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1.  Suppression of SCARA5 by Snail1 is essential for EMT-associated cell migration of A549 cells 
Oncogenesis  2013;2(9):e73-.
Accumulating evidence indicates that epithelial-to-mesenchymal transition (EMT) might be a key event for cancer progression. The upregulation of Snail1, one of the most extensively studied EMT regulators, has been implicated in cancer metastasis, but the underlying mechanisms remain unclear. This study aims to identify that Snail1 targets regulating EMT-associated cancer cell migration. Human lung carcinoma A549 cells were treated with transforming growth factor beta 1 (TGF-β1), and EMT-associated phenotypic and functional alterations were monitored. TGF-β1 induced typical EMT-like morphological changes, ‘cadherin switching' and cell migration in A549 cells. TGF-β1 stimulation induced rapid and persistent upregulation of Snail1. Moreover, Snail1 upregulation was required for EMT-associated cell migration. Several metastasis suppressors with putative Snail1-binding sites in their promoters were dramatically repressed in A549 cells during TGF-β1-induced EMT. Gain- and loss-of Snail1 function experiments demonstrated that scavenger receptor class A member 5 (SCARA5) was negatively regulated by Snail1. Importantly, SCARA5 downregulation was essential for EMT-induced migration in A549 cells. The chromatin immunoprecipitation assay revealed that Snail1 could bind to the E-box elements in SCARA5 promoter, implying that SCARA5 is a direct Snail1 target modulating cancer cell mobility during EMT. In addition, we showed that DNA methyltransferase 1 was physically associated with Snail1 to silence SCARA5 expression with an unidentified DNA methylation-independent mechanism, suggesting the complexity of Snail1-mediated epigenetic regulation. Collectively, our data demonstrated that EMT-regulator Snail1 suppresses the expression of SCARA5 to promote cancer progression, highlighting the possibility to target Snail1 and SCARA5 for cancer treatment.
doi:10.1038/oncsis.2013.37
PMCID: PMC3816226  PMID: 24061576
Snail1; TGF-β1; EMT; migration; SCARA5; lung cancer
2.  Repression of PTEN Phosphatase by Snail1 Transcriptional Factor during Gamma Radiation-Induced Apoptosis▿  
Molecular and Cellular Biology  2008;28(5):1528-1540.
The product of the Snail1 gene is a transcriptional repressor required for triggering the epithelial-to-mesenchymal transition. Furthermore, ectopic expression of Snail1 in epithelial cells promotes resistance to apoptosis. In this study, we demonstrate that this resistance to γ radiation-induced apoptosis caused by Snail1 is associated with the inhibition of PTEN phosphatase. In MDCK cells, mRNA levels of the p53 target gene PTEN are induced after γ radiation; the transfection of Snail1 prevents this up-regulation. Decreased mRNA levels of PTEN were also detected in RWP-1 cells after the ectopic expression of this transcriptional factor. Snail1 represses and associates to the PTEN promoter as detected both by the electrophoretic mobility shift assay and chromatin immunoprecipitation experiments performed with either endogenous or ectopic Snail1. The binding of Snail1 to the PTEN promoter increases after γ radiation, correlating with the stabilization of Snail1 protein, and prevents the association of p53 to the PTEN promoter. These results stress the critical role of Snail1 in the control of apoptosis and demonstrate the regulation of PTEN phosphatase by this transcriptional repressor.
doi:10.1128/MCB.02061-07
PMCID: PMC2258777  PMID: 18172008
3.  Snail transcription factor negatively regulates maspin tumor suppressor in human prostate cancer cells 
BMC Cancer  2012;12:336.
Background
Maspin, a putative tumor suppressor that is down-regulated in breast and prostate cancer, has been associated with decreased cell motility. Snail transcription factor is a zinc finger protein that is increased in breast cancer and is associated with increased tumor motility and invasion by induction of epithelial-mesenchymal transition (EMT). We investigated the molecular mechanisms by which Snail increases tumor motility and invasion utilizing prostate cancer cells.
Methods
Expression levels were analyzed by RT-PCR and western blot analyses. Cell motility and invasion assays were performed, while Snail regulation and binding to maspin promoter was analyzed by luciferase reporter and chromatin immunoprecipitation (ChIP) assays.
Results
Snail protein expression was higher in different prostate cancer cells lines as compared to normal prostate epithelial cells, which correlated inversely with maspin expression. Snail overexpression in 22Rv1 prostate cancer cells inhibited maspin expression and led to increased migration and invasion. Knockdown of Snail in DU145 and C4-2 cancer cells resulted in up-regulation of maspin expression, concomitant with decreased migration. Transfection of Snail into 22Rv1 or LNCaP cells inhibited maspin promoter activity, while stable knockdown of Snail in C4-2 cells increased promoter activity. ChIP analysis showed that Snail is recruited to the maspin promoter in 22Rv1 cells.
Conclusions
Overall, this is the first report showing that Snail can negatively regulate maspin expression by directly repressing maspin promoter activity, leading to increased cell migration and invasion. Therefore, therapeutic targeting of Snail may be useful to re-induce expression of maspin tumor suppressor and prevent prostate cancer tumor progression.
doi:10.1186/1471-2407-12-336
PMCID: PMC3437215  PMID: 22857708
Snail; Maspin; Prostate cancer
4.  Snail promotes CXCR2 ligand dependent tumor progression in NSCLC 
Purpose
As a transcriptional repressor of E-cadherin, Snail has predominantly been associated with epithelial-mesenchymal transition (EMT), invasion, and metastasis. However, other important Snail-dependent malignant phenotypes have not been fully explored. Here, we investigate the contributions of Snail to the progression of non-small cell lung cancer (NSCLC).
