The expression of Snail family members has been associated with the acquisition of resistance to several types of programmed cell death. For instance, both Snail1 and Snail2 (Slug) protect hematopoietic cells from γ radiation-induced apoptosis (21
). We show here that the overexpression of Snail1 also preserves epithelial MDCK and RWP-1 cells from this type of apoptosis. The percentage of cells presenting characteristics of programmed cell death 48 h after inducing DNA damage is much lower in MDCK cells overexpressing Snail1 than in control cells. Similar results have been published by other authors using the same cellular model, which induced apoptosis by the withdrawal of survival factors or by other proapoptotic signals (44
). These authors have also shown that Snail1 induces an activation of PI3K and Akt, a pathway that confers resistance to apoptosis. However, the mechanism underlying such a Snail1 effect has not been clarified yet. Our data show increased levels of active Akt in MDCK-Snail1 and RWP-1-Snail1 cells with regard to the respective controls and identify a critical effector of this pathway, PTEN, as a direct Snail1 target gene.
In our assays, MDCK-Snail1 cells respond normally to γ radiation, with an arrest in cell proliferation, and accumulate in G2
phase. This arrest is accompanied by an up-regulation of active p53 that is not affected by the expression of Snail1. These results are in agreement with previous results showing that ectopic expression of Snail1 in mouse embryo fibroblasts does not modify the expression of p53 after γ radiation (33
). Moreover, in our MDCK cells, the transfection of Snail1 does not prevent the up-regulation of two p53 target genes, p21 (Fig. ) and Puma (Fig. ), further indicating that p53 activity is not affected by Snail1. However, other authors have indicated that Snail1 can alter the response of MCF-7 cells to the genotoxic stress induced by adriamycin, preventing the increase in p53 (24
). The reasons for this discrepancy are unknown, although it is possible that Snail1 might act on genes involved in adriamycin export. Alternatively, it is possible that the factor responsible for p53 repression is not Snail1 by itself, but another transcriptional repressor specifically induced by Snail1 in MCF-7 and not in other cells.
Our results show that Snail1 prevents the up-regulation of PTEN phosphatase, an inhibitor of the PI3K/Akt pathway (48
). The role of this pathway and PTEN in the modulation of apoptosis has been clearly demonstrated by several studies. The reexpression of PTEN in several carcinoma cell lines can induce apoptosis directly or in cooperation with apoptotic stimuli (38
); therefore, the ectopic manipulation of PTEN levels in MDCK cells affects the capability of the cells to undergo apoptosis. For instance, the depletion of PTEN by an interferent RNA increases the resistance of MDCK cells to γ radiation-induced apoptosis (Fig. ), indicating the role of PTEN and, therefore, of Snail1 in the control of cell death. However, even in cells without PTEN expression, Snail1 causes a further increase in resistance to apoptosis, indicating that Snail1 is also acting on another cellular element. Experiments performed with RWP-1 cells also confirmed this conclusion. Therefore, our studies indicate that although the repression of PTEN by Snail1 contributes to the resistance to cell death, Snail1 is also acting on other factors involved in the regulation of this cellular event.
We have also determined that Snail1 repression of the PTEN promoter is specific, since Snail2 (Slug) presents very little activity on this promoter and other repressors with a similar specificity, such as Zeb1, are also inactive. Like what has been reported for other genes controlled by Snail1 (4
), the effect of Snail1 on this promoter is dependent on the integrity of two 5′-CACCTG-3′ boxes present in the proximal human promoter. The mutation of these two elements precludes not only the association of recombinant Snail1 to this sequence but also the repression by Snail1 of PTEN promoter activity. One box was present in PTEN promoters from all mammals studied, such as mouse, rat, rabbit, dog, and others, suggesting that probably only one of these elements present in the human PTEN promoter is involved in repression. Perhaps because of the existence of just one functional binding element, the effect of Snail1 is lower than that measured on the E-cadherin promoter (4
) (contains three E boxes) but comparable to the effect of this repressor on the promoter of other targets such as the vitamin D receptor (28
ChIP analysis demonstrated that Snail1 binds to the PTEN promoter. The association of Snail1 to this promoter precludes the binding of p53, a transcriptional activator of PTEN during apoptosis. The similarities existent between the regulation of PTEN and that of Puma during the process of cell death are noteworthy, although they also present relevant differences. Both genes are induced after γ irradiation by p53, and this induction is prevented in cells expressing Snail1 (in the case of PTEN) or expressing Snail2 (in the case of Puma) (46
). It is also remarkable that both genes show a high specificity for their corresponding repressors, since Snail2 cannot repress the PTEN promoter (Fig. ) and Snail1 neither binds to the Puma promoter nor affects Puma mRNA up-regulation in response to γ irradiation (Fig. and data not shown).
