ZEB1 represses major regulators of epithelial differentiation
To identify ZEB1 target genes in cancer cells we examined the genome-wide transcriptional repertoire of ZEB1 in the highly metastatic MDA-MB-231 breast cancer cell line, which has been widely used as an invasion and metastasis cell model (Lacroix and Leclercq, 2004
). Knockdown of ZEB1 by small interfering RNAs (siRNAs) has previously been shown to induce E-cadherin expression and to re-establish epithelial features (Eger et al., 2005
). To determine global gene expression changes in MDA-MB-231 cells following a 3 days knockdown of ZEB1, which reduced ZEB1 mRNA levels by 80–90% (), we performed Affymetrix GeneChip analyses (Human Genome U133 Plus 2.0). ZEB1 knockdown caused upregulation of ~200 genes (repressed by ZEB1) and downregulation of ~30 genes (activated by ZEB1) (complete microarray data will be published elsewhere). ZEB1 mRNA level was strongly reduced, confirming the efficiency of the knockdown, while other E-cadherin repressors were not affected (data not shown).
Figure 1 RNAi-mediated knockdown of ZEB1 reactivates epithelial gene expression. (a) RNAi-mediated knockdown of ZEB1 and Snail1 in MDA-MB-231 cells. Transcript levels of ZEB1 and Snail1 were determined by real-time PCR using the TaqMan system with assay-on-demand (more ...)
In this study, we focused on potential ZEB1 targets involved in epithelial differentiation. Besides E-cadherin, ZEB1 depletion also induced re-expression of a multitude of genes crucial for epithelial cell–cell adhesion and differentiation (). Potential ZEB1 targets include the cell polarity genes Crumbs3, Pals1-associated tight junction protein (PATJ) and human lethal giant larvae homologue 2 (LLGL2; HUGL2); members of the classical cadherin superfamily (placental (P-) and retinal (R-) cadherin); components of tight junctions (occludin, JAM1, claudin 7, tricellulin and shroom), desmosomes (desmoplakin, plakophilin 3, desmocollin 2 and desmoglein 2) and gap junctions (connexin 26 and 31); epithelial-specific adhesion molecules with Ig-like domains (epithelial V-like antigen 1); the apically localized protein Mucin 1 and various genes involved in vesicular trafficking and transcytosis in epithelial cells, including the yeast homologue CDC50p implicated in the polarized establishment of actin patches at bud sites (). Transcript levels were confirmed by reverse transcription (RT)–PCR in three independent siRNA experiments (, one representative experiment is shown).
Functional classification of epithelial-specific genes repressed by ZEB1. Fold-change indicates relative increase in transcript levels after ZEB1-specific compared to unspecific (scrambled) siRNA treatments
Knockdown of Snail1 is not sufficient to establish epithelial features
We have recently demonstrated that, unlike ZEB1, the expression of Snail1 did not tightly correlate with repression of E-cadherin in a panel of 20 breast cancer cell lines (Eger et al., 2005
). To compare directly the functions of ZEB1 and Snail1 in MDA-MB-231 cells, we abrogated expression of Snail1 by siRNA treatment. Although transcript levels of Snail1 were reduced by ~90% (), the downregulation of Snail1 was not sufficient to activate expression of E-cadherin Crumbs3 and HUGL2 (). Consequently, MDA-MB-231 cells remained fibroblastoid (data not shown). Thus, ZEB1 rather than Snail1 controls epithelial plasticity of MDA-MB-231 cells.
Knockdown of ZEB1 partially restores epithelial polarity
De-repression of Crumbs3, HUGL2 and PATJ upon ZEB1 knockdown also induced strong upregulation of respective proteins (). Upregulated Crumbs3 and HUGL2 accumulated in the cytoplasm and at the plasma membrane in MDA-MB-231 cells (). PATJ accumulated in the nucleus but failed to localize to the membrane (). As PATJ has been implicated in the biogenesis of tight junctions (Michel et al., 2005
; Shin et al., 2005
), we analysed tight junction formation after ZEB1 knockdown. The tight junction marker ZO1 was detected at cell–cell contact sites (), indicating that PATJ may be dispensable for initial stages of tight junction formation. Altogether, ZEB1 depletion caused de novo
expression of cell polarity proteins, their partial translocation to the plasma membrane and formation of rudimentary peripheral tight junction complexes.