Experimental Design
Immunohistochemistry was performed to quantify and localize Snail in human lung cancer tissues, and tissue microarray analysis (TMA) was utilized to correlate these findings with survival. NSCLC cell lines gene-modified to stably over-express Snail were evaluated in vivo in two severe combined immunodeficiency (SCID) murine tumor models. Differential gene expression between Snail over-expressing and control cell lines was evaluated using gene expression microarray analysis.
Results
Snail is up-regulated in human NSCLC tissue, and high levels of Snail expression correlate with decreased survival (p<0.026). In a heterotopic model, mice bearing Snail over-expressing tumors developed increased primary tumor burden (p=0.008). In an orthotopic model, mice bearing Snail over-expressing tumors also demonstrated a trend toward increased metastases. In addition, Snail over-expression led to increased angiogenesis in primary tumors as measured by MECA-32 (p<0.05) positivity and CXCL8 (p=0.002) and CXCL5 (p=0.0003) concentrations in tumor homogenates. Demonstrating the importance of these pro-angiogenic chemokines, the Snail-mediated increase in tumor burden was abrogated with CXCR2 blockade. Gene expression analysis also revealed Snail-associated differential gene expression with the potential to affect angiogenesis and diverse aspects of lung cancer progression.
Conclusion
Snail up-regulation plays a role in human NSCLC by promoting tumor progression mediated by CXCR2 ligands.
doi:10.1158/1078-0432.CCR-09-1558
PMCID: PMC2783274  PMID: 19887480
Snail; lung cancer; angiogenesis; CXCL8; CXCL5
5.  A PHD12–Snail2 repressive complex epigenetically mediates neural crest epithelial-to-mesenchymal transition 
The Journal of Cell Biology  2012;198(6):999-1010.
Snail2 and the adaptor protein PHD12 are recruited to the Cad6b promoter by Sin3A and result in promoter deacetylation, revealing the nature of the in vivo Snail repressive complex that regulates neural crest EMT.
Neural crest cells form within the neural tube and then undergo an epithelial to mesenchymal transition (EMT) to initiate migration to distant locations. The transcriptional repressor Snail2 has been implicated in neural crest EMT via an as of yet unknown mechanism. We report that the adaptor protein PHD12 is highly expressed before neural crest EMT. At cranial levels, loss of PHD12 phenocopies Snail2 knockdown, preventing transcriptional shutdown of the adhesion molecule Cad6b (Cadherin6b), thereby inhibiting neural crest emigration. Although not directly binding to each other, PHD12 and Snail2 both directly interact with Sin3A in vivo, which in turn complexes with histone deacetylase (HDAC). Chromatin immunoprecipitation revealed that PHD12 is recruited to the Cad6b promoter during neural crest EMT. Consistent with this, lysines on histone 3 at the Cad6b promoter are hyperacetylated before neural crest emigration, correlating with active transcription, but deacetylated during EMT, reflecting the repressive state. Knockdown of either PHD12 or Snail2 prevents Cad6b promoter deacetylation. Collectively, the results show that PHD12 interacts directly with Sin3A/HDAC, which in turn interacts with Snail2, forming a complex at the Cad6b promoter and thus revealing the nature of the in vivo Snail repressive complex that regulates neural crest EMT.
doi:10.1083/jcb.201203098
PMCID: PMC3444776  PMID: 22986495
6.  14-3-3 binding sites in the Snail protein are essential for Snail-mediated transcriptional repression and epithelial-mesenchymal differentiation 
Cancer research  2010;70(11):4385-4393.
The Snail transcription factor is a repressor and a master regulator of epithelial-mesenchymal transition events (EMT) in normal embryonic development and during tumor metastases. Snail directly regulates genes affecting cell adhesion, motility and polarity. Invasive tumor cells express high levels of Snail and it is a marker for aggressive disease and poor prognosis. Transcriptional repression and EMT induction by Snail requires binding to its obligate corepressor, the LIM protein Ajuba. It is unclear how this complex is assembled and maintained on Snail target genes. Here we define functional 14-3-3 binding motifs in Snail and Ajuba which selectively bind 14-3-3 protein isoforms. In Snail, a NH2-terminal motif in the repression domain cooperates with a COOH-terminal, high affinity motif for binding to 14-3-3 proteins. Coordinate mutation of both motifs abolishes 14-3-3 binding and inhibits Snail-mediated gene repression and EMT differentiation. Snail, 14-3-3 proteins, and Ajuba form a ternary complex which is readily detected via ChIP at the endogenous E-cadherin promoter. Collectively, these data show that 14-3-3 proteins are new components of the Snail transcriptional repression machinery and mediate its important biological functions.
doi:10.1158/0008-5472.CAN-10-0070
PMCID: PMC2894621  PMID: 20501852
Snail; 14-3-3; Ajuba; epithelial-mesenchymal transition; transcriptional repression
7.  Polycomb Complex 2 Is Required for E-cadherin Repression by the Snail1 Transcription Factor▿ †  
Molecular and Cellular Biology  2008;28(15):4772-4781.
