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1.  Activation of synovial fibroblasts in rheumatoid arthritis: lack of expression of the tumour suppressor PTEN at sites of invasive growth and destruction 
Arthritis Research  1999;2(1):59-64.
In the present study, we searched for mutant PTEN transcripts in aggressive rheumatoid arthritis synovial fibroblasts (RA-SF) and studied the expression of PTEN in RA. By automated sequencing, no evidence for the presence of mutant PTEN transcripts was found. However, in situ hybridization on RA synovium revealed a distinct expression pattern of PTEN, with negligible staining in the lining layer but abundant expression in the sublining. Normal synovial tissue exhibited homogeneous staining for PTEN. In cultured RA-SF, only 40% expressed PTEN. Co-implantation of RA-SF and normal human cartilage into severe combined immunodeficiency (SCID) mice showed only limited expression of PTEN, with no staining in those cells aggressively invading the cartilage. Although PTEN is not genetically altered in RA, these findings suggest that a lack of PTEN expression may constitute a characteristic feature of activated RA-SF in the lining, and may thereby contribute to the invasive behaviour of RA-SF by maintaining their aggressive phenotype at sites of cartilage destruction.
PTEN is a novel tumour suppressor which exhibits tyrosine phosphatase activity as well as homology to the cytoskeletal proteins tensin and auxilin. Mutations of PTEN have been described in several human cancers and associated with their invasiveness and metastatic properties. Although not malignant, rheumatoid arthritis synovial fibroblasts (RA-SF) exhibit certain tumour-like features such as attachment to cartilage and invasive growth. In the present study, we analyzed whether mutant transcripts of PTEN were present in RA-SF. In addition, we used in situ hybridization to study the expression of PTEN messenger (m)RNA in tissue samples of RA and normal individuals as well as in cultured RA-SF and in the severe combined immunodeficiency (SCID) mouse model of RA.
Synovial tissue specimens were obtained from seven patients with RA and from two nonarthritic individuals. Total RNA was isolated from synovial fibroblasts and after first strand complementary (c)DNA synthesis, polymerase chain reaction (PCR) was performed to amplify a 1063 base pair PTEN fragment that encompassed the coding sequence of PTEN including the phosphatase domain and all mutation sites described so far. The PCR products were subcloned in Escherichia coli, and up to four clones were picked from each plate for automated sequencing. For in situ hybridization, digoxigenin-labelled PTEN-specific RNA probes were generated by in vitro transcription. For control in situ hybridization, a matrix metalloproteinase (MMP)-2-specific probe was prepared. To investigate the expression of PTEN in the absence of human macrophage or lymphocyte derived factors, we implanted RA-SF from three patients together with normal human cartilage under the renal capsule of SCID mice. After 60 days, mice were sacrificed, the implants removed and embedded into paraffin.
PCR revealed the presence of the expected 1063 base pair PTEN fragment in all (9/9) cell cultures (Fig. 1). No additional bands that could account for mutant PTEN variants were detected. Sequence analysis revealed 100% homology of all RA-derived PTEN fragments to those from normal SF as well as to the published GenBank sequence (accession number U93051). However, in situ hybridization demonstrated considerable differences in the expression of PTEN mRNA within the lining and the sublining layers of RA synovial membranes. As shown in Figure 2a, no staining was observed within the lining layer which has been demonstrated to mediate degradation of cartilage and bone in RA. In contrast, abundant expression of PTEN mRNA was found in the sublining of all RA synovial tissues (Figs 2a and b). Normal synovial specimens showed homogeneous staining for PTEN within the thin synovial membrane (Fig. 2c). In situ hybridization using the sense probe gave no specific staining (Fig. 2d). We also performed in situ hybridization on four of the seven cultured RA-SF and followed one cell line from the first to the sixth passage. Interestingly, only 40% of cultured RA-SF expressed PTEN mRNA (Fig. 3a), and the proportion of PTEN expressing cells did not change throughout the passages. In contrast, control experiments using a specific RNA probe for MMP-2 revealed mRNA expression by nearly all cultured cells (Fig. 3b). As seen before, implantation of RA-SF into the SCID mice showed considerable cartilage degradation. Interestingly, only negligible PTEN expression was found in those RA-SF aggressively invading the cartilage (Fig. 3c). In situ hybridization for MMP-2 showed abundant staining in these cells (Fig. 3d).
Although this study found no evidence for mutations of PTEN in RA synovium, the observation that PTEN expression is lacking in the lining layer of RA synovium as well as in more than half of cultured RA-SF is of interest. It suggests that loss of PTEN function may not exclusively be caused by genetic alterations, yet at the same time links the low expression of PTEN to a phenotype of cells that have been shown to invade cartilage aggressively.
