The miR-200 Family Inhibits Epithelial-Mesenchymal Transition and Cancer Cell Migration by Direct Targeting of E-cadherin Transcriptional Repressors ZEB1 and ZEB2

Abstract

MicroRNAs are small non-coding RNA molecules that can regulate gene expression by interacting with multiple mRNAs and inducing either translation suppression or degradation of mRNA. Recently, several miRNAs were identified as either promoters or suppressors of metastasis. However, it is unclear in which step(s) of the multistep metastatic cascade these miRNAs play a defined functional role. To study the functional importance of miRNAs in epithelial-mesenchymal transition (EMT), a process thought to initiate metastasis by enhancing the motility of tumor cells, we used a well established in vitro EMT assay: transforming growth factor-β-induced EMT in NMuMG murine mammary epithelial cells. We found that members of the miR-200 family, organized as two clusters in the genome, were repressed during EMT. Overexpression of each miRNA individually or as clusters in NMuMG cells hindered EMT by enhancing E-cadherin expression through direct targeting of ZEB1 and ZEB2, which encode transcriptional repressors of E-cadherin. In the 4TO7 mouse carcinoma cell line, which expresses low levels of endogenous E-cadherin and displays a mesenchymal phenotype, ectopic expression of the miR-200 family miRNAs significantly increased E-cadherin expression and altered cell morphology to an epithelial phenotype. Furthermore, ectopic expression of each miR-200 miRNA cluster significantly reduced the in vitro motility of 4TO7 cells in migration assays. These results suggested that loss of expression of the miR-200 family members may play a critical role in the repression of E-cadherin by ZEB1 and ZEB2 during EMT, thereby enhancing migration and invasion during cancer progression. MicroRNAs are small non-coding RNA molecules that can regulate gene expression by interacting with multiple mRNAs and inducing either translation suppression or degradation of mRNA. Recently, several miRNAs were identified as either promoters or suppressors of metastasis. However, it is unclear in which step(s) of the multistep metastatic cascade these miRNAs play a defined functional role. To study the functional importance of miRNAs in epithelial-mesenchymal transition (EMT), a process thought to initiate metastasis by enhancing the motility of tumor cells, we used a well established in vitro EMT assay: transforming growth factor-β-induced EMT in NMuMG murine mammary epithelial cells. We found that members of the miR-200 family, organized as two clusters in the genome, were repressed during EMT. Overexpression of each miRNA individually or as clusters in NMuMG cells hindered EMT by enhancing E-cadherin expression through direct targeting of ZEB1 and ZEB2, which encode transcriptional repressors of E-cadherin. In the 4TO7 mouse carcinoma cell line, which expresses low levels of endogenous E-cadherin and displays a mesenchymal phenotype, ectopic expression of the miR-200 family miRNAs significantly increased E-cadherin expression and altered cell morphology to an epithelial phenotype. Furthermore, ectopic expression of each miR-200 miRNA cluster significantly reduced the in vitro motility of 4TO7 cells in migration assays. These results suggested that loss of expression of the miR-200 family members may play a critical role in the repression of E-cadherin by ZEB1 and ZEB2 during EMT, thereby enhancing migration and invasion during cancer progression. MicroRNAs are a large family of small (21–23-nt) 4The abbreviations used are: nt, nucleotide; EMT, epithelial-mesenchymal transition; TGF, transforming growth factor; miRNA, microRNA; pre-miR, pre-microRNA; UTR, untranslated region; qRT-PCR, quantitative reverse transcription-PCR. RNAs that exhibit a high degree of structural and functional conservation throughout metazoan species. miRNAs are initially synthesized by polymerase II as long primary transcripts, which are subsequently processed into ∼70-nt stem-loop pre-microRNAs by Drosha RNase III endonuclease (1Lee Y. Ahn C. Han J. Choi H. Kim J. Yim J. Lee J. Provost P. Radmark O. Kim S. Kim V.N. Nature. 