The transcriptional factor Snail1 is a repressor of E-cadherin (CDH1) gene expression essential for triggering epithelial-mesenchymal transition. Snail1 represses CDH1, directly binding its promoter and inducing the synthesis of the Zeb1 repressor. In this article, we show that repression of CDH1 by Snail1, but not by Zeb1, is dependent on the activity of Polycomb repressive complex 2 (PRC2). Embryonic stem (ES) cells null for Suz12, one of the components of PRC2, show higher levels of Cdh1 mRNA than control ES cells. In tumor cells, interference of PRC2 activity prevents the ability of Snail1 to downregulate CDH1 and partially derepresses CDH1. Chromatin immunoprecipitation assays demonstrated that Snail1 increases the binding of Suz12 to the CDH1 promoter and the trimethylation of lysine 27 in histone H3. Moreover, Snail1 interacts with Suz12 and Ezh2, as shown by coimmunoprecipitation experiments. In conclusion, these results demonstrate that Snail1 recruits PRC2 to the CDH1 promoter and requires the activity of this complex to repress E-cadherin expression.
doi:10.1128/MCB.00323-08
PMCID: PMC2493371  PMID: 18519590
8.  The LIM Protein AJUBA Recruits Protein Arginine Methyltransferase 5 To Mediate SNAIL-Dependent Transcriptional Repression▿  
Molecular and Cellular Biology  2008;28(10):3198-3207.
The SNAIL transcription factor contains C-terminal tandem zinc finger motifs and an N-terminal SNAG repression domain. The members of the SNAIL family have recently emerged as major contributors to the processes of development and metastasis via the regulation of epithelial-mesenchymal transition events during embryonic development and tumor progression. However, the mechanisms by which SNAIL represses gene expression are largely undefined. Previously we demonstrated that the AJUBA family of LIM proteins function as corepressors for SNAIL and, as such, may serve as a platform for the assembly of chromatin-modifying factors. Here, we describe the identification of the protein arginine methyltransferase 5 (PRMT5) as an effector recruited to SNAIL through an interaction with AJUBA that functions to repress the SNAIL target gene, E-cadherin. PRMT5 binds to the non-LIM region of AJUBA and is translocated into the nucleus in a SNAIL- and AJUBA-dependent manner. The depletion of PRMT5 in p19 cells stimulates E-cadherin expression, and the SNAIL, AJUBA, and PRMT5 ternary complex can be found at the proximal promoter region of the E-cadherin gene, concomitant with increased arginine methylation of histones at the locus. Together, these data suggest that PRMT5 is an effector of SNAIL-dependent gene repression.
doi:10.1128/MCB.01435-07
PMCID: PMC2423142  PMID: 18347060
9.  A p53/miRNA-34 axis regulates Snail1-dependent cancer cell epithelial–mesenchymal transition 
The Journal of Cell Biology  2011;195(3):417-433.
Expression of the essential EMT inducer Snail1 is inhibited by miR-34 through a p53-dependent regulatory pathway.
Snail1 is a zinc finger transcriptional repressor whose pathological expression has been linked to cancer cell epithelial–mesenchymal transition (EMT) programs and the induction of tissue-invasive activity, but pro-oncogenic events capable of regulating Snail1 activity remain largely uncharacterized. Herein, we demonstrate that p53 loss-of-function or mutation promotes cancer cell EMT by de-repressing Snail1 protein expression and activity. In the absence of wild-type p53 function, Snail1-dependent EMT is activated in colon, breast, and lung carcinoma cells as a consequence of a decrease in miRNA-34 levels, which suppress Snail1 activity by binding to highly conserved 3′ untranslated regions in Snail1 itself as well as those of key Snail1 regulatory molecules, including β-catenin, LEF1, and Axin2. Although p53 activity can impact cell cycle regulation, apoptosis, and DNA repair pathways, the EMT and invasion programs initiated by p53 loss of function or mutation are completely dependent on Snail1 expression. These results identify a new link between p53, miR-34, and Snail1 in the regulation of cancer cell EMT programs.
doi:10.1083/jcb.201103097
PMCID: PMC3206336  PMID: 22024162
10.  Snail2 directly represses cadherin6B during epithelial-to-mesenchymal transitions of the neural crest 
Development (Cambridge, England)  2007;134(8):1481-1490.
The neural crest, a transient population of migratory cells, forms the craniofacial skeleton and peripheral nervous system, among other derivatives in vertebrate embryos. The transcriptional repressor Snail2 is thought to be crucial for the epithelial-to-mesenchymal transition (EMT) that promotes neural crest delamination from the neural tube; however, little is known about its downstream targets. To this end, we depleted avian Snail2 in the premigratory neural crest using morpholino antisense oligonucleotides and examined effects on potential targets by quantitative PCR. Several dorsal neural tube genes were upregulated by alleviating Snail2 repression; moreover, the cell adhesion molecule cadherin6B was derepressed within 30 minutes of blocking Snail2 translation. Examination of the chick cadherin6B genomic sequence reveals that the regulatory region contains three pairs of clustered E boxes, representing putative Snail2 binding sites. Furthermore, in vivo and in vitro biochemical analyses demonstrate that Snail2 directly binds to these sites and regulates cadherin6B transcription. These results are the first to describe a direct target of Snail2 repression in vivo and in the context of the EMT that characterizes neural crest development.
doi:10.1242/dev.02834
PMCID: PMC2595139  PMID: 17344227
Snail2 (Slug); cadherin6B; Neural crest; Epithelial-to-mesenchymal transitions; E boxes
11.  Phosphorylation Regulates the Subcellular Location and Activity of the Snail Transcriptional Repressor 
Molecular and Cellular Biology  2003;23(14):5078-5089.