Figure 2 Cell polarity proteins are upregulated and partially redistributed to the plasma membrane upon ZEB1 depletion. (a) Immunolocalization of Crumbs3 (CRB3), HUGL2, PATJ and ZO1 in MDA-MB-231 cells treated with unspecific (si-Control) or ZEB1-specific (si-ZEB1) (more ...)
Ectopic E-cadherin expression is not sufficient to induce epithelial genes
Expression of E-cadherin in undifferentiated cancer cells rescues epithelial architecture and affects several signalling pathways (Wheelock and Johnson, 2003
). Since E-cadherin upregulation is a hallmark of ZEB1 downregulation in MDA-MB-231 cells (Eger et al., 2005
) (, ), the observed changes in gene expression upon ZEB1 depletion may be indirectly caused by increased E-cadherin levels. To address this issue, we stably expressed E-cadherin in MDA-MB-231 cells. In 90% of the cells, E-cadherin was detected in the cytoplasm (, E-cadherin-1) and cells were unable to develop pronounced epithelial features (, E-cadherin-1). Only 10% of cells formed ‘epitheloid’ cell clusters in which E-cadherin accumulated at cell–cell contact sites (, E-cadherin-2). RT–PCR analyses revealed that transcription of the ZEB1 target genes (Crumbs3, HUGL2 and PATJ) was unaffected upon expression of E-cadherin (). Crumbs3 and HUGL2 proteins were neither upregulated nor redistributed, even in the 10% of E-cadherin expressing ‘epitheloid’ cells ().
ZEB1 directly repressed promoter activity of cell polarity genes
Next, we examined whether ZEB1 can directly repress the promoters of Crumbs3, HUGL2 and PATJ. Since ZEB1 represses transcription via binding to E-box elements (5′-CACCTG-3′) (Grooteclaes and Frisch, 2000
; Eger et al., 2005
), we screened the proximal promoter regions of Crumbs3 and HUGL2 for the presence of E-box consensus sites and cloned relevant fragments into reporter constructs. For human Crumbs3, we generated three reporter constructs spanning different, albeit overlapping promoter regions (CRB3-prom 1–3, ), which cover ~3.5 kbp (−3421 to +74) and contain 20 E-box consensus sites mostly arranged in three clusters (). For HUGL2, a reporter construct containing a ~900 bp fragment with four E-box consensus sites was generated (−743 to +135) ().
Figure 3 ZEB1 directly represses the proximal promoter of cell polarity genes. (a) Scheme of the Crumbs3 promoter. E-boxes are indicated as vertical black bars. (b) ZEB1 and Snail repress the human Crumbs3 promoter. The relative repression of promoter fragments (more ...)
Epithelial MCF7 cells expressing endogenous Crumbs3 and HUGL2 were transfected with the reporter plasmids together with a ZEB1 expression vector or empty control vector and a constitutive β-galactosidase expression plasmid for normalization of transfection efficiency. Unlike the control, ZEB1 repressed the activity of the two proximally located Crumbs3 promoter fragments CRB3-prom1 (−1402/ +74) and CRB3-prom2 (−1958/ +74) as well as the HUGL2 promoter fragment HUGL2-prom (−743/ +135) (). ZEB1 expression had no effect on the more distally located Crumbs3 promoter region (CRB3-prom3; −3421/−1402) (). Ectopic expression of Snail1 also repressed promoter activities of both genes (), but unlike ZEB1, Snail1 preferentially repressed the activity of the most upstream promoter fragment (CRB3-prom3; −3421/−1402).
To test whether ZEB1 can directly interact with the endogenous promoters in vivo, we performed chromatin immunoprecipitations (ChIP). In line with the reporter analyses, chromatin fragments containing the first or the second proximal E-box clusters of the Crumbs3 promoter were efficiently pulled down by ZEB1 antibodies (Abs) (, ChIP1 and ChIP2), whereas fragments located at the most distal E-box cluster were barely detectable above background levels (, ChIP3). Similarly, ZEB1 physically interacted with the proximal PATJ promoter, which contained two E-box consensus sites (). To demonstrate specificity, we knocked down ZEB1 via siRNA before ChIP. Reduction of ZEB1 significantly decreased coprecipitated Crumbs3 and PATJ promoter fragment levels (; lower panels, siRNA-ChIP). Thus, ZEB1 can directly repress Crumbs3 and PATJ by binding to defined proximal promoter segments.