It has been proposed that the tyrosine phosphatase activity of PTEN is responsible for its tumour suppressor activity by counteracting the actions of protein tyrosine kinases. As some studies have demonstrated an upregulation of tyrosine kinase activity in RA synovial cells, it might be speculated that the lack of PTEN expression in aggressive RA-SF contributes to the imbalance of tyrosine kinases and phosphatases in this disease. However, the extensive amino-terminal homology of the predicted protein to the cytoskeletal proteins tensin and auxilin suggests a complex regulatory function involving cellular adhesion molecules and phosphatase-mediated signalling. The tyrosine phosphatase TEP1 has been shown to be identical to the protein encoded by PTEN, and gene transcription of TEP1 has been demonstrated to be downregulated by transforming growth factor (TGF)-β. Therefore, it could be hypothesized that TGF-β might be responsible for the downregulation of PTEN. However, the expression of TGF-β is not restricted to the lining but found throughout the synovial tissue in RA. Moreover, in our study the percentage of PTEN expressing RA-SF remained stable for six passages in culture, whereas molecules that are cytokine-regulated in vivo frequently change their expression levels when cultured over several passages. Also, cultured RA-SF that were implanted into SCID mice and deeply invaded the cartilage did not show significant expression of PTEN after 60 days. The drop in the percentage of PTEN expressing cells from the original cell cultures to the SCID mouse implants is of interest as this observation goes along with data from previous studies that have shown the prominent expression of activation-related molecules in the SCID mice implants that in vivo are found predominantly in the lining layer. Therefore, our data point to endogenous mechanisms rather than to the influence of exogenous human cytokines or factors in the downregulation of PTEN. Low expression of PTEN may belong to the features that distinguish between the activated phenotype of RA-SF and the sublining, proliferating but nondestructive cells.
PMCID: PMC17804  PMID: 11219390
rheumatoid arthritis; synovial membrane; fibroblasts; PTEN tumour suppressor; severe combined immunodeficiency (SCID) mouse model; cartilage destruction; in situ hybridization
2.  Involvement of TGFβ-Induced Phosphorylation of the PTEN C-Terminus on TGFβ-Induced Acquisition of Malignant Phenotypes in Lung Cancer Cells 
PLoS ONE  2013;8(11):e81133.
Transforming growth factor β (TGFβ) derived from the tumor microenvironment induces malignant phenotypes such as epithelial-mesenchymal transition (EMT) and aberrant cell motility in lung cancers. TGFβ-induced translocation of β-catenin from E-cadherin complexes into the cytoplasm is involved in the transcription of EMT target genes. PTEN (phosphatase and tensin homologue deleted from chromosome 10) is known to exert phosphatase activity by binding to E-cadherin complexes via β-catenin, and recent studies suggest that phosphorylation of the PTEN C-terminus tail might cause loss of this PTEN phosphatase activity. However, whether TGFβ can modulate both β-catenin translocation and PTEN phosphatase activity via phosphorylation of the PTEN C-terminus remains elusive. Furthermore, the role of phosphorylation of the PTEN C-terminus in TGFβ-induced malignant phenotypes has not been evaluated. To investigate whether modulation of phosphorylation of the PTEN C-terminus can regulate malignant phenotypes, here we established lung cancer cells expressing PTEN protein with mutation of phosphorylation sites in the PTEN C-terminus (PTEN4A). We found that TGFβ stimulation yielded a two-fold increase in the phosphorylated -PTEN/PTEN ratio. Expression of PTEN4A repressed TGFβ-induced EMT and cell motility even after snail expression. Our data showed that PTEN4A might repress EMT through complete blockade of β-catenin translocation into the cytoplasm, besides the inhibitory effect of PTEN4A on TGFβ-induced activation of smad-independent signaling pathways. In a xenograft model, the tumor growth ratio was repressed in cells expressing PTEN4A. Taken together, these data suggest that phosphorylation sites in the PTEN C-terminus might be a therapeutic target for TGFβ-induced malignant phenotypes in lung cancer cells.
PMCID: PMC3838341  PMID: 24278390
3.  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.
PMCID: PMC3206336  PMID: 22024162
4.  Snail1 transcriptional repressor binds to its own promoter and controls its expression 
Nucleic Acids Research  2006;34(7):2077-2084.