2003; 425: 415-419Crossref PubMed Scopus (4088) Google Scholar) and are transported out of the nucleus by exportin 5 (2Yi R. Qin Y. Macara I.G. Cullen B.R. Genes Dev. 2003; 17: 3011-3016Crossref PubMed Scopus (2268) Google Scholar). Pre-microRNAs are further processed in the cytoplasm by Dicer to yield the final ∼22-nt mature miRNAs (3Hutvagner G. McLachlan J. Pasquinelli A.E. Balint E. Tuschl T. Zamore P.D. Science. 2001; 293: 834-838Crossref PubMed Scopus (2217) Google Scholar). Binding of miRNA to target mRNAs with perfect or near perfect complementarity induces mRNA degradation, whereas imperfect complementarity often induces translational repression. It is believed that 7–8 nt in the 5′ end of miRNAs, referred to as the seed sequence, are critical for efficient targeting. miRNAs have been implicated in regulating complex physiological processes such as embryogenesis (4Wienholds E. Kloosterman W.P. Miska E. Alvarez-Saavedra E. Berezikov E. de Bruijn E. Horvitz H.R. Kauppinen S. Plasterk R.H. Science. 2005; 309: 310-311Crossref PubMed Scopus (1358) Google Scholar), organ development (5Yi R. O'Carroll D. Pasolli H.A. Zhang Z. Dietrich F.S. Tarakhovsky A. Fuchs E. Nat. Genet. 2006; 38: 356-362Crossref PubMed Scopus (460) Google Scholar), and oncogenesis (6Esquela-Kerscher A. Slack F.J. Nat. Rev. Cancer. 2006; 6: 259-269Crossref PubMed Scopus (6346) Google Scholar, 7Johnson S.M. Grosshans H. Shingara J. Byrom M. Jarvis R. Cheng A. Labourier E. Reinert K.L. Brown D. Slack F.J. Cell. 2005; 120: 635-647Abstract Full Text Full Text PDF PubMed Scopus (3157) Google Scholar). However, the functional roles of a vast majority of miRNAs remain unknown. Recently, several groups have used a variety of model systems to identify different miRNAs as promoters or suppressors of metastasis (8Huang Q. Gumireddy K. Schrier M. le Sage C. Nagel R. Nair S. Egan D.A. Li A. Huang G. Klein-Szanto A.J. Gimotty P.A. Katsaros D. Coukos G. Zhang L. Pure E. Agami R. Nat. Cell Biol. 2008; 10: 202-210Crossref PubMed Scopus (888) Google Scholar, 9Ma L. Teruya-Feldstein J. Weinberg R.A. Nature. 2007; 449: 682-688Crossref PubMed Scopus (2271) Google Scholar, 10Tavazoie S.F. Alarcon C. Oskarsson T. Padua D. Wang Q. Bos P.D. Gerald W.L. Massague J. Nature. 2008; 451: 147-152Crossref PubMed Scopus (1654) Google Scholar, 11Zhu S. Wu H. Wu F. Nie D. Sheng S. Mo Y.Y. Cell Res. 2008; 18: 350-359Crossref PubMed Scopus (1000) Google Scholar, 12Asangani I.A. Rasheed S.A. Nikolova D.A. Leupold J.H. Colburn N.H. Post S. Allgayer H. Oncogene. 2008; 27: 2128-2136Crossref PubMed Scopus (1632) Google Scholar). Although these studies clearly implicate these miRNAs in metastasis, it is unclear which step(s) in the multistep metastatic progression these miRNAs regulate. In the present study, we sought to define a role for miRNAs in regulating the initiating step in metastasis, epithelial-mesenchymal transition (EMT). Applying a classical model system of inducing EMT in NMuMG cells (normal murine mammary epithelial cells), we found that members of the miR-200 family, existing as two clusters in the genome, are significantly repressed during EMT, suggesting a role as suppressors of EMT. We further discovered that members of the miR-200 family hinder EMT by positively regulating E-cadherin expression through direct targeting of ZEB1 and ZEB2. Furthermore, ectopic expression of the miR-200 family in 4TO7 mammary carcinoma cells, which express low endogenous levels of these miRNAs, induced mesenchymal-epithelial transition by up-regulating E-cadherin