The Snail gene product is a transcriptional repressor of E-cadherin expression and an inducer of the epithelial-to-mesenchymal transition in several epithelial tumor cell lines. This report presents data indicating that Snail function is controlled by its intracellular location. The cytosolic distribution of Snail depended on export from the nucleus by a CRM1-dependent mechanism, and a nuclear export sequence (NES) was located in the regulatory domain of this protein. Export of Snail was controlled by phosphorylation of a Ser-rich sequence adjacent to this NES. Modification of this sequence released the restriction created by the zinc finger domain and allowed nuclear export of the protein. The phosphorylation and subcellular distribution of Snail are controlled by cell attachment to the extracellular matrix. Suspended cells presented higher levels of phosphorylated Snail and an augmented extranuclear localization with respect to cells attached to the plate. These findings show the existence in tumor cells of an effective and fine-tuning nontranscriptional mechanism of regulation of Snail activity dependent on the extracellular environment.
doi:10.1128/MCB.23.14.5078-5089.2003
PMCID: PMC162233  PMID: 12832491
12.  Central role of Snail1 in the regulation of EMT and resistance in cancer: a target for therapeutic intervention 
Snail1 is the founding member of the Snail superfamily of zinc-finger transcription factors, which also includes Snail2 (Slug) and Snail3 (Smuc). The superfamily is involved in cell differentiation and survival, two processes central in cancer research. Encoded by the SNAI1 gene located on human chromosome 20q13.2, Snail1 is composed of 264 amino acids and usually acts as a transcriptional repressor. Phosphorylation and nuclear localization of Snail1, governed by PI3K and Wnt signaling pathways crosstalk, are critical in Snail1’s regulation. Snail1 has a pivotal role in the regulation of epithelial-mesenchymal transition (EMT), the process by which epithelial cells acquire a migratory, mesenchymal phenotype, as a result of its repression of E-cadherin. Snail1-induced EMT involves the loss of E-cadherin and claudins with concomitant upregulation of vimentin and fibronectin, among other biomarkers. While essential to normal developmental processes such as gastrulation, EMT is associated with metastasis, the cancer stem cell phenotype, and the regulation of chemo and immune resistance in cancer. Snail1 expression is a common sign of poor prognosis in metastatic cancer, and tumors with elevated Snail1 expression are disproportionately difficult to eradicate by current therapeutic treatments. The significance of Snail1 as a prognostic indicator, its involvement in the regulation of EMT and metastasis, and its roles in both drug and immune resistance point out that Snail1 is an attractive target for tumor growth inhibition and a target for sensitization to cytotoxic drugs.
doi:10.1186/s13046-014-0062-0
PMCID: PMC4237825  PMID: 25084828
Cancer; EMT; Metastasis; Resistance; Snail; Stem cells
13.  Peroxisome Proliferator-Activated Receptor-γ Inhibits Transformed Growth of Non-Small Cell Lung Cancer Cells through Selective Suppression of Snail12 
Neoplasia (New York, N.Y.)  2010;12(3):224-234.
Work from our laboratory and others has demonstrated that activation of the nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) inhibits transformed growth of non-small cell lung cancer (NSCLC) cell lines in vitro and in vivo. We have demonstrated that activation of PPARγ promotes epithelial differentiation of NSCLC by increasing expression of E-cadherin, as well as inhibiting expression of COX-2 and nuclear factor-κB. The Snail family of transcription factors, which includes Snail (Snail1), Slug (Snail2), and ZEB1, is an important regulator of epithelial-mesenchymal transition, as well as cell survival. The goal of this study was to determine whether the biological responses to rosiglitazone, a member of the thiazolidinedione family of PPARγ activators, are mediated through the regulation of Snail family members. Our results indicate that, in two independent NSCLC cell lines, rosiglitazone specifically decreased expression of Snail, with no significant effect on either Slug or ZEB1. Suppression of Snail using short hairpin RNA silencing mimicked the effects of PPARγ activation, in inhibiting anchorage-independent growth, promoting acinar formation in three-dimensional culture, and inhibiting invasiveness. This was associated with the increased expression of E-cadherin and decreased expression of COX-2 and matrix metaloproteinases. Conversely, overexpression of Snail blocked the biological responses to rosiglitazone, increasing anchorage-independent growth, invasiveness, and promoting epithelial-mesenchymal transition. The suppression of Snail expression by rosiglitazone seemed to be independent of GSK-3 signaling but was rather mediated through suppression of extracellular signal-regulated kinase activity. These findings suggest that selective regulation of Snail may be critical in mediating the antitumorigenic effects of PPARγ activators.
PMCID: PMC2838440  PMID: 20234816
14.  Snail Recruits Ring1B to Mediate Transcriptional Repression and Cell Migration in Pancreatic Cancer Cells 
Cancer research  2014;74(16):4353-4363.