The product of Snail1 gene is a transcriptional repressor of E-cadherin expression and an inductor of the epithelial–mesenchymal transition in several epithelial tumour cell lines. Transcription of Snail1 is induced when epithelial cells are forced to acquire a mesenchymal phenotype. In this work we demonstrate that Snail1 protein limits its own expression: Snail1 binds to an E-box present in its promoter (at −146 with respect to the transcription start) and represses its activity. Therefore, mutation of the E-box increases Snail1 transcription in epithelial and mesenchymal cells. Evidence of binding of ectopic or endogenous Snail1 to its own promoter was obtained by chromatin immunoprecipitation (ChIP) experiments. Studies performed expressing different forms of Snail1 under the control of its own promoter demonstrate that disruption of the regulatory loop increases the cellular levels of Snail protein. These results indicate that expression of Snail1 gene can be regulated by its product and evidence the existence of a fine-tuning feed-back mechanism of regulation of Snail1 transcription.
PMCID: PMC1440880  PMID: 16617148
5.  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.
PMCID: PMC2493371  PMID: 18519590
6.  Role of Snail Activation in Alcohol-induced iNOS-mediated Disruption of Intestinal Epithelial Cell Permeability 
Chronic alcohol use results in many pathological effects including alcoholic liver disease (ALD). ALD pathogenesis requires endotoxemia. Our previous studies showed that increased intestinal permeability is the major cause of endotoxemia and that this gut leakiness is dependent on alcohol stimulation of inducible nitric oxide synthase (iNOS) in both alcoholic subjects and rodent models of alcoholic steatohepatitis (ASH). The mechanism of the alcohol-induced, iNOS-mediated disruption of the intestinal barrier function is not known. We have recently shown that alcohol stimulates activation of the transcription factor Snail and biomarkers of epithelial mesenchymal transition. Since activated Snail disrupts tight junctional proteins , we hypothesized that activation of Snail by iNOS might be one of the key signaling pathways mediating alcohol stimulated intestinal epithelial cell hyperpermeability.
We measured intestinal permeability in alcohol-fed C57BL/6 control and iNOS KO mice and measured Snail protein expression in the intestines of these mice. We then examined intestinal epithelial permeability using the Caco-2 cell model of the intestinal barrier ± siRNA inhibition of Snail. We assessed Snail activation by alcohol in Caco-2 cells ± inhibition of iNOS with L-NIL or siRNA. Finally, we assessed Snail activation by alcohol ± inhibition with siRNA for p21-activated kinase (PAK1).
Our data show that chronic alcohol feeding promotes intestinal hyperpermeability in wild type BL/6 but not in iNOS KO mice. Snail protein expression was increased in the intestines of alcohol-treated wild type mice but not in iNOS KO mice. SiRNA inhibition of Snail significantly inhibited alcohol-induced hyperpermeability in Caco-2 cell monolayers. Alcohol stimulation of SnailpS246 activation was blocked by inhibition of iNOS with L-NIL or with siRNA. SiRNA inhibition of PAK1 significantly inhibited alcohol-mediated activation of Snail in Caco-2 cells.
Our data confirmed our prior results and further demonstrated that alcohol-induced gut leakiness in rodents and intestinal epithelial cell monolayers is iNOS dependent. Our data also support a novel role for Snail activation in alcohol-induced, iNOS mediated intestinal hyperpermeability and that PAK1 is responsible for activation of Snail at Ser246 with alcohol stimulation. Identification of these mechanisms for alcohol-induced intestinal hyperpermeability may provide new therapeutic targets for prevention and treatment of alcohol-induced leaky gut, endotoxemia and endotoxin- associated complications of alcoholism such as ALD.
PMCID: PMC3698619  PMID: 21535025
alcohol; iNOS; intestinal permeability; Snail; epithelial-mesenchymal transition
7.  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.
PMCID: PMC3494124  PMID: 23226767
bone morphogenetic protein-7; Snail; mesenchymal-epithelial transition; liver fibrosis
8.  Epithelial–Mesenchymal Transition (EMT) Induced by TNF-α Requires AKT/GSK-3β-Mediated Stabilization of Snail in Colorectal Cancer 
PLoS ONE  2013;8(2):e56664.
