Our research focused on studying the mechanism(s) by which Snail transcription factor may contribute to cancer progression in prostate cancer. One of the ways by which Snail can lead to cancer progression is through induction of epithelial-mesenchymal transition (EMT), which involves the loss of epithelial markers such as E-cadherin, and acquisition of mesenchymal markers such as vimentin [2
]. Snail can negatively regulate a number of tumor suppressors including E-cadherin, claudins, and occludin, by binding to E-boxes in the promoter region [2
]. This communication studied the relationship between Snail and maspin tumor suppressor, to discover a new mechanism by which maspin may be downregulated during prostate tumor progression.
Maspin tumor suppressor has been shown to be downregulated in breast and gastric cancer through promoter methylation [15
]. Maspin expression is also lost with prostate tumor progression, through inactivation of a positive Ets response element and activation of a negative HRE response element recognized by AR [18
]. Recently, interleukin-6 (IL-6) signaling has been shown to downregulate maspin expression [27
The present study correlates Snail expression with prostate cancer, as Snail protein was absent in normal immortalized prostate epithelial cells (PrEC), however it was then expressed in our LNCaP progression model (LNCaP, C4-2), DU145 prostate cancer cell lines, though undetectable in 22Rv1 cells. Conversely, maspin expression was high in PrEC and low in the prostate cancer cell lines. The inverse relationship between Snail and maspin led us to investigate whether Snail may be negatively regulating maspin expression. Indeed, we found that when Snail is overexpressed in 22Rv1 cells, maspin expression was decreased, while migratory and invasive potential increased. Conversely, when Snail expression was inhibited with siRNA or shRNA in DU145 or C4-2 cells, respectively, maspin expression increased, while migratory potential decreased. This study reports evidence for the first time, that Snail oncogene can negatively regulate maspin tumor suppressor. Since maspin is silenced epigenetically in some cancers, studies aim at preventing tumor progression by reinducing maspin expression with methylation inhibitors such as 5- aza-2 ′-deoxycytidine and histone deacetylase inhibitors [28
]. These are general inhibitors that would lead to non-specific demethylation. We provide a novel mechanism by which therapeutic targeting of Snail in the future, may prevent tumor cell migration by reinducing maspin expression.
We have also utilized LNCaP and 22Rv1 cells transiently or stably transfected with Snail to show that Snail does significantly reduce maspin promoter activity, while knockdown of endogenous Snail in C4-2 cells increased maspin promoter activity. To elucidate the mechanism, we have found 8 E-boxes within the maspin promoter and showed that Snail directly binds to the maspin promoter in 22Rv1 cells. Our data suggest that Snail may repress maspin independently of AR since knockdown of Snail resulted in decreased maspin expression in both AR-negative DU145 and AR-positive C4-2 cells. It is also possible that Snail may negatively regulate maspin by recruiting histone deacteylases (HDACs). Although Snail has been shown to directly bind to the E-cadherin promoter, it can also repress E-cadherin epigenetically by recruiting a corepressor, Ajuba LIM domain protein resulting in histone modifications and promoter methylation [31
]. It was reported that receptor activator of NF-kappa B ligand (RANKL) signaling to Ikappa B kinase alpha (IKKalpha) represses maspin expression in prostate epithelial cells, associated with nuclear translocation of IKKalpha [33
]. We have previously shown that Snail can induce the expression of RANKL [19
], so it is possible that Snail may be repressing maspin through the RANKL-IKKalpha pathway. Alternatively, p53 has been shown to bind to maspin promoter leading to activation of its transcription [34
], while Snail interacts directly with the DNA binding domain of p53 diminishing its tumor suppressive function [36
], therefore, it seems plausible that Snail may inhibit maspin via p53 pathway. Thus although we report one step in which Snail directly binds to maspin promoter to inhibit its promoter activity and expression, this does not exclude other possibilities by which Snail may negatively regulate maspin.