Transcriptional repressor Snail is a master regulator of epithelial–mesenchymal transition (EMT), yet the epigenetic mechanism governing Snail to induce EMT is not well understood. Here, we report that in pancreatic ductal adenocarcinoma (PDAC), elevated levels of the ubiquitin E3 ligase Ring1B and Snail, along with elevated monoubiquitination of H2A at K119 (H2AK119Ub1), are highly correlated with poor survival. Mechanistic investigations identified Ring1B as a Snail-interacting protein and showed that the carboxyl zinc fingers of Snail recruit Ring1B and its paralog Ring1A to repress its target promoters. Simultaneous depletion of Ring1A and Ring1B in pancreatic cancer cells decreased Snail binding to the target chromatin, abolished H2AK119Ub1 modification, and thereby compromised Snail-mediated transcriptional repression and cell migration. We found that Ring1B and the SNAG-associated chromatin modifier EZH2 formed distinct protein complexes with Snail and that EZH2 was required for Snail-Ring1A/B recruitment to the target promoter. Collectively, our results unravel an epigenetic mechanism underlying transcriptional repression by Snail, suggest Ring1A/B as a candidate therapeutic target, and identify H2AK119Ub1 as a potential biomarker for PDAC diagnosis and prognosis. Cancer Res; 74(16); 4353-63. ©2014 AACR
doi:10.1158/0008-5472.CAN-14-0181
PMCID: PMC4285394  PMID: 24903147
15.  Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction – A model for cross-modulation 
BMC Cancer  2009;9:235.
Background
A feature of epithelial to mesenchymal transition (EMT) relevant to tumour dissemination is the reorganization of actin cytoskeleton/focal contacts, influencing cellular ECM adherence and motility. This is coupled with the transcriptional repression of E-cadherin, often mediated by Snail1, Snail2 and Zeb1/δEF1. These genes, overexpressed in breast carcinomas, are known targets of growth factor-initiated pathways, however it is less clear how alterations in ECM attachment cross-modulate to regulate these pathways. EGF induces EMT in the breast cancer cell line PMC42-LA and the kinase inhibitor staurosporine (ST) induces EMT in embryonic neural epithelial cells, with F-actin de-bundling and disruption of cell-cell adhesion, via inhibition of aPKC.
Methods
PMC42-LA cells were treated for 72 h with 10 ng/ml EGF, 40 nM ST, or both, and assessed for expression of E-cadherin repressor genes (Snail1, Snail2, Zeb1/δEF1) and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR) and immunofluorescence +/- cycloheximide. Actin and focal contacts (paxillin) were visualized by confocal microscopy. A public database of human breast cancers was assessed for expression of Snail1 and Snail2 in relation to outcome.
Results
When PMC42-LA were treated with EGF, Snail2 was the principal E-cadherin repressor induced. With ST or ST+EGF this shifted to Snail1, with more extreme EMT and Zeb1/δEF1 induction seen with ST+EGF. ST reduced stress fibres and focal contact size rapidly and independently of gene transcription. Gene expression analysis by MT-PCR indicated that ST repressed many genes which were induced by EGF (EGFR, CAV1, CTGF, CYR61, CD44, S100A4) and induced genes which alter the actin cytoskeleton (NLF1, NLF2, EPHB4). Examination of the public database of breast cancers revealed tumours exhibiting higher Snail1 expression have an increased risk of disease-recurrence. This was not seen for Snail2, and Zeb1/δEF1 showed a reverse correlation with lower expression values being predictive of increased risk.
Conclusion
ST in combination with EGF directed a greater EMT via actin depolymerisation and focal contact size reduction, resulting in a loosening of cell-ECM attachment along with Snail1-Zeb1/δEF1 induction. This appeared fundamentally different to the EGF-induced EMT, highlighting the multiple pathways which can regulate EMT. Our findings add support for a functional role for Snail1 in invasive breast cancer.
doi:10.1186/1471-2407-9-235
PMCID: PMC2717979  PMID: 19604397
16.  Inhibiting interactions of lysine demethylase LSD1 with Snail/Slug blocks cancer cell invasion 
Cancer research  2012;73(1):235-245.
The process of epithelial-mesenchymal transition (EMT) which is required for cancer cell invasion is regulated by a family of E-box binding transcription repressors which include Snail (SNAI) and Slug (SNAI2). Snail appears to repress the expression of the EMT marker E-cadherin by epigenetic mechanisms dependent on the interaction of its N-terminal SNAG domain with chromatin modifying proteins including lysine specific demethylase 1 (LSD1/KDM1A). We assessed whether blocking Snail/Slug-LSD1 interaction by treatment with Parnate, an enzymatic inhibitor of LSD1, or TAT-SNAG, a cell-permeable peptide corresponding to the SNAG domain of Slug, suppresses the motility and invasiveness of cancer cells of different origin and genetic background. We show here that either treatment blocked Slug-dependent repression of the E-cadherin promoter and inhibited the motility and invasion of tumor cell lines without any effect on their proliferation. These effects correlated with induction of epithelial and repression of mesenchymal markers and were phenocopied by LSD1 or Slug down-regulation. Parnate treatment also inhibited bone marrow homing/engraftment of Slug-expressing K562 cells. Together, these studies support the concept that targeting Snail/Slug-dependent transcription repression complexes may lead to the development of novel drugs selectively inhibiting the invasive potential of cancer cells.
