207-2211Crossref PubMed Scopus (97) Google Scholar). BM is composed of a number of different proteins. LN, the main component of BM, is comprised of at least 15 different LN trimers. LN-5 is composed of α3, β3, and γ2 chains and is present in most epithelial BMs. LN-5 interacts with integrin α6β4 and plays a crucial role in maintaining the stability of epithelial cells (16Ryan M.C. Lee K. Miyashita Y. Carter W.G. J. Cell Biol. 1999; 145: 1309-1323Crossref PubMed Scopus (262) Google Scholar). Oral squamous carcinoma cells that overexpress snail are deficient in LN-5 synthesis (17Takkunen M. Grenman R. Hukkanen M. Korhonen M. García de Herreros A. Virtanen I. J. Histochem. Cytochem. 2006; 54: 1263-1275Crossref PubMed Scopus (88) Google Scholar). Indeed, we found the expressions of LN-5 and its receptor proteins integrin α3, α6, and β4 were strongly suppressed in snail-expressing cells. However, cell detachment was not enhanced by inhibition of the expression of LN-5. These findings indicate that snail must affect the expressions of other molecules besides LN-5 to induce cell detachment. Collagen IV is also a major component of BM. It has been suggested that Collagen IV α5/α6 chains might protect against rapid cancer progression, as it has been shown that the normal production and assembly of BM is disrupted during malignant cancer progression (18Ikeda K. Iyama K. Ishikawa N. Egami H. Nakao M. Sado Y. Ninomiya Y. Baba H. Am. J. Pathol. 2006; 168: 856-865Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Integrins, heterodimeric transmembrane receptor complexes, interact with specific ECM proteins and thereby transmit signals to the cells. Enhanced expression of integrin αVβ3, in either a solid tumor or a melanoma, has been shown to correlate with tumor malignancy and with enhanced tumor cell growth and invasion (19Hood J.D. Bednarski M. R. S. R.A. R. 2002; PubMed Scopus Google Scholar, M. R.A. T. G. Cell. Full Text PDF PubMed Scopus Google Scholar). enhanced activity of integrin leads to tumor cell migration and to matrix I. J. C. F. C. M. A. N. P. J. 2002; Scopus Google Scholar). in cells in different of metastatic of these the of to multiple Nat. Cancer. 2002; 2: PubMed Scopus Google Scholar, T. M. Oncogene. 2003; PubMed Scopus Google Scholar). these cells from the of from these and have cell migration and invasion in snail-expressing a role for snail expression in the of cell attachment or detachment has not been This is the that that snail enhances cell detachment from and attachment to ECM proteins at least in by the expression of integrins and BM proteins. and and A431 were in with fetal calf serum MDCK and A431 cells were with of human snail expression or by a as M. Olmeda D. Vinyals A. Valero E. Cubillo E. Llorens A. Cano A. Fabra A. J. Cell Sci. 2005; 118: 3371-3385Crossref PubMed Scopus (201) Google Scholar), and were and We used MDCK cells in this of we the cells show normal epithelial cell However, of the of that canine proteins, we also used human A431 cells. The cell by was in medium with cells were with and of the conditioned medium of these cells was K. Y. T. K. K. S. K. T. J. Cell PubMed Scopus Google Scholar). and against E-cadherin, integrins α3, and and were from integrin was from against was from against was from fibronectin was from collagen IV was from collagen was from osteopontin was from LN-5 was from and were from were as or of FN and in phosphate-buffered saline was on the of The was to for either or at were with bovine serum albumin in Cell were in and cells were on to cells were and cells were with with a of or for the cells were and the or the cells was with The was at 2003; 24: PubMed Scopus Google Scholar). The number of the cells that to the was by a number and inhibition the and at a of were with the cell for of the cells to the cells were on and for were from the by with or at for the or was to the cells to of the the number of cells was as The are also as a of cells to cells. were in with a were in the of the for and with that was to the the was with the cells in the were and the cells were by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide as H. T. M. Y. S. R. T. M. T. M. M. T. S. Cancer Res. 2002; 62: Google Scholar). RNA were from the cells with the and was reverse The were as for specific The for were in and of dehydrogenase was used as an for in a for in a cells were in in with cells were with an against LN-α3 The of the was M. H. A. Biol. Cell. 2006; PubMed Google Scholar). for was and the cells were for an The cells were and for methacrylate was to of a in and to This was and MDCK/snail cells were either on or for and cells from were in and at for from were and in a The were with of propidium iodide in at for 15 The number of cells was by and MDCK/snail cells were either on or for at The medium was from cell and was for from a M. S. S. S. S. H. J. Exp. 2000; PubMed Scopus Google Scholar). for were to and expressed as a were in integrin αV with the of the was from the luciferase The luciferase expression the to to were and M. Y. M. PubMed Scopus Google Scholar). were to of and of were both firefly and Renilla luciferase were the luciferase were to the The were in were in with an as H. T. M. Y. S. R. T. M. T. M. M. T. S. Cancer Res. 2002; 62: Google Scholar) with of cells in with was the FN or osteopontin was to of with in the at a of medium of cells or medium was to of with in of the cells in the were The cells that to the of the were to the was by were to significant at Snail Cell from observed that the transfection of snail MDCK cells enhanced cell detachment from We detachment of MDCK/snail cells and it to that of