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Summary: Snail1 and ZEB1 are transcriptional repressors that drive tumor initiation and metastasis in animal models. Snail1 and ZEB1 are frequently coexpressed in tumor cell lines, suggesting that these factors may cooperate to promote tumor progression. However, coexpression of these transcriptional repressors in primary human cancer specimens has not been investigated. Previous studies assessed expression in primary breast cancers of Snail1 messenger RNA, which does not reflect Snail1 activity because Snail1 is subject to posttranslational modifications that inhibit its nuclear localization/activity. In the current study, using breast tumor cell lines of known Snail1 and ZEB1 expression status, we developed immunohistochemistry protocols for detecting nuclear Snail1 and nuclear ZEB1 proteins. Using these protocols, we assessed nuclear Snail1 and nuclear ZEB1 expressions in primary human breast cancers of varying subtypes (n = 78). Nuclear Snail1 and estrogen receptor α expressions were inversely associated in primary breast cancers, and nuclear Snail1 was expressed in approximately 80% of triple-negative breast cancers (lacking estrogen receptor α, progesterone receptor, and human epidermal growth factor receptor 2 overexpression). In contrast, nuclear ZEB1 was expressed at a significantly lower frequency in these breast cancers. Notably, nuclear Snail1 protein was detected in 45% of ductal carcinoma in situ specimens (n = 29), raising the important possibility that nuclear Snail1 expression in early stage breast lesions may predict future development of invasive breast cancer. Collectively, our studies demonstrate frequent expression of nuclear Snail1, but not nuclear ZEB1, in invasive, triple-negative breast cancers as well as in intraductal carcinomas. [Copyright &y& Elsevier]

Snail! and Zebi are E-cadherin-transcriptional repressors induced during epithelial mesenchymal transition (EMT). In this article we have analyzed the factors controlling Zebi expression during EMT. In NMuMG cells treated with TGF-13, Snaill RNA and protein are induced 1 h after addition of the cytokine preceding Zeb1 up-regulation that requires 6-8 h. Zeb1 gene expression is caused by increased RNA levels but also by enhanced protein stability and is markedly dependent on Snaill because depletion of this protein prevents Zeb1 protein and RNA up-regulation. In addition to Snaill, depletion of the Twist transcriptional factor retards Zeb1 stimulation by TGF-β or decreases Zeb1 expression in other cellular models indicating that this factor is also required for Zebi expression. Accordingly, Snaill and Twist cooperate in the induction of Zeb1: co- transfection of both cDNAs is required for the maximal expression of ZEB1 mRNA. Unexpectedly, the expression of Snail! and Twist shows a mutual dependence although to a different extent; whereas Twist depletion retards Snail! up-regulation by TGF-β, Snail1 is necessary for the rapid increase in Twist protein and later up-regulation of Twisti mRNA induced by the cytokine. Besides this effect on Twist, Snail! also induces the nuclear translocation of Etsi, another factor required for Zebi expression. Both Twist and Ets1 bind to the ZEB1 promoter although to different elements: whereas Ets1 interacts with the proximal promoter, Twist does it with a 700-bp sequence upstream of the transcription start site. These results indicate that Snail! controls Zeb1 expression at multiple levels and acts cooperatively with Twist in the ZEB1 gene transcription induction. [ABSTRACT FROM AUTHOR]

The transcription factor SNAIL1 is a master regulator of epithelial to mesenchymal transition. SNAIL1 is a very unstable protein, and its levels are regulated by the E3 ubiquitin ligase β-TrCP1 that interacts with SNAIL1 upon its phosphorylation by GSK-3β. Here we show that SNAIL! polyubiquitylation and degradation may occur in conditions precluding SNAIL1 phosphorylation by GSK-3β, suggesting that additional E3 ligases participate in the control of SNAIL1 protein stability. In particular, we demonstrate that the F-box E3 ubiquitin ligase FBX114 interacts with SNAIL 1 and promotes its ubiquitylation and proteasome degradation independently of phosphorylation by GSK-3β. In vivo, inhibition of FBXI14 using short hairpin RNA stabilizes both ectopically expressed and endogenous SNAIL1. Moreover, the expression of FBXI14 is potently down-regulated during hypoxia, a condition that increases the levels of SNAIL! protein but not SNAIL1 mRNA. FBX14 mRNA is decreased in tumors with a high expression of two proteins up-regulated in hypoxia, carbonic anhydrase 9 and TWIST 1. In addition, Twist1 small interfering RNA prevents hypoxia-induced Fbxl14 down-regulation and SNAIL1 stabilization in NMuMG cells. Altogether, these results demonstrate the existence of an alternative mechanism controlling SNAIL1 protein levels relevant for the induction of SNAIL1 during hypoxia. [ABSTRACT FROM AUTHOR]