Chronic inflammation-promoted metastasis has been considered as a major challenge in cancer therapy. Pro-inflammatory cytokine TNFα can induce cancer invasion and metastasis associated with epithelial–mesenchymal transition (EMT). However, the underlying mechanisms are not entirely clear. In this study, we showed that TNFα induces EMT in human HCT116 cells and thereby promotes colorectal cancer (CRC) invasion and metastasis. TNFα-induced EMT was characterized by acquiring mesenchymal spindle-like morphology and increasing the expression of N-cadherin and fibronectin with a concomitant decrease of E-cadherin and Zona occludin-1(ZO-1). TNFα treatment also increased the expression of transcription factor Snail, but not Slug, ZEB1 and Twist. Overexpression of Snail induced a switch from E-cadherin to N-cadherin expression in HCT116 cells, which is a characteristic of EMT. Conversely, knockdown of Snail significantly attenuated TNFα-induced EMT in HCT116 cells, suggesting that Snail plays a crucial role in TNFα-induced EMT. Interestingly, exposure to TNFα rapidly increased Snail protein expression and Snail nuclear localization but not mRNA level upregulation. Finally, we demonstrated that TNFα elevated Snail stability by activating AKT pathway and subsequently repressing GSK-3β activity and decreasing the association of Snail with GSK-3β. Knockdown of GSK-3β further verified our finding. Taken together, these results revealed that AKT/GSK-3β-mediated stabilization of Snail is required for TNFα-induced EMT in CRC cells. Our study provides a better understanding of inflammation-induced CRC metastasis.
PMCID: PMC3576347  PMID: 23431386
9.  Snail transcription factor negatively regulates maspin tumor suppressor in human prostate cancer cells 
BMC Cancer  2012;12:336.
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.
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.
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.
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.
PMCID: PMC3437215  PMID: 22857708
Snail; Maspin; Prostate cancer
10.  Snail promotes CXCR2 ligand dependent tumor progression in NSCLC 
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.
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.
Snail up-regulation plays a role in human NSCLC by promoting tumor progression mediated by CXCR2 ligands.
PMCID: PMC2783274  PMID: 19887480
Snail; lung cancer; angiogenesis; CXCL8; CXCL5
11.  Nuclear Snail1 and nuclear ZEB1 protein expression in invasive and intraductal human breast carcinomas 
Human Pathology  2011;42(8):1125-1131.
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 expression 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.
PMCID: PMC3315281  PMID: 21315410
Snail1; ZEB1; Breast cancer; Estrogen receptor; Ductal carcinoma in situ
12.  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.
PMCID: PMC3816226  PMID: 24061576
Snail1; TGF-β1; EMT; migration; SCARA5; lung cancer
13.  Mouse Snail family transcription repressors regulate chondrocyte, extracellular matrix, type II collagen, and aggrecan 
The Journal of Biological Chemistry  2003;278(43):41862-41870.
Snail family genes are conserved among species during evolution and encode transcription factors expressed at different stages of development in different tissues. These genes are involved in a broad spectrum of biological functions; cell differentiation, cell motility, cell cycle regulation, and apoptosis. However, little is known about the target genes involved in these functions. Here we show that mouse Snail family members, Snail (Sna) and Slug (Slugh), are involved in chondrocyte differentiation by controlling the expression of type II collagen (Col2a1) and aggrecan. In situ hybridization analysis of developing mouse limb demonstrated that Snail and Slug mRNAs were highly expressed in hypertrophic chondrocytes. Inversely, the expression of collagen type II mRNA disappeared during hypertrophic differentiation. Snail and Slug mRNA expression was downregulated during differentiation of the mouse chondrogenic cell line ATDC5 and overexpression of exogenous Snail or Slug in ATDC5 cells inhibited expression of collagen type II and aggrecan mRNA. Reporter analysis revealed Snail and Slug suppressed the promoter activity of Col2a1, and the E-boxes in the promoter region were the responsible element. Gel shift assay demonstrated the binding of Snail to the E-box. Since type II collagen and aggrecan are major functional components of extracellular matrix in cartilage, these results suggest an important role for Snail-related transcription repressors during chondrocyte differentiation.
PMCID: PMC2253659  PMID: 12917416
Aggrecans; Animals; Cell Differentiation; genetics; Cell Line; Chondrocytes; cytology; metabolism; Collagen Type II; genetics; DNA-Binding Proteins; physiology; E-Box Elements; Embryo, Mammalian; Extracellular Matrix; Extracellular Matrix Proteins; Gene Expression Regulation, Developmental; Lectins, C-Type; Mice; Promoter Regions (Genetics); Proteoglycans; RNA, Messenger; analysis; Repressor Proteins; physiology; Tibia; Transcription Factors; physiology; Snail; transcription factor; chondrocyte; collagen type II; aggrecan
14.  PTEN inhibits BMI1 function independently of its phosphatase activity 
Molecular Cancer  2009;8:98.
