Introduction
Increasing evidence indicates that aberrant activation of the embryonic programme ‘epithelial–mesenchymal transition’ (EMT) promotes tumour cell invasion and metastasis (
Berx et al, 2007). EMT allows detachment of cells from each other and increases cell mobility, both of which are necessary for tumour cell dissemination. Metastases often recapitulate the differentiated phenotype of the primary tumour; therefore, EMT seems to be transiently activated by the inductive tumour environment at the invasive tumour edge, but is reversed in growing metastases (
Brabletz et al, 2001,
2005). Activators of EMT, such as transforming growth factor (TGF)β, tumour necrosis factor α (TNFα) and hepatocyte growth factor, are produced by infiltrating cells or the tumour cells themselves, and trigger expression of EMT‐inducing transcriptional repressors (
Thiery & Sleeman, 2006). These include members of the Snail family, the basic helix–loop–helix family, Goosecoid and members of the ZFH family (zinc‐finger E‐box binding homeobox (ZEB)1 and ZEB2;
Barrallo‐Gimeno & Nieto, 2005;
Hugo et al, 2007;
Peinado et al, 2007). Recently, we described that ZEB1 is a crucial EMT activator in human colorectal and breast cancer, and suppresses expression of basement membrane components (
Spaderna et al, 2006) and cell polarity factors (
Aigner et al, 2007;
Spaderna et al, 2008). Expression of ZEB1 promotes metastasis of tumour cells in a mouse xenograft model, indicating a role of ZEB1 in invasion and metastasis of human tumours (
Spaderna et al, 2008).
MicroRNAs (miRNAs) are small non‐coding RNAs that can silence their cognate target genes by specifically binding and cleaving messenger RNAs or inhibiting their translation (
Bartel, 2004). miRNAs regulate diverse cellular processes and some miRNAs have been shown to function as either tumour suppressors or oncogenes (
Esquela‐Kerscher & Slack, 2006). Recent important examples are the oncogenic miR‐10b, which promotes metastasis (
Ma et al, 2007), and miR‐335/miR‐126, which suppress metastasis in breast cancer (
Tavazoie et al, 2008).
Owing to these important regulatory functions of miRNAs, it is of prime interest to know how their expression is regulated by upstream factors. Here, we address this point and focus on the activation and stabilization of EMT in cancer cells. We investigated whether aberrant expression of the crucial EMT activator ZEB1 and the control of potential EMT‐regulatory miRNAs are linked and can synergize to promote malignant tumour progression.
Discussion
By applying a miRNA expression array screen for various human cancer cells, we detected several miRNAs suppressed by the EMT inducer ZEB1. The most prominent effect was on members of the miR‐200 family. ZEB1 directly suppressed transcription of two members closely linked on human chromosome 12, miR‐141 and 200c, by binding to at least two highly conserved sites in their putative promoter. In confirmation with the data published during the course of our work (
Hurteau et al, 2007;
Gregory et al, 2008;
Park et al, 2008), the detected miRNAs induced a mesenchymal to epithelial transition (MET) and inhibited EMT, migration and invasion of undifferentiated cancer cells. We further identified putative target genes, which are known promoters of EMT and malignant tumour progression. One target of miR‐200c is ZEB1 itself, indicating an EMT‐enhancing feedforward loop in invading cancer cells. This regulatory loop might be stabilized further by downregulation of miR141, as one of its putative targets is TGFβ2. There is increasing evidence that ZEB1 has crucial effects on various processes of malignant tumour progression (
Peinado et al, 2007) and promotes metastasis (
Spaderna et al, 2008). In the light of the important role of ZEB1 and other EMT inducers, such as Snail (
Olmeda et al, 2007), Twist (
Yang et al, 2004) and of EMT as a whole in tumour progression, our data, indicating that ZEB1 promotes an EMT‐stabilizing feedforward loop by suppressing specific miRNAs, add functional evidence for the molecular mechanisms underlying these processes.
Our work addressed the clinically relevant question of how putative tumour‐suppressive miRNAs can be inactivated in cancer progression. The fact that both the ZEB1 binding sites and the overall structure of the
miR‐200c and
miR‐141 genes are highly conserved in vertebrates from zebrafish to human suggests that the tumour cells use a long‐established regulatory mechanism of miRNA expression. Moreover, the second miRNA cluster of the miR‐200 family on human chromosome 1p36 also contains highly conserved putative ZEB1 binding sites in the upstream sequence, indicating that the whole miR‐200 family can be suppressed by ZEB1 (
supplementary Fig 3E online). In addition, both the strong transcriptional inhibition of the two miRNAs by ZEB1 and their putative tumour‐suppressive effect were detected in tumour cell lines of various important cancer entities, namely pancreatic, colorectal and breast cancer. A clinical relevance is indicated by the fact that both miRNAs are lost in the highly aggressive basal type of breast cancer, which, in contrast to the luminal and ductal invasive type, is poorly differentiated, shows no expression of oestrogen and progesterone receptors, and has a worse clinical prognosis (
Sempere et al, 2007).