expression and inhibited migration of these tumor cells. These results strongly suggested an important role of the miR-200 family miRNAs in repressing epithelial-mesenchymal transition and cancer progression. Cell Lines and Cell Culture—NMuMG, 4TO7, and HeLa cells were maintained in Dulbeccoʼns modified Eagleʼns medium supplemented with 10% fetal bovine serum. NMuMG medium was further supplemented with 10 μg/ml insulin (Sigma). RNA Extraction and Quantitative Real-time PCR—Total RNA was extracted using the miRVana miRNA isolation kit (Ambion). For miRNA analysis, mature miRNAs were reverse-transcribed, and real-time PCR was performed using TaqMan microRNA assays (Applied Biosystems). All data were normalized to U6 expression. For mRNA analysis, real-time PCR was performed using Power SYBR® green PCR master mix (Applied Biosystems) on an ABI 7900HT series PCR machine Applied Biosystems, and data were normalized to GAPDH expression and further normalized to the negative control unless otherwise indicated. ZEB1 and ZEB2 3′-UTR Luciferase Reporter Assays—The 3′-UTRs for both ZEB1 and ZEB2 were PCR-amplified from genomic DNA extracted from NMuMG cells. PCR primers used to amplify the Zeb1 3′-UTR include 5′-AAAAATCCGGGTGTGCCTGA-3′ (forward) and 5′-AACTGCTTTCTACTGCTCTG-3′ (reverse), whereas the primers used to amplify the Zeb2 3′-UTR include 5′-CAGTTCAGCCAAGACAGAGT-3′ (forward) and 5′-TTCGAGCATGGTCATTTTC-3′ (reverse). Amplified 3′-UTRs were cloned downstream of the firefly luciferase coding region in the pMIR-REPORT™ (Ambion). HeLa or 4TO7 cells were seeded in 24-well plates 24 h prior to transfection. The following day, 200 ng of reporter plasmid along with 200 ng of control Renilla-luciferase plasmid were co-transfected using Lipofectamine 2000 (Invitrogen). Cells were collected 24 h after transfection and assayed for luciferase activity using the Glomax 96 luminometer (Promega). To assess the effect of miRNAs on reporter activity, 50 pm of synthetic precursor miRNAs (pre-miRs) (Ambion) were co-transfected. All experiments were performed in triplicates. Immunoblot Analysis—For EMT assays, NMuMG cells were seeded in 6-well plates, and the next day, cells were transfected with synthetic miRNAs individually or as clusters. 24 h after transfection, cells were treated with 200 pm TGFβ1 (R&D systems) for 48 h. Cells were lysed in prechilled lysis buffer, and 10 μg of protein from the supernatant was loaded per lane and resolved by SDS-polyacrylamide electrophoresis. Protein was transferred onto nitrocellulose membranes, blocked, and probed with mouse anti-E-cadherin (BD Biosciences), mouse anti-N-cadherin (BD Biosciences), or mouse anti-β-actin (AbCam). Immunofluorescence—NMuMG cells were transfected in 6-well plates with synthetic pre-microRNAs (pre-miRs). 4–6 h after transfection, the cells were dissociated and seeded onto gelatin-coated glass coverslips placed in 24-well plates and stimulated with recombinant TGFβ for an additional 48 h. 4TO7 cells were similarly treated except that stimulation with TGFβ did not take place. 72 h after transfection, media were aspirated, and cells were fixed with ice-cold methanol for 10 min, permeabilized with 0.2% Triton for 3 min, and blocked in 10% goat serum for 1 h at room temperature. E-cadherin was probed with mouse-anti-E-cadherin for 1 h at room temperature followed by detection with a rhodamine-conjugated goat anti-mouse secondary antibody for 1 h at room temperature. Hoechst dye (1 μg/ml) was subsequently used to stain nuclei. Cells were observed on a Zeiss microscope, and pictures were taken using an Axiocam Icc3 camera. Transwell Migration Assays—4TO7 cells were transfected with pre-miRs for 48 h and subsequently dissociated. 1 × 105 cells were resuspended in serum-free media and placed in inserts containing 8-μm pores. These inserts were placed in wells with serum-containing media. 