PTEN is the second most mutated tumor suppressor gene other than p53. It suppresses tumorigenesis by dephosphorylating phosphatidylinositol (3,4,5)-triphosphate (PIP3) to phosphatidylinositol (4,5)-biphosphate (PIP2), thereby directly inhibiting phosphatidylinositol 3 kinase (PI3K)-mediated tumorigenic activities. Consistent with this model of action, cytosolic PTEN is recruited to the plasma membrane to dephosphorylate PIP3. While nuclear PTEN has been shown to suppress tumorigenesis by governing genome integrity, additional mechanisms may also contribute to nuclear PTEN-mediated tumor suppression. The nuclear protein BMI1 promotes stem cell self-renewal and tumorigenesis and PTEN inhibits these events, suggesting that PTEN may suppress BMI1 function.
We investigated whether PTEN inhibits BMI1 function during prostate tumorigenesis. PTEN binds to BMI1 exclusively in the nucleus. This interaction does not require PTEN's phosphatase activity, as phosphatase-deficient PTEN mutants, PTEN/C124S (CS), PTEN/G129E (GE), and a C-terminal PTEN fragment (C-PTEN) excluding the catalytic domain, all associate with BMI1. Furthermore, the residues 186-286 of C-PTEN are sufficient for binding to BMI1. This interaction reduces BMI1's function. BMI1 enhances hTERT activity and reduces p16INK4A and p14ARF expression. These effects were attenuated by PTEN, PTEN(CS), PTEN(GE), and C-PTEN. Furthermore, knockdown of PTEN in DU145 cells increased hTERT promoter activity, which was reversed when BMI1 was concomitantly knocked-down, indicating that PTEN reduces hTERT promoter activity via inhibiting BMI1 function. Conversely, BMI1 reduces PTEN's ability to inhibit AKT activation, which can be attributed to its interaction with PTEN in the nucleus, making PTEN unavailable to dephosphorylate membrane-bound PIP3. Furthermore, BMI1 appears to co-localize with PTEN more frequently in clinical prostate tissue samples from patients diagnosed with PIN (prostatic intraepithelial neoplasia) and carcinoma compared to normal prostate epithelium. While PTEN co-localized with BMI1 in 2.4% of normal prostate epithelial cells, co-localization was observed in 37.6% and 18.5% of cells in PIN and carcinoma, respectively. Collectively, we demonstrate that PTEN inhibits BMI1 function via binding to BMI1 in a phosphatase independent manner.
We demonstrate that nuclear PTEN reduces BMI1 function independently of its phosphatase activity. It was recently observed that nuclear PTEN also suppresses tumorigenesis. Our results, therefore, provide a plausible mechanism by which nuclear PTEN prevents tumorigenesis.
PMCID: PMC2777864  PMID: 19903340
15.  Alcohol Stimulates Activation of Snail, Epidermal Growth Factor Receptor Signaling, and Biomarkers of Epithelial-mesenchymal Transition in Colon and Breast Cancer Cells 
Alcohol consumption is associated with the risk of progressive cancers including colon and breast cancer. The mechanisms for the alcohol-induced aggressive behavior of these epithelial cancer cells have not been fully identified. Epithelial-mesenchymal transition (EMT) is a developmental program recently shown to play a role in cancer progression and metastases. We hypothesized that alcohol might promote cancer progression by inducing EMT in cancer cells and tested this hypothesis by assessing alcohol-stimulated changes in phenotypic markers of EMT as well as the EMT transcription factor Snail and its related cell signaling.
Colon and breast cancer cell lines and a normal intestinal epithelial cell line were tested as well as colonic mucosal biopsy samples from alcoholic subjects. Cells were treated with alcohol and assessed for EMT-related changes using immunofluorescent microscopy, western blotting, reporter assays, RT-PCR, and knockdown of Snail with siRNA.
We show alcohol upregulated the signature EMT phenotypic marker vimentin as well as MMP-2, MMP-7, and MMP-9 and cell migration in colon and breast cancer cells -all characteristics of EMT. Alcohol also stimulated nuclear localization of Snail phosphorylated at Ser246, transcription from a Snail reporter plasmid, and Snail mRNA expression by RT-PCR. Snail siRNA knockdown prevented alcohol-stimulated vimentin expression. In vivo, Snail expression was significantly elevated in colonic mucosal biopsies from alcoholics. Also, we found alcohol stimulated activation of epidermal growth factor receptor (EGFR) signaling and an EGFR inhibitor blocked alcohol-induced cell migration and Snail mRNA expression.
Collectively, our data support a novel mechanism for alcohol promoting cancer progression through stimulating the EMT program in cancer cells via an EGFR-Snail mediated pathway. This study reveals new pathways for alcohol-mediated promotion of cancer that could be targeted for therapy or prevention of alcohol-related cancers.