cells. were to cell-cell for at the cells were with for to The cells were with and the of cells to cells was of MDCK/snail cells by In of cells were at this We also found that of A431/snail cells of cells by It has been that cell-cell the of to cell proteins that cell attachment to the E-cadherin is to the main component of that this the effect of proteins on the of we the detachment in MDCK/snail or cells were in the of the was to the of the to the were by Over of MDCK/snail cells were of with of cells of to the proteins in attachment is not the MDCK/snail cells show cell detachment with cells. Snail the of ECM the rapid detachment of snail-expressing cells was to changes in the ECM by these cells. We the of or MDCK/snail cells to the ECM by either cell MDCK/snail cells to the ECM by both the and MDCK/snail with a However, cells were to the ECM by MDCK/snail cells to ECM This that the ECM by the MDCK/snail cells must in The expression of ECM proteins in binding to the receptors of cells might in MDCK/snail cells. The ECM with deoxycholic to of cells that with might the proteins that in the of cells. the expression of receptors for ECM proteins in MDCK/snail thereby MDCK/snail cells to the ECM from both cell to a The major expressed and by epithelial cells are LN, and We and expressions of these molecules in and A431/snail cells. was in MDCK/snail A431/snail cells but was expressed in and as with an against The expressions of the and were also in MDCK/snail and the expressions of LN-α3 and γ2 were in A431/snail cells when by and In agreement with a the expression of as by was enhanced in MDCK/snail and in A431/snail cells with cells and cells In the of expression of was in and MDCK/snail cells and or A431/snail cells when by and We also found not LN-5 expression but also the expressions of receptor proteins for LN-5 such as integrin and were strongly suppressed in MDCK/snail cells as by and or by The expression of integrin β4 was also suppressed in A431/snail cells as by both and and by These indicate that snail the expression of specific ECM proteins and We also the expressions of collagen integrin integrin and and found were in snail-expressing cells not the expression of integrins and ECM proteins. and effect of snail on the expression of integrins and ECM proteins. and MDCK/snail cells and and A431/snail cells were in and to with the was as an for of expression was by of The are as the of the of from MDCK/snail cells to that in cells and the from A431/snail to that in cells or cells. of snail on expression of integrins and ECM proteins in MDCK cells and in A431 cells of dehydrogenase was used as an The for are in and of LN-α3 with Cell the reduction in LN-5 expression might for the changes in cell detachment from the ECM observed in snail-expressing LN-5 synthesis was inhibited by of to the LN-α3 The expression of LN-α3 was in cells that been of to LN-α3 with that in cells with The of the LN-α3 cells with cells not We the effect of of LN-α3 on cell detachment. was no in detachment LN-α3 and cells These suggest that the observed reduction of LN-5 in snail-expressing cells is not for the cell detachment in these cells. we the effect of other matrix proteins on cell detachment. and A431/snail cells were on the with ECM proteins, and detachment was for and or The detachment of and MDCK/snail cells by was suppressed in the with collagen and We also found the detachment of A431/snail cells was suppressed in the with collagen IV and the expression of collagen IV was in MDCK/snail cells this reduction might for detachment of MDCK/snail cells at least in the detachment of cells by was the of detachment of MDCK/snail cells were and This that the of ECM proteins might enhanced in MDCK/snail cells. Snail has been to induce the expression of a of to in the of BM proteins. we observed expression of in snail-expressing cells and showed no significant effect on detachment of MDCK/snail cells IV the detachment of MDCK/snail and A431/snail cells. of ECM proteins on cell detachment of MDCK cells. The of were with of collagen collagen or and MDCK/snail cells were these and for or in These cells were with for The cells were and the cells on the were with and with and cells on of ECM proteins on cell detachment of A431 cells. and A431/snail cells were was as cells on effect of on detachment of MDCK/snail cells. were or with at for and to detachment cell Snail to snail has been to as a Snail expression against cell of the A. Nieto M.A. 2005; PubMed Scopus Google Scholar) and against cell induced by the of factors or signals S. A. F. I. Nieto M.A. Dev. 2004; PubMed Scopus Google Scholar). of contact to induces snail enhances the detachment of we snail expression might confer resistance to the to cells were by on We the of cell by as a of the cells to the propidium iodide of MDCK/snail cells showed a or of anoikis that of cells These suggested that snail is to protect cells against were when cell was by Snail Cell to that snail enhances cell detachment from the ECM but not anoikis in for the snail-expressing tumor cells to and in the must to the at a different Indeed, MDCK/snail cells to cells attachment to or was enhanced in MDCK/snail and A431/snail cells with and cells when a and The of which the binding of FN to its cell the attachment of MDCK/snail cells Snail the of of cell to as as inhibition of cell by suggested that snail might the expression of We the expression of integrins in MDCK/snail and A431/snail cells to that in and cells. The expression of integrin was in A431/snail cells at and and in MDCK/snail cells at RNA the RNA the expression of the receptors integrin αV was also