12 h after seeding, serum-containing media were aspirated, and trypsin was placed into the wells to trypsinize the cells that had passed through the pores, which were counted using a hemocytometer. Down-regulation of miR-200 Family miRNAs during EMT—To study EMT, we used a classical model system: TGFβ1-induced EMT in the NMuMG mouse mammary epithelial cell line. Within 72 h of TGFβ treatment, NMuMG cells undergo a dramatic morphological change, from compact, cobblestone-like epithelial structures to fibroblastoid spindle-shaped cells, together with significant disintegration of cell-cell adhesions (Fig. 1A). This morphological transition is accompanied by E-cadherin down-regulation with reduced membrane localization (Fig. 1, A and B) and N-cadherin up-regulation (Fig. 1B) with increased localization to the membrane (Fig. 1A). These hallmark shifts at the morphological and molecular levels indicate a successful EMT program in NMuMG cells. Two miR-200 family miRNAs, miR-200b and miR-200c, have been previously correlated with the E-cadherin expression in epithelial cells (13Christoffersen N.R. Silahtaroglu A. Orom U.A. Kauppinen S. Lund A.H. RNA (Cold Spring Harbor). 2007; 13: 1172-1178Google Scholar, 14Hurteau G.J. Carlson J.A. Spivack S.D. Brock G.J. Cancer Res. 2007; 67: 7972-7976Crossref PubMed Scopus (373) Google Scholar). Three additional miRNAs, miR-200a, miR-141, and miR-429, also belong to the miR-200 family. qRT-PCR analysis revealed that the expression of all miR-200 family miRNAs, except miR-141, were strongly down-regulated in NMuMG cells during TGFβ-induced EMT (Fig. 1C). The expression of each miRNA was reduced to less than 30% of the pretreatment level within 24 h of TGFβ exposure. We did not observe detectable expression of miR-141 in NMuMG cells. The miR-200 family of miRNAs were mapped to two separate clusters of less than 2000 bp each in the mouse genome (Fig. 2D, upper panel). The first cluster (Cluster 1) contains miR-200a, miR-200b, and miR-429 and is located in mouse chromosome 4. Analysis of expression levels of miR-200a, miR-200b, and miR-429 in several cell lines revealed that the three members of this cluster are co-expressed (r = 0.97, data not shown), suggesting that a common promoter is likely to drive the expression of all three miRNAs. The second cluster (Cluster 2), consisting of miR-200c and miR-141, is located in a 500-bp region of chromosome 6. The five miR-200 family miRNAs contain very similar seed sequences (Fig. 1D, lower panel). The seed sequence of miR-200b, miR-200c, and miR-429, AAUACU, differs only by one nucleotide to the seed sequence of miR-200a and miR-141, AACACU. Overexpression of miR-200 Members Individually or in Combination Represses EMT and Enhances E-cadherin Expression—Because miRNAs in the miR-200 family were similarly down-regulated during EMT, we tested the functional role of each miRNA in the regulation of E-cadherin expression and the control of EMT in NMuMG cells by transient overexpression of synthetic pre-miRs. Successful transfection was achieved in over 90% of the cell populations for up to 3 days as assessed by fluorescence-activated cell sorting analysis of a cy3-labeled control pre-miR (data not shown). NMuMG cells transfected with the negative control pre-miR responded to TGFβ treatment with more than 90% reduction of E-cadherin expression (Fig. 1E) and morphological changes characteristic of EMT (Fig. 1F). In contrast, overexpression of pre-miRs corresponding to each of the five miR-200 miRNAs led to significant resistance of E-cadherin to transcriptional repression during EMT (Fig. 1E). miR-200b, miR-200c, and miR-429 have the most robust effect in maintaining E-cadherin expression, whereas miR-200a and miR-141 have a relatively modest effect (Fig. 1E). Transfection of all five miRNAs, or miRNAs in two individual