PMCID: PMC3689303  PMID: 19860811
Snail; epithelial-mesenchymal transition; epidermal growth factor receptor; alcohol; cancer
16.  Epithelial-Mesenchymal Transition Is a Critical Step in Tumorgenesis of Pancreatic Neuroendocrine Tumors  
Cancers  2012;4(1):281-294.
The transcription factors Snail, Slug and Twist repress E-cadherin and induce epithelial-mesenchymal transition (EMT), a process exploited by invasive cancer cells. In this study, we evaluated the role of EMT in the tumorgenesis of neuroendocrine tumors of the pancreas (PNETs) in vitro, in vivo and human tumor specimen. Expression of EMT markers was analyzed using immunohistochemistry and real-time PCR. For in vitro studies, BON-1 cells were analyzed regarding expression of EMT markers before and after transfection with siRNA against Slug or Snail, and cell aggregation assays were performed. To asses in vivo effects, Rip1Tag2 mice were treated with vehicle or the snail-inhibitor polythlylenglykol from week 5-10 of age. The resected pancreata were evaluated by weight, tumor cell proliferation and apoptosis. Snail and Twist was expressed in 61 % and 64% of PNETs. This was associated with loss of E-cadherin. RT-PCR revealed conservation of the EMT markers Slug and Snail in BON-1 cells. Transfection with siRNA against Slug was associated with upregulation of E-cadherin, enhanced cell-cell adhesion and inhibition of cell proliferation. Snail-inhibition in vivo by PEG was associated with increased apoptosis, decreased tumor cell proliferation and dramatic reduced tumor volume in Rip1Tag2 mice. The presented data show that EMT plays a key role in tumorgenesis of PNETs. The activation of Snail in a considerable subset of human PNETs and the successful effect of Snail inhibition by PEG in islet cell tumors of transgenic mice provides first evidence of Snail as a drug target in PNETs.
PMCID: PMC3712679  PMID: 24213240
EMT; E-cadherin; snail; slug; twist; Rip1Tag2; PEG
17.  A Somatically Diversified Defense Factor, FREP3, Is a Determinant of Snail Resistance to Schistosome Infection 
Schistosomiasis, a neglected tropical disease, owes its continued success to freshwater snails that support production of prolific numbers of human-infective cercariae. Encounters between schistosomes and snails do not always result in the snail becoming infected, in part because snails can mount immune responses that prevent schistosome development. Fibrinogen-related protein 3 (FREP3) has been previously associated with snail defense against digenetic trematode infection. It is a member of a large family of immune molecules with a unique structure consisting of one or two immunoglobulin superfamily domains connected to a fibrinogen domain; to date fibrinogen containing proteins with this arrangement are found only in gastropod molluscs. Furthermore, specific gastropod FREPs have been shown to undergo somatic diversification. Here we demonstrate that siRNA mediated knockdown of FREP3 results in a phenotypic loss of resistance to Schistosoma mansoni infection in 15 of 70 (21.4%) snails of the resistant BS-90 strain of Biomphalaria glabrata. In contrast, none of the 64 control BS-90 snails receiving a GFP siRNA construct and then exposed to S. mansoni became infected. Furthermore, resistance to S. mansoni was overcome in 22 of 48 snails (46%) by pre-exposure to another digenetic trematode, Echinostoma paraensei. Loss of resistance in this case was shown by microarray analysis to be associated with strong down-regulation of FREP3, and other candidate immune molecules. Although many factors are certainly involved in snail defense from trematode infection, this study identifies for the first time the involvement of a specific snail gene, FREP3, in the phenotype of resistance to the medically important parasite, S. mansoni. The results have implications for revealing the underlying mechanisms involved in dictating the range of snail strains used by S. mansoni, and, more generally, for better understanding the phenomena of host specificity and host switching. It also highlights the role of a diversified invertebrate immune molecule in defense against a human pathogen. It suggests new lines of investigation for understanding how susceptibility of snails in areas endemic for S. mansoni could be manipulated and diminished.
Author Summary
Schistosomiasis, a neglected tropical disease, owes its continued success to freshwater snails that support production of prolific numbers of human-infective cercariae. Encounters between schistosomes and snails do not always result in the snail becoming infected, in part because snails can mount immune responses that prevent schistosome development. Understanding the factors important for snail resistance to schistosome infection will facilitate new lines of investigation to 1) understand the underlying basis of compatibility between schistosomes and snails in endemic areas and how this affects transmission dynamics and control efforts; and 2) to reveal ways to manipulate natural snail populations to enhance their resistance to schistosome infections. Here, we present the first evidence that a snail immune molecule, fibrinogen related protein 3 (FREP3), is important for successful defense against schistosome infections in Biomphalaria snails. In addition, we demonstrate that FREP3 is a target suppressed by trematode parasites to facilitate their establishment within the snail.