Both miRNAs affect the expression of different molecules, which all work in the same proinvasive manner, as is known for TGFβ2, ZEB1, cofilin and leptin receptor. The differential function of the two miRNAs can synergize, as they are coexpressed, possibly through coactivation by a common promoter. The intriguing fact is that both miRNAs inhibit members of their own repressing pathway: miR‐200c targets ZEB1 and miR‐141 targets TGFβ2. Thus, ZEB1 becomes a crucial regulator, as its aberrant expression in cancer might start a self‐enhancing feedforward loop by downregulating its own inhibitors miR‐141 and miR‐200c (
Fig 4F). Moreover, if the initial signal breaks down (for example, tumour environmental TGFβ), such a loop might as well re‐enforce expression of the miRNAs, thereby re‐inducing an epithelial phenotype. This might explain the strong phenotypic heterogeneity often seen within individual tumours and metastases. Recently,
Liu et al (2008) showed that ZEB1 is crucial for TGFβ‐mediated EMT in various steps of organ development. This important role of ZEB1 points out that the predicted regulatory loop might also have a physiological role in separating mesenchymal from epithelial tissue in development and organogenesis.
In conclusion, we suggest that ZEB1 is a crucial promoter of tumour progression by reducing transcription of both mRNAs and miRNAs. Thus, ZEB1 is a central molecular regulator of a miRNA‐mediated feedforward loop, which can re‐enforce EMT.
Methods
miRNA expression microarray screen. A 50 μg portion of total RNA including small RNAs isolated from 4 × 10
7 cells using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) was shipped to Capital Bio (Beijing, China). An expression screening was carried out using CapitalBio Mammalian miRNA Array V2.0 containing 743 human, rat and mouse non‐redundant miRNA probes. The microarray data have been deposited on ArrayExpress (
http://www.ebi.ac.uk/arrayexpress/) with the accession code E‐TABM‐461.
DNA constructs. For the hsa‐miR‐200c promoter reporter plasmid nucleotides −683 to −67, and for the spacer reporter nucleotides +66 to +403 (relative to first nucleotide of miR‐200c stem–loop) were cloned into pGL3basic (Promega, Mannheim, Germany). For the 3′UTR reporter plasmids, nucleotides +3,399 to +3,953 of human ZEB1 complementary DNA and nucleotides +1,427 to +1,695 of human TGFβ2 cDNA were amplified and cloned downstream of the luciferase gene in the pMIR‐REPORT vector (Ambion, Austin, TX, USA).
Cell culture and standard assays. All cell lines were purchased from ATCC (Manassas, VA, USA). Standard cell culture, transient transfections, reporter assays, electromobility shift assays, immunoblots, transient short interfering RNA (siRNA)‐mediated knockdown and quantitative real‐time reverse transcription–PCR were carried out as described previously (
Brabletz et al, 1999,
2004;
Hlubek et al, 2001). For TGFβ/TNFα stimulation 3 × 10
4 cells per well were seeded in a 12‐well plate, transfected at day 1 as indicated, and stimulated with 2 ng/ml TGFβ and 10 ng/ml TNFα for 5 days.
Specific assays. miRNA modulation: A total of 5 × 104 cells per well were seeded in a 12‐well plate. After 24 h, cells were transfected with 30 pmol oligonucleotides for miR‐141, miR‐200c or control miRNA‐16 (Ambion, Austin, TX, USA) using Oligofectamine™ Reagent (Invitrogen, Carlsbad, CA, USA) for overexpression, or with 420 nM of specific anti‐miRs (Ambion) for inhibition. Cells were cultivated for days before further use. Cell invasion was evaluated using the Chemicon Cell Invasion Assay Kit as described previously (Chemicon International, Millipore, Schwalbach, Germany), using 20,000 transiently (miRNA or siRNA) transfected cells.
Cell migration assay: Cells were transfected with miRNAs and controls as described (Spaderna et al, 2006). After reaching confluence, cells were scratched with a pipette tip and the migration potential was observed for up to 50 h.
Quantitative real‐time PCR for miRNAs: RNA from cultured cells was extracted using the mirVana™ PARIS™ Kit (Ambion, Austin, TX, USA). Total RNA of formalin‐fixed, paraffin‐embedded samples of breast carcinomas retrieved from the archives of the Department of Pathology, University of Erlangen was extracted after microdissection using the Total Nucleic Acid Isolation Kit for FFPE (Ambion, Austin, TX, USA). Specific quantitative real‐time PCR experiments were carried out using TaqMan® MicroRNA Assays for miR‐141, miR‐200c and control miRNA‐16 (Applied Biosystems, Foster City, CA, USA) on a Roche LightCycler 480.
ChIP analysis: The ChIP IT kit (Active Motif, Carlsbad, CA, USA) was applied according to the manufacturer's instructions. A 3 μg portion of control rabbit antiserum or antisera against ZEB1 was used for immunoprecipitation.