clusters, achieved a similar effect on E-cadherin expression as that of miR-200b, miR-200c, or miR-429 alone. Furthermore, expression of miRNAs as clusters (Fig. 1F) or individually (data not shown) significantly reduced the of mesenchymal during the TGFβ-induced EMT of NMuMG cells (Fig. 1F). In the cells, the of cells and the loss of cell-cell was the transition to fibroblastoid morphology was with clusters of cells maintaining the cobblestone-like epithelial (Fig. 1F). analysis of cells revealed a significant level of E-cadherin expression and the of The of N-cadherin expression in cells, was not by miR-200 overexpression (data not shown). these results indicate that all five members of the miR-200 family are of the epithelial-mesenchymal in NMuMG cells by maintaining high levels of E-cadherin expression. Overexpression of the miR-200 Family the of the 4TO7 Cell we tested the of the miR-200 family of miRNAs to reverse the mesenchymal of metastatic cancer cells. 4TO7 is a mammary carcinoma cell from a mammary tumor in a mouse Cancer Res. Google Scholar). The 4TO7 cell is and is of from mammary in J. S.A. S. R.A. C. P. A. Weinberg R.A. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). the 4TO7 cells a morphology (Fig. with a very low level of E-cadherin expression (Fig. and miR-200 family miRNA expression (data not shown). Overexpression of the two miR-200 clusters, individually or in for 3 days in 4TO7 cells a dramatic in from a spindle-shaped mesenchymal to a epithelial (Fig. The epithelial of 4TO7 cells was after 48 a at 72 h and to mesenchymal at h after transfection, to a degradation of transfected pre-miRs. analysis revealed a significant of E-cadherin expression. E-cadherin is on the membrane in miR-200 overexpression cells and cells, suggesting that E-cadherin can to the of epithelial in 4TO7 cells. of E-cadherin ZEB1 and ZEB2 by miR-200 Family identify likely direct of miR-200 miRNAs, we the data Cell. 2005; 120: Full Text Full Text PDF PubMed Scopus Google Scholar) for miR-200 target in the mRNA sequences of suppressors of E-cadherin expression, and ZEB2. ZEB2 is to the most likely target gene of the miR-200 family 3′-UTR contains at two for and five for (Fig. ZEB1 to the family as ZEB2, is also to contain at two target for To the direct targeting of ZEB1 and ZEB2 by we cloned 3′-UTR sequences downstream of a firefly luciferase reporter of the reporter plasmid along with miR-200a, miR-200b, miR-429, as well as each or both of the clusters in 4TO7 cells, in a significantly reduced expression, suggesting that these miRNAs are likely to target ZEB2 (Fig. 2D, panel). In reporter assays using the ZEB1 only miR-200b and both clusters were to significantly luciferase reporter expression, the lower of miR-200 in the ZEB1 3′-UTR cloned into the luciferase reporter reporter results were from the HeLa cell (Fig. 2D, panel). Reporter results were less in NMuMG cells (data not shown), to the high level of miR-200 expression in this epithelial cell line. To for the targeting of endogenous ZEB1 and ZEB2 by we ZEB1 and ZEB2 mRNA levels in NMuMG cells transfected with the miR-200 family members or in clusters. The transfected cells were induced to undergo EMT with TGFβ1 Although ZEB1 and ZEB2 levels increased during EMT in control cells, overexpression of each of the miR-200 family members significantly reduced the of both ZEB1 and ZEB2 it is likely that the miR-200 family members E-cadherin expression by the expression of transcriptional suppressors of E-cadherin. To direct targeting of ZEB1 and ZEB2 is also in the up-regulation of E-cadherin in 4TO7 mammary tumor cells, we performed qRT-PCR analysis at 48 h following the transfection of miR-200 clusters. A dramatic in E-cadherin expression was accompanied by a significant in ZEB1 and ZEB2 mRNA levels (Fig. analysis revealed targeting of ZEB1 and ZEB2 within 