PMCID: PMC3313920  PMID: 22479663
18.  Characterization of the SNAG and SLUG Domains of Snail2 in the Repression of E-Cadherin and EMT Induction: Modulation by Serine 4 Phosphorylation 
PLoS ONE  2012;7(5):e36132.
Snail1 and Snail2, two highly related members of the Snail superfamily, are direct transcriptional repressors of E-cadherin and EMT inducers. Previous comparative gene profiling analyses have revealed important differences in the gene expression pattern regulated by Snail1 and Snail2, indicating functional differences between both factors. The molecular mechanism of Snail1-mediated repression has been elucidated to some extent, but very little is presently known on the repression mediated by Snail2. In the present work, we report on the characterization of Snail2 repression of E-cadherin and its regulation by phosphorylation. Both the N-terminal SNAG and the central SLUG domains of Snail2 are required for efficient repression of the E-cadherin promoter. The co-repressor NCoR interacts with Snail2 through the SNAG domain, while CtBP1 is recruited through the SLUG domain. Interestingly, the SNAG domain is absolutely required for EMT induction while the SLUG domain plays a negative modulation of Snail2 mediated EMT. Additionally, we identify here novel in vivo phosphorylation sites at serine 4 and serine 88 of Snail2 and demonstrate the functional implication of serine 4 in the regulation of Snail2-mediated repressor activity of E-cadherin and in Snail2 induction of EMT.
PMCID: PMC3342263  PMID: 22567133
19.  Nuclear ubiquitination by FBXL5 modulates Snail1 DNA binding and stability 
Nucleic Acids Research  2013;42(2):1079-1094.
The zinc finger transcription factor Snail1 regulates epithelial to mesenchymal transition, repressing epithelial markers and activating mesenchymal genes. Snail1 is an extremely labile protein degraded by the cytoplasmic ubiquitin-ligases β-TrCP1/FBXW1 and Ppa/FBXL14. Using a short hairpin RNA screening, we have identified FBXL5 as a novel Snail1 ubiquitin ligase. FBXL5 is located in the nucleus where it interacts with Snail1 promoting its polyubiquitination and affecting Snail1 protein stability and function by impairing DNA binding. Snail1 downregulation by FBXL5 is prevented by Lats2, a protein kinase that phosphorylates Snail1 precluding its nuclear export but not its polyubiquitination. Actually, although polyubiquitination by FBXL5 takes place in the nucleus, Snail1 is degraded in the cytosol. Finally, FBXL5 is highly sensitive to stress conditions and is downregulated by iron depletion and γ-irradiation, explaining Snail1 stabilization in these conditions. These results characterize a novel nuclear ubiquitin ligase controlling Snail1 protein stability and provide the molecular basis for understanding how radiotherapy upregulates the epithelial to mesenchymal transition-inducer Snail1.
PMCID: PMC3902928  PMID: 24157836
20.  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.
PMCID: PMC2894621  PMID: 20501852
Snail; 14-3-3; Ajuba; epithelial-mesenchymal transition; transcriptional repression
21.  Tbx3 represses PTEN and is over-expressed in head and neck squamous cell carcinoma 
BMC Cancer  2012;12:481.
Despite advances in diagnostic and treatment strategies, head and neck squamous cell cancer (HNSCC) constitutes one of the worst cancer types in terms of prognosis. PTEN is one of the tumour suppressors whose expression and/or activity have been found to be reduced in HNSCC, with rather low rates of mutations within the PTEN gene (6-8%). We reasoned that low expression levels of PTEN might be due to a transcriptional repression governed by an oncogene. Tbx2 and Tbx3, both of which are transcriptional repressors, have been found to be amplified or over-expressed in various cancer types. Thus, we hypothesize that Tbx3 may be over expressed in HNSCC and may repress PTEN, thus leading to cancer formation and/or progression.
Using immunohistochemistry and quantitative PCR (qPCR), protein and mRNA levels of PTEN and Tbx3 were identified in samples excised from cancerous and adjacent normal tissues from 33 patients who were diagnosed with HNSCC. In addition, HeLa and HEK cell lines were transfected with a Tbx3 expressing plasmid and endogenous PTEN mRNA and protein levels were determined via qPCR and flow cytometry. Transcription assays were performed to demonstrate effects of Tbx3 on PTEN promoter activity. Mann–Whitney, Spearman’s Correlation and Wilcoxon signed-rank tests were used to analyze the data.