doi:10.1158/0008-5472.CAN-12-1739
PMCID: PMC3537890  PMID: 23054398
17.  Bone morphogenetic protein-7 regulates Snail signaling in carbon tetrachloride-induced fibrosis in the rat liver 
The aim of this study was to explore the molecular mechanism of the bone morphogenetic protein-7 (BMP-7) downregulation of Snail-mediated E-cadherin repression and mesenchymal-epithelial transition (MET) induction, since little is presently known about this issue. In this study, our aim was to elucidate the underlying mechanism by which cells acquire liver fibrosis characteristics after epithelial-mesenchymal transition (EMT). Cell cultures were exposed to Snail alone or in the presence of BMP-7; control cultures were exposed to medium only. The expression of the mRNA encoding α-smooth muscle actin (α-SMA), Snail and E-cadherin in rat liver epithelial cells was determined by real-time quantitative PCR (RT-PCR) and the main results were confirmed by ELISA. Cell differentiation was determined by analysis of the expression of α-SMA, Snail and E-cadherin by western blotting and co-immunoprecipitation. We demonstrated Snail-induced upregulation of mRNAs encoding α-SMA and downregulation of mRNAs encoding E-cadherin in rat liver epithelial cells when compared with unstimulated cells, and confirmed these results at the protein level. BMP-7 downregulated Snail-induced α-SMA and upregulated E-cadherin release compared with untreated and Snail-treated cells. In summary, we demonstrated that BMP-7 induces MET through decreased downregulation of Snail. In addition, Snail1 directly regulates Nanog promoter activity. Notch signaling is also involved in this process.
doi:10.3892/etm.2012.720
PMCID: PMC3494124  PMID: 23226767
bone morphogenetic protein-7; Snail; mesenchymal-epithelial transition; liver fibrosis
18.  Snail2 is an essential mediator of Twist1-induced epithelial-mesenchymal transition and metastasis 
Cancer research  2011;71(1):245-254.
To metastasize, carcinoma cells must attenuate cell-cell adhesion to disseminate into distant organs. A group of transcription factors, including Twist1, Snail1, Snail2, ZEB1, and ZEB2, have been shown to induce Epithelial-Mesenchymal Transition (EMT), thus promoting tumor dissemination. However, it is unknown whether these transcription factors function independently or coordinately to activate the EMT program. Here we report that direct induction of Snail2 is essential for Twist1 to induce EMT. Snail2 knockdown completely blocks the ability of Twist1 to suppress E-cadherin transcription. Twist1 binds to an evolutionarily conserved E-box on the proximate Snail2 promoter to induce its transcription. Snail2 induction is essential for Twist1-induced cell invasion and distant metastasis in mice. In human breast tumors, the expression of Twist1 and Snail2 is highly correlated. Together, our results show that Twist1 needs to induce Snail2 to suppress the epithelial branch of the EMT program and that Twist1 and Snail2 act together to promote EMT and tumor metastasis.
doi:10.1158/0008-5472.CAN-10-2330
PMCID: PMC3025803  PMID: 21199805
tumor metastasis; E-cadherin; Epithelial-Mesenchymal Transition; Snail2; Twist1
19.  Down-regulation of the transcription factor snail in the placentas of patients with preeclampsia and in a rat model of preeclampsia 
Background
Placental malfunction in preeclampsia is believed to be a consequence of aberrant differentiation of trophoblast lineages and changes in utero-placental oxygenation. The transcription factor Snail, a master regulator molecule of epithelial-mesenchymal transition in embryonic development and in cancer, is shown to be involved in trophoblast differentiation as well. Moreover, Snail can be controlled by oxidative stress and hypoxia. Therefore, we examined the expression of Snail and its downstream target, e-cadherin, in human normal term, preterm and preeclamptic placentas, and in pregnant rats that developed preeclampsia-like symptoms in the response to a 20-fold increase in sodium intake.
Methods
Western blotting analysis was used for comparative expression of Snail and e- cadherin in total protein extracts. Placental cells expressing Snail and e-cadherin were identified by immunohistochemical double-labeling technique.
Results
The levels of Snail protein were decreased in human preeclamptic placentas by 30% (p < 0.01) compared to normal term, and in the rat model by 40% (p < 0.001) compared to control placentas. In preterm placentas, the levels of Snail expression varied, yet there was a strong trend toward statistical significance between preterm and preeclamptic placentas. In humans, e-cadherin protein level was 30% higher in preeclamptic (p < 0.05) placentas and similarly, but not significantly (p = 0.1), high in the preterm placentas compared to normal term. In the rat model of preeclampsia, e-cadherin was increased by 60% (p < 0.01). Immunohistochemical examination of human placentas demonstrated Snail-positive staining in the nuclei of the villous trophoblasts and mesenchymal cells and in the invasive trophoblasts of the decidua. In the rat placenta, the majority of Snail positive cells were spongiotrophoblasts of the junctional zone, while in the labyrinth, Snail-positive sinusoidal giant trophoblasts cells were found in some focal areas located close to the junctional zone.