in MDCK/snail and A431/snail cells and The suggest that snail the expression of specific of the as as Snail the of is to the transcription of E-cadherin. We the effect of snail on the expression of integrin αV was of its promoter The promoter activity of integrin αV was in the MDCK/snail cells with the cells the of the promoter that is for the effect of induction by we MDCK/snail cells with luciferase expression in the from to in resulted in a in luciferase However, of the to from resulted in a in luciferase activity This the binding for the transcription factors and Snail of MDCK toward and the of has been that expression of integrin enhances the of breast to The also showed the migration of tumor cells toward a of cells N. J. Cancer Res. 2006; PubMed Scopus Google Scholar). we found an in the expression of integrin αV in snail-expressing we snail expression might also the activity of these cells. of MDCK/snail cells toward an ligand for integrin in was with the migration of MDCK/snail cells toward or the conditioned medium of was enhanced the migration of MDCK/snail cells to osteopontin and was suppressed by function integrin the migration of MDCK/snail cells to FN was by this These findings suggest that snail cell migration to osteopontin and but not by enhancement of the expression of integrin the expression of integrin of FN receptors R. Y. J.D. J. D. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), was enhanced in MDCK/snail this of integrin might the migration of these cells to snail has been to the expression of or and as an of cell and tumor a role for snail in the of has not been This is the that snail enhances not cell detachment but also cell attachment. that snail enhances cell detachment that the ECM proteins and cell receptors in snail-expressing cells. The that the of cells to ECM by MDCK/snail cells is that to the ECM by the also a in the production of ECM in snail-expressing cells. It has been that cells not the major in BM (17Takkunen M. Grenman R. Hukkanen M. Korhonen M. García de Herreros A. Virtanen I. J. Histochem. Cytochem. 2006; 54: 1263-1275Crossref PubMed Scopus (88) Google Scholar). We confirmed the reduction of and in MDCK/snail and A431/snail cells. We that the in the expression of these proteins must play a role in cell detachment in snail-expressing cells. However, we found that suppression of LN-α3 expression by transient transfection of cell detachment. It is that a of LN-5 is for of cells that have expressions of receptors for LN-5 such as integrin and MDCK/snail cells have expressions of integrin and in to might show significant enhancement of detachment. of cells from the ECM induces a cell Snail has been associated with resistance to cell by M. Cell Biol. 2004; 24: PubMed Scopus Google Scholar). In to in the expression of snail did not confer resistance to cell-cell in tumor cells was to the anoikis J. N. Shimada H. T. Cancer Res. PubMed Scopus Google Scholar). The reduction of E-cadherin in snail-expressing cells might the these cells are to The detachment of snail-expressing cells from the with to that it for snail-expressing cells to in However, in snail expression not enhanced detachment from the ECM but also enhanced cell attachment to This attachment to FN was in the of that it was by cell integrin In agreement with this the expression of the FN receptors integrin αV and were in MDCK/snail and A431/snail cells. snail expression the tumor cells to specific ECM thereby a for tumor cell in It is not MDCK/snail showed enhancement of detachment these cells have enhancement of expressions of FN and integrin αV and We that enhancement of expression of FN and integrin αV or might not for the effect of of other ECM proteins. The promoter activity of integrin αV was in snail-expressing cells that the effect of snail on integrin expression was by its transcriptional of in the promoter we the the integrin αV promoter to that was by this the binding for the transcription factors and This is with a that and proximal are crucial for snail of the MMP-9 promoter (11Birchmeier C. Birchmeier W. Brand-Saberi B. Acta Anat. (Basel). 1996; 156: 17-26Crossref Scopus (232) Google Scholar). are also present in the promoters of other such as as as integrin the expressions of which were in MDCK/snail cells in this However, also in the promoter and of the that showed expression in snail-expressing cells such as integrin α6, α3, and The is present in the promoters of integrin and Snail has also been shown to the expression of E-cadherin by binding to that to the are present in the promoter and of the such as integrin α3, α6, and and integrin snail the expression of genes, it might with other factors for the of different These other factors to cell The expression of integrin in tumor is strongly of a potential role for this receptor in tumor progression for invasive that to N. J. Cancer Res. 2006; PubMed Scopus Google Scholar, I. J. C. F. C. M. A. N. P. J. 2002; PubMed Scopus Google Scholar). these findings as snail-expressing cells that showed enhanced expression of integrin αV also enhanced cell migration to the conditioned medium of cells as as toward is an ligand to the integrin in we the that the factor in the of cells is a matrix from it is that snail-expressing have a to to in is to the the of snail expression and the of The enhancement of integrin expression in snail-expressing cells a for cancer of binding integrin and its by induce tumor cell of snail might migration and to specific such as and protect cells from in The of an integrin for the of with snail-expressing We for with and Sato for
Haraguchi et al. (Tue,) studied this question.