Snail1 protein is up-regulated in response to DNA damage at a posttranslational level. Our results indicate that the accumulation of Snail1 is mainly a consequence of the modification of Ser246. The phosphorylation of this residue by PAK1 prevents Snail1 export from the nucleus and its subsequent degradation (49
). This protein kinase (the γ isoform) is activated after DNA damage in fibroblasts (34
). Moreover, PAK1 has been shown to down-regulate several proapoptotic pathways (reviewed in reference 25
). Therefore, it is likely that the modification of Ser246 in Snail1 after γ irradiation is catalyzed by this kinase.
It is noteworthy that this increase in Snail1 protein is transient. For instance, in MDCK cells 8 h after irradiation, the total levels of the protein are lower than those before this insult. However, this down-regulation is not reflected in a concomitant decrease in Snail1-PTEN promoter association. This apparent discrepancy might be explained by the fact that the binding of Snail1 to DNA stabilizes this protein (M. Escrivà and A. Garcia de Herreros, unpublished observations), probably because it prevents its export from the nucleus. Therefore, we expect that PTEN promoter-bound Snail1 is not an efficient target for the nuclear export machinery and the repression of the activity of this promoter is maintained even after the cellular levels of Snail1 have returned to the basal levels.
In spite of its important role in the modulation of apoptosis, not much is known about the mechanisms controlling the expression of PTEN, other than the transcription of this gene is sensitive to the direct binding and activation of p53 of its promoter (39
). It has been also reported that PTEN levels are negatively regulated by transforming growth factor β and NF-κB (26
), two factors that stimulate Snail1 transcription in MDCK and other epithelial cells (2
). Mutations and deletions in the PTEN gene have previously been described for a wide variety of tumors, although in some advanced carcinomas, such as prostatic and endometrial carcinomas, as well as melanomas, the silencing of this gene seems to be controlled epigenetically (35
). It is possible that Snail1 is responsible for this inhibition, considering that this gene is expressed in advanced tumors (3
). However, the limited expression of Snail1 to specific areas of epithelial tumors (13
) suggests that Snail1 might be involved in the down-regulation of PTEN in cells that are undergoing an EMT, more than in the permanent silencing of this gene. Similarly, since Snail1 is expressed only in a subset of fibroblasts (activated fibroblasts) (13
), the negative effect of Snail1 on PTEN expression might be limited to these cells.
As indicated above, a consequence of PTEN repression is the activation of Akt observed after the transfection of Snail1. Curiously, the overexpression of Akt has also been shown to induce EMT through the NF-κB-dependent activation of Snail1 (16
). These results suggest the existence of a positive feedback loop wherein Snail1 might induce its own transcription. Actually, results from our lab indicate that Snail1 can stimulate the activity of its own promoter in a cell-specific manner (M. Escrivà, S. Peiró, and A. Garcia de Herreros, unpublished observations). This positive feedback loop would be coordinated with the negative self-regulation already described for this gene, since Snail1 is also capable of repressing its own synthesis, both directly, through the binding to its own promoter (32
), and indirectly, through the inhibition of Egr-1, an activator of its transcription (17
). The existence of transcriptional feedback loops has previously been described, and they seem to be particularly relevant for cell pathways implicated in embryo development (14
). A possible consequence of the operation of the positive and negative feedback controls of a gene is the appearance of oscillatory patterns of expression (14
). In this respect, it is remarkable that oscillations in the levels of Snail1 RNA have been detected in the presomitic mesoderm (8
). In any case, this positive feedback loop might help coordinate and integrate the signals provided by factors of the fibroblast growth factor and transforming growth factor superfamilies required for the induction of Snail1 during the development and subsequent triggering of EMT (3
Moreover, in addition to its role in the modulation of apoptosis, PTEN reconstitution or overexpression inhibits cell migration (48
). PTEN-null mouse fibroblasts show increased rates of migration, a property that is reversed by the reintroduction of PTEN (27
). PTEN also prevents tumoral cell invasion (42
). It has been suggested that the effects of PTEN are due not only to its lipid phosphatase activity but also to its tyrosine phosphatase activity on focal adhesion kinase and Shc (48
). In any case, since the overexpression of Snail1 in different cell lines induces migration and invasion, the possibility that the down-regulation of PTEN is relevant for these effects is worth being studied.