Conclusion
We demonstrated that human preeclampsia and the salt-induced rat model of preeclampsia are associated with the reduced levels of Snail protein in placenta. Down-regulation of the transcription factor Snail in placental progenitor cell lineages, either by intrinsic defects and/or by extrinsic and maternal factors, may affect normal placenta development and function and thus contribute to the pathology of preeclampsia.
doi:10.1186/1477-7827-10-15
PMCID: PMC3298516  PMID: 22360878
Preeclampsia; Placenta; Snail; Trophoblast; E-cadherin
20.  Snail-Mediated Regulation of Reactive Oxygen Species in ARCaP Human Prostate Cancer Cells 
Reactive oxygen species increases in various diseases including cancer and has been associated with induction of epithelial-mesenchymal transition (EMT), as evidenced by decrease in cell adhesion-associated molecules like E-cadherin, and increase in mesenchymal markers like vimentin. We investigated the molecular mechanisms by which Snail transcription factor, an inducer of EMT, promotes tumor aggressiveness utilizing ARCaP prostate cancer cell line. An EMT model created by Snail overexpression in ARCaP cells was associated with decreased E-cadherin and increased vimentin. Moreover, Snail-expressing cells displayed increased concentration of reactive oxygen species (ROS), specifically, superoxide and hydrogen peroxide, in vitro and in vivo. Real time PCR profiling demonstrated increased expression of oxidative stress-responsive genes, such as aldeyhyde oxidase I, in response to Snail. The ROS scavenger, N-acetyl cysteine partially reversed Snail-mediated EMT after 7 days characterized by increased E-cadherin levels and decreased ERK activity, while treatment with the MEK inhibitor, UO126, resulted in a more marked effect by 3 days, characterized by cells returning back to the epithelial morphology and increased E-cadherin. In conclusion, this study shows for the first time that Snail transcription factor can regulate oxidative stress enzymes and increase ROS-mediated EMT regulated in part by ERK activation. Therefore, Snail may be an attractive molecule for therapeutic targeting to prevent tumor progression in human prostate cancer.
doi:10.1016/j.bbrc.2010.11.044
PMCID: PMC3021188  PMID: 21093414
Snail; EMT; ROS; prostate cancer
21.  Snail controls the mesenchymal phenotype and drives erlotinib resistance in Oral epithelial and HNSCC cells 
PURPOSE
The presence of regional metastases in HNSCC patients is a common and adverse event associated with poor prognosis. Understanding the molecular mechanisms that mediate HNSCC metastasis may enable identification of novel therapeutic targets. Our recent work on human HNSCC tissues underlies Snail’s role as a molecular prognostic marker for HNSCC. Snail positivity is significantly predictive of poorly differentiated, lymphovascular invasive, as well as regionally metastatic tumors. We recently reported the role of Snail in the inflammation-induced promotion of EMT in HNSCC. However, other important Snail-dependent malignant phenotypes have not been fully explored. Here, we investigate the capacity of Snail to drive EMT in human oral epithelial cell lines, and its ability to confer drug resistance.
EXPERIMENTAL DESIGN
Snail was overexpressed HNSCC and oral epithelial cell lines. AIG assays, wound healing assays, invasion & migration assays, spheroid modeling, and cell survival assays were performed.
RESULTS
The overexpression of Snail in human HNSCC and oral epithelial cell lines drives EMT. The sole transfection of Snail confers the expression of a mesenchymal molecular signature including down-regulation of the epithelial adherens, such as E-cadherin and β-catenin, and induction of mesenchymal markers, Snail overexpressing cell lines demonstrate rapid growth in Anchorage-independent growth assays; a decreased capacity to form tight spheroids; increased resistance to erlotinib; and have an increased capacity for invasion.
CONCLUSION
Snail controls the mesenchymal phenotype and drives erlotinib resistance in HNSCC cells. Snail may prove to be a useful marker in predicting EGFR inhibitor responsiveness.
doi:10.1177/0194599812446407
PMCID: PMC4167686  PMID: 22568942
22.  Repression of Na,K-ATPase β1-Subunit by the Transcription Factor Snail in Carcinoma 
Molecular Biology of the Cell  2004;15(3):1364-1373.
The Na,K-ATPase consists of two essential α- and β-subunits and regulates the intracellular Na+ and K+ homeostasis. Although the α-subunit contains the catalytic activity, it is not active without functional β-subunit. Here, we report that poorly differentiated carcinoma cell lines derived from colon, breast, kidney, and pancreas show reduced expression of the Na,K-ATPase β1-subunit. Decreased expression of β1-subunit in poorly differentiated carcinoma cell lines correlated with increased expression of the transcription factor Snail known to down-regulate E-cadherin. Ectopic expression of Snail in well-differentiated epithelial cell lines reduced the protein levels of E-cadherin and β1-subunit and induced a mesenchymal phenotype. Reduction of Snail expression in a poorly differentiated carcinoma cell line by RNA interference increased the levels of Na,K-ATPase β1-subunit. Furthermore, Snail binds to a noncanonical E-box in the Na,K-ATPase β1-subunit promoter and suppresses its promoter activity. These results suggest that down-regulation of Na,K-ATPase β1-subunit and E-cadherin by Snail are associated with events leading to epithelial to mesenchymal transition.
doi:10.1091/mbc.E03-09-0646
PMCID: PMC363145  PMID: 14699059
23.  Loss of 4E-BP1 function induces EMT and promotes cancer cell migration and invasion via cap-dependent translational activation of snail 
Oncotarget  2014;5(15):6015-6027.
