EMT occurs frequently during normal development in processes such as mesoderm and neural crest cell formation. During tumor progression, EMT is also crucial for loss of cell polarity of epithelial cells, thus facilitating migratory and invasive behavior. The involvement of the transcription factor SIP1/ZEB2 during EMT in developmental processes was indicated by the phenotype of the SIP1 knock-out mouse (17
). Loss of SIP1 expression was correlated with loss of the migratory capacities of neural crest cells. Retroviral insertion mutagenesis suggested that SIP1 could contribute to oncogenic transformation (9
). Furthermore, the upregulation of ZEB-family members during EMT was recently demonstrated (33
). To study the role of SIP1 in more detail in EMT-like processes, we generated human cell lines with conditional SIP1 expression. In these Tet-on cell systems, adding Dox to the cell culture medium resulted in nuclear expression of SIP1. A drastic morphological change was induced in these epithelial cells as a consequence of exogenous SIP1 expression. As the transmembrane cell adhesion protein E-cadherin is a direct target of SIP1, we analyzed the expression of the different components of the cadherin–catenin complex. Downregulation of protein and mRNA levels was only detected for E-cadherin. The complexing αE-catenin was altered only at the protein expression level, probably as a consequence of loss of E-cadherin expression (34
). Different p120ctn isoforms were inversely regulated during the SIP1-induced EMT-like process. The upregulation of p120ctn isoform 1 (~120 kDa) and the downregulation of isoform 3 (~100 kDa) indicate putative specific roles for different isoforms in epithelial and mesenchymal states. A similar shift in p120ctn isoform expression was seen in FosER cells, in which EMT is induced as a consequence of FosER activation by estradiol addition (35
). This shift is in line with the previously observed predominant expression of 100 kDa and 120 kDa isoforms in epithelial cells and in highly motile fibroblastoid cells, respectively (36
). The functional difference between these isoforms remains to be elucidated. The other E-cadherin-binding Armadillo protein β-catenin showed no decrease in mRNA and protein expression levels, but β-catenin was no longer expressed at the cell–cell contacts. SIP1 expression resulted in the redistribution of β-catenin to the cytoplasm and possibly also to the nucleus.
The E-cadherin promoter was previously identified as a direct target of SIP1. SIP1 binds to the E2-boxes (CACCTG) present in the E-cadherin promoter, resulting in downregulation of the promotor's activity (7
). Global gene expression analysis using the in vitro
SIP1-induced cell models revealed that SIP1 expression results in downregulation of major constituents of different cell junctional complexes, such as tight junctions, adherens junctions, desmosomes and gap junctions. Interestingly we found by Q-RT–PCR analyis that SIP1 downregulates several cell junction genes on the transcript level. The fact that expression of a SIP1 mutant, with one missense mutation in each of the zinc finger clusters, has no effect on mRNA expression levels of these regulated genes, suggests that downregulation by SIP1 is mediated mainly via promoter regulation. Both zinc finger clusters are indeed needed for SIP1-dependent promoter repression via E-box-binding (7
). Cloning of the promoter regions of the regulated genes connexin 26 (in the gap junctions), P-cadherin (at the adherens junction) and claudin 4 (in the tight junctions) revealed the presence of several SIP1-binding sequences in each of them. Mutation of these elements in the cloned connexin 26 promoter showed the importance of the integrity of these sequences in the SIP1-dependent suppressive activity. Furthermore, we could demonstrate physical interaction at the chromatin level between SIP1 and the promoter regions of E-cadherin, plakophilin 2, connexin 26 and ZO-3
, all of which contain SIP1-binding sites.
The change in expression and distribution of those SIP1 target genes during EMT could be explained as a secondary consequence of repression of E-cadherin. A crucial role for E-cadherin in epithelial cell polarity has been well documented (37
). However, exogenous E-cadherin expression in mesenchymal cells expressing EMT inducers such as Snail cannot restore the epithelial phenotype (19
). Moreover, downregulation of the tight junction components, occludins and claudins, by Snail was linked to repression of their promoter activity (18
). Hence, we have to conclude that E-cadherin and other junctional genes are simultaneously downregulated as part of the SIP1 driven reprogramming during EMT. It remains enigmatic though why these different junctional genes are repressed in a coordinated fashion. We do know that some of these genes can be regarded as NACos, proteins that can localize both to the nucleus and adhesion complexes (42
). Such proteins have the intriguing potential to coordinate the regulation of cell adhesion and transcription. The function of several NACos proteins belonging to the Armadillo family such as PKP2
, β-catenin and p120ctn seem to be affected in epithelial cells with SIP1 expression. The desmosomal PKP2
has been reported to be present also in the nucleus at all times. This protein seems to be part of particles containing RNA polymerases (43
). Moreover the transcription factor Snail is also able to repress PKP2
expression very potently (19
). This strong downregulation of PKP2
suggests that inhibiting the potential role of PKP2
in adhesion and/or transcriptional regulation could be essential in the process of EMT. It is at present not clear if the SIP1-induced isoform switching has functional consequences for a particular nuclear role of p120ctn (23
). P120ctn can interact with Kaiso and has as such the potential to influence beta-catenin/TCF signaling (44
). On the other hand, downregulation of E-cadherin could result in loss of β-catenin sequestration to sites of cell–cell adhesion, enabling β-catenin/TCF mediated transcription. However SIP1 did not induce clear nuclear β-catenin localization nor enhanced WNT signaling (data not shown).
Enhanced SIP1 expression has so far been reported in a distinct set of cancers comprising gastric, hepatocellular, ovarian and breast carcinomas (8
). Here, the described candidate SIP1 target genes have been documented to show abberant expression in a variety of human cancer types (47
). This suggests that repression of these genes could be due to enhanced SIP1 expression, although this has to be examined in detail in the near future.
Taken together, the present results identify the SIP1 protein as an important mediator of epithelial dedifferentiation through direct downregulation of a distinct set of constituents of adherens junctions, tight junctions, desmosomes and gap junctions, which are key determinants of the epithelial phenotype, including epithelial cell polarity.