24 h in 4TO7 cells, which was maintained after 48 h. However, ZEB1 and ZEB2 targeting was after 72 h after transfection, with the to the mesenchymal at h after transfection. miR-200 Family miRNAs Migration of 4TO7 of E-cadherin and of mesenchymal increased are thought to critical during the transition of to the of E-cadherin and of epithelial in 4TO7 mammary carcinoma cells transfected with the miR-200 family miRNAs, we sought to the effect of these miRNAs in cell an in vitro migration overexpression of each of the miR-200 clusters strongly reduced growth migration of the 4TO7 cells (Fig. EMT is a process in the of and such as the and However, during only a of cells the to undergo transient EMT, for during Cell Biol. 2003; PubMed Scopus Google Scholar). tumor cells are to such as EMT to a such as motility and J. S.A. S. R.A. C. P. A. Weinberg R.A. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. H. H. T. J. PubMed Scopus Google Scholar, J. E. S. J. PubMed Scopus (460) Google Scholar). EMT is to molecular of this process may as for studies have the miR-200 family with the epithelial and the family. of the miR-200 family was found to in epithelial S. RNA (Cold Spring Harbor). 2005; Scholar, J. G. Miska Alvarez-Saavedra E. J. D. A. R.H. T. Horvitz H.R. Nature. 2005; 834-838Crossref PubMed Scopus Google Scholar) and correlated with ZEB1 and ZEB2 expression during development T. M. T. H. D. Y. Dev. 2006; PubMed Scopus Google Scholar). In the family been implicated in EMT, and metastasis Y. D. M. Cancer Res. 2006; PubMed Scopus Google Scholar, S. O. F. G. A. S. A. T. T. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, G. T. M. T. Y. T. K. S. K. J. 2005; 27: Google Scholar, Y. A. T. A. Oncogene. 2007; PubMed Scopus Google Scholar). However, the functional of miR-200 in EMT and tumor as well as direct targeting of ZEB1 and ZEB2 by the miR-200 family, not been in these In present study, we identified the miR-200 family as suppressors of EMT through direct targeting of ZEB1 and ZEB2, well transcriptional repressors of E-cadherin. The miR-200 family is of five members as two clusters, and ZEB1 and ZEB2 were likely both are EMT and contain target for of the miR-200 family ZEB2 had target for all five miRNAs, the data that ZEB1 had only two target for miR-200b, and To the results that overexpression of miR-200a and also significantly reduced the expression of endogenous ZEB1 (Fig. to that the 3′-UTR of ZEB1 may in additional target for these miRNAs. a found that the for ZEB1 was and revealed several target for miR-200a and miR-141 in the ZEB1 3′-UTR P.A. A. G. Y. G.J. Nat. Cell Biol. 2008; Google Scholar). We found that ectopic expression of the miR-200 family as clusters, or hindered EMT progression in NMuMG cells by ZEB1 and ZEB2 expression levels low and E-cadherin expression levels high and these cells the miR-200 family maintained a cobblestone-like epithelial phenotype. expression of each miR-200 miRNA was to hinder EMT miR-200a and miR-141 were the of E-cadherin. overexpression of miRNAs from the cluster or from both clusters did not to have a effect in regulating ZEB2, or at in the in vitro Although we found that the miR-200 family can hinder EMT, overexpression was not to this suggesting that miRNAs may also in regulating EMT. expression of the miR-200 family in 4TO7 carcinoma cells a and significant in ZEB1 and ZEB2 the miR-200 family in 4TO7 cells induced a dramatic morphological from a mesenchymal a more epithelial with of Furthermore, overexpression of miR-200 significantly growth a hallmark of metastatic cancer cells. This that ectopic expression of the miR-200 family can the mesenchymal-epithelial transition and tumor cell ZEB1 and ZEB2 have been implicated in the progression of tumor the miR-200 family may an important for targeting of metastatic We members of the for and We are to and for and