ZEB1 enhances the migratory potential of cells
Next, we performed transpore migration assays to determine whether knockdown of ZEB1 also affects cell motility in vitro. MDA-MB-231 cells treated with ZEB1-specific siRNA for 3 days were seeded onto Transwell filter inserts and cell migration through pores was analysed 24h later. ZEB1 depletion impaired cell motility by 80% (). The reduced motility is not only a result of E-cadherin, upregulation, since ectopic expression of E-cadherin in MDA-MB-231 cells barely affected motility (). Interestingly, also Snail1 depletion affected transmigration of MDA-MB-231 cells through filters (), although they retained a fibroblastoid morphology.
Figure 4 ZEB1 and Snail depletion reduced the motility of MDA-MB-231 cells in vitro. MDA-MB-231, treated with control or ZEB1 and Snail-specific siRNAs for 3 days, and MDA-MB-231 cells stably transfected with empty or E-cadherin-expressing plasmids, were seeded (more ...)
Expression of ZEB1 in human tumours correlated with tumour cell dedifferentiation and invasion
Our findings suggested that aberrant upregulation of ZEB1 in human tumours may induce cancer cell dissemination. To test this hypothesis, we screened paraffin-embedded human colon and breast neoplasm specimens immunohistochemically for ZEB1, E-cadherin, cytokeratin and HUGL2 expression.
In colon cancer samples, the bulk tumour area stained positively for E-cadherin, cytokeratin and HUGL2 ( and Supplementary Figure 1
, arrows). ZEB1 expression was not detected in differentiated tumour cells but in many stroma cells adjacent to the tumour areas ( and Supplementary Figure 1
, arrowheads). In normal colon tissue, ZEB1-positive stroma cells were mostly absent (Supplementary Figure 1
, arrowheads). Unspecific goat IgG controls did not reveal specific signals (Supplementary Figure 2
, cytokeratin/goat IgG double stainings).
Figure 5 ZEB1 expression in colon and breast tumours correlates with cancer cell dedifferentiation. (a) Serial tumour sections were immunohistochemically stained for ZEB1, E-cadherin or cytokeratin (brown). In addition, single sections were double stained for (more ...)
As EMT-like phenotypic conversions at the tumour–host interface are prominent features of colorectal cancer progression (Brabletz et al., 2001
), we screened tumour–host interfaces for ZEB1 expression. In eight out of ten colon tumours, we found strong ZEB1 expression at tumour margins exhibiting tumour cell dedifferentiation. ZEB1-positive cells expressed low levels of cytokeratin, formed loosely attached clusters or invaded the tumour stroma as single cells ( and Supplementary Figure 3
, arrows). ZEB1-positive cancer cells were clearly distinguishable from tumour-associated stroma cells by perinuclear and/or cytoplasmic cytokeratin remnants and by nuclear morphology ( and Supplementary Figure 1
, ZEB1/cytokeratin, arrows). In addition, we found co-expression of ZEB1 and β
-catenin in a significant number of invasive tumour cells (Supplementary Figure 4
, lower panel, arrows).
We also examined ZEB1 expression in eight ductal and five lobular breast cancer specimens. In all ductal carcinomas, ZEB1 expression inversely correlated with cancer cell differentiation. Nuclear accumulation of ZEB1 was always associated with low expression of cytokeratin (, Ductal-Dediff., arrows) and weakened intercellular adhesion, while differentiated tumour areas were strongly positive for cytokeratin and lacked ZEB1 (, Ductal-Diff., arrow). In three ductal carcinomas, ZEB1 was highly expressed in tumour cells invading host tissue as loosely associated group or single cells, reminiscent of infiltrating tumour cells in invasive lobular breast cancer (, Mixed, arrows). Accordingly, the most striking upregulation of ZEB1 was found in breast cancer specimens exhibiting all histological criteria of invasive lobular carcinomas (, Lobular).