The cap-dependent translation is frequently deregulated in a variety of cancers associated with tumor progression. However, the molecular basis of the translation activation for metastatic progression of cancer remains largely elusive. Here, we demonstrate that activation of cap-dependent translation by silencing the translational repressor 4E-BP1 causes cancer epithelial cells to undergo epithelial-mesenchymal transition (EMT), which is associated with selective upregulation of the EMT inducer Snail followed by repression of E-cadherin expression and promotion of cell migratory and invasive capabilities as well as metastasis. Conversely, inhibition of cap-dependent translation by a dominant active mutant 4E-BP1 effectively downregulates Snail expression and suppresses cell migration and invasion. Furthermore, dephosphorylation of 4E-BP1 by mTORC1 inhibition or directly targeting the translation initiation also profoundly attenuates Snail expression and cell motility, whereas knockdown of 4E-BP1 or overexpression of Snail significantly rescues the inhibitory effects. Importantly, 4E-BP1-regulated Snail expression is not associated with its changes in the level of transcription or protein stability. Together, these findings indicate a novel role of 4E-BP1 in the regulation of EMT and cell motility through translational control of Snail expression and activity, and suggest that targeting cap-dependent translation may provide a promising approach for blocking Snail-mediated metastatic potential of cancer.
PMCID: PMC4171609  PMID: 24970798
4E-BP1; mTORC1; Snail; EMT; migration; invasion
24.  Snail cooperates with KrasG12D to promote pancreatic fibrosis 
Molecular cancer research : MCR  2013;11(9):1078-1087.
Patients with pancreatic cancer, which is characterized by an extensive collagen-rich fibrotic reaction, often present with metastases. A critical step in cancer metastasis is epithelial to mesenchymal transition, which can be orchestrated by Snail family of transcription factors. To understand the role of Snail (Snai1) in pancreatic cancer, we generated transgenic mice expressing Snail in the pancreas. While there was robust Snail expression, no phenotypic changes were observed. Since chronic pancreatitis can contribute to pancreatic cancer development, Snail-expressing mice were treated with cerulein to induce pancreatitis. Although there was significant tissue injury, no difference in pancreatitis was observed between control and Snail-expressing mice. As Kras mutation is necessary for tumor development in mouse models of pancreatic cancer, we generated mice expressing both mutant KrasG12D and Snail (Kras+/Snail+). Compared to control mice (Kras+/Snail−), Kras+/Snail+ mice developed acinar ectasia and more advanced acinar to ductal metaplasia. The Kras+/Snail+ mice exhibited increased fibrosis, increased pSmad2 levels and TGF-β2 expression, and activation of pancreatic stellate cells. To further understand how Snail promoted fibrosis, we established an in vitro model to examine the effect of Snail expression in pancreatic cancer cells on stellate cell collagen production. Snail expression in pancreatic cancer cells increased TGF-β2 levels and conditioned media from Snail-expressing pancreatic cancer cells increased collagen production by stellate cells. Additionally, inhibiting TGF-β signaling in stellate cells attenuated the conditioned media-induced collagen production by stellate cells. Together these results suggest that Snail contributes to pancreatic tumor development by promoting fibrotic reaction through increased TGF-β signaling.
doi:10.1158/1541-7786.MCR-12-0637
PMCID: PMC3778055  PMID: 23761168
Snail; Fibrosis; Pancreatic Cancer; Stellate Cells; TGF-β
25.  PROINFLAMMATORY MEDIATORS UP-REGULATE SNAIL IN HEAD AND NECK SQUAMOUS CELL CARCINOMA 
PURPOSE
Inflammatory cytokines have been implicated in the progression of HNSCC. Herein we investigate the mechanisms by which IL-1β might contribute to EMT in HNSCC.
EXPERIMENTAL DESIGN
We evaluated the effect of IL-1β on the molecular events of EMT in surgical specimens and HNSCC cell lines. We examined the correlation with tumor histologic features, and a SCID xenograft model was used to assess the effects of Snail overexpression.
RESULTS
COX-2-dependent pathways contribute to the modulation of E-cadherin expression in HNSCC. An inverse relationship between COX-2 and E-cadherin was demonstrated in situ by double immunohistochemical staining of human HNSCC tissue sections. Treatment of HNSCC cells with IL-1β, caused the downregulation of E-cadherin expression and upregulation of COX-2 expression. This effect was blocked in the presence of COX-2 shRNA. IL-1β -treated HNSCC cell lines demonstrated a significant decrease in E-cadherin mRNA and an increase in the mRNA expression of the transcriptional repressor Snail. IL-1β exposure led to enhanced Snail binding at the chromatin level. ShRNA-mediated knockdown of Snail interrupted the capacity of IL-1β to downregulate E-cadherin. In a SCID xenograft model, HNSCC Snail overexpressing cells demonstrated significantly increased primary and metastatic tumor burdens.
CONCLUSIONS
IL-1β modulates Snail and thereby regulates COX-2-dependent E-cadherin expression in HNSCC. This is the first report indicating the role of Snail in the inflammation-induced promotion of EMT in HNSCC. This newly defined pathway for transcriptional regulation of E-cadherin in HNSCC has important implications for targeted chemoprevention and therapy.
doi:10.1158/1078-0432.CCR-09-0011
PMCID: PMC2782528  PMID: 19789323

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