We demonstrate that in HNSCC samples, Tbx3 mRNA levels are increased with respect to their normal tissue counterparts (p<0.001), whereas PTEN mRNA levels are significantly reduced in cancer tissues. Moreover, Tbx3 protein is also increased in HNSCC tissue sections. Over-expression of Tbx3 in HeLa and HEK cell lines causes reduction in endogenous PTEN mRNA and protein levels. In addition, transcription activity assays reveal that Tbx3 is capable of repressing both the basal and induced promoter activity of PTEN.
We show that Tbx3 is up-regulated in tissue samples of HNSCC patients and that Tbx3 represses PTEN transcription. Thus, our data not only reveals a new mechanism that may be important in cancer formation, but also suggests that Tbx3 can be used as a potential biomarker in cancer.
PMCID: PMC3517435  PMID: 23082988
Tbx3; PTEN; Cancer; Squamous cell carcinoma
22.  The Role of DDX3 in Regulating Snail 
Biochimica et biophysica acta  2011;1813(3):438-447.
DDX3, a DEAD box protein family member, appears to promote the progression of some cancers, which may partly result from its impedance of death receptor-mediated apoptosis. We found that another mechanism by which DDX3 may aid cancer progression is by promoting increased levels of the transcription factor Snail. Snail represses expression of cellular adhesion proteins, leading to increased cell migration and metastasis of many types of cancer. Knockdown of DDX3 levels by shRNA reduced basal levels of Snail in HeLa cells and MCF-7 cells, and this was associated with reduced cell proliferation and migration. Snail protein and mRNA levels were increased by treatment with the HDAC inhibitors sodium butyrate or trichostatin A, and these increases were attenuated in cells with DDX3 knocked down. Treatment of cells with camptothecin was discovered to increase Snail protein levels, and this increase was diminished in cells with DDX3 knocked down. Analysis of 31 patient glioblastoma multiforme (GBM) samples revealed a significant correlation between the levels of DDX3 and Snail. Thus, DDX3 is required for basal Snail expression and increases in Snail induced by HDAC inhibitors or camptothecin, indicating that this action of DDX3 may contribute to its promotion of the progression of some cancers.
PMCID: PMC3046234  PMID: 21237216
DDX3; Snail; HDAC; camptothecin; metastasis
23.  PTEN Represses RNA Polymerase I Transcription by Disrupting the SL1 Complex†  
Molecular and Cellular Biology  2005;25(16):6899-6911.
PTEN is a tumor suppressor whose function is frequently lost in human cancer. It possesses a lipid phosphatase activity that represses the activation of PI3 kinase/Akt signaling, leading to decreased cell growth, proliferation, and survival. The potential for PTEN to regulate transcription of the large rRNAs by RNA polymerase I (RNA Pol I) was investigated. As increased synthesis of rRNAs is a hallmark of neoplastic transformation, the ability of PTEN to control the transcription of rRNAs might be crucial for its tumor suppressor function. The expression of PTEN in PTEN-deficient cells represses RNA Pol I transcription, while decreasing PTEN expression enhances transcription. PTEN-mediated repression requires its lipid phosphatase activity and is independent of the p53 status of the cell. This event can be uncoupled from PTEN′s ability to regulate the cell cycle. RNA Pol I is regulated through PI3 kinase/Akt/mammalian target of rapamycin/S6 kinase, and the expression of constitutively activated S6 kinase is able to abrogate transcription repression by PTEN. No change in the expression of the RNA Pol I transcription components, upstream binding factor or SL1, was observed upon PTEN expression. However, chromatin immunoprecipitation assays demonstrate that PTEN differentially reduces the occupancy of the SL1 subunits on the rRNA gene promoter. Furthermore, PTEN induces dissociation of the SL1 subunits. Together, these results demonstrate that PTEN represses RNA Pol I transcription through a novel mechanism that involves disruption of the SL1 complex.
PMCID: PMC1190253  PMID: 16055704
24.  PTEN status switches cell fate between premature senescence and apoptosis in glioma exposed to ionizing radiation 
Cell Death and Differentiation  2010;18(4):666-677.
Loss of the tumor suppressor phosphatase and tensin homolog (PTEN) has frequently been observed in human gliomas, conferring AKT activation and resistance to ionizing radiation (IR) and drug treatments. Recent reports have shown that PTEN loss or AKT activation induces premature senescence, but many details regarding this effect remain obscure. In this study, we tested whether the status of PTEN determined fate of the cell by examining PTEN-deficient U87, U251, and U373, and PTEN-proficient LN18 and LN428 glioma cells after exposure to IR. These cells exhibited different cellular responses, senescence or apoptosis, depending on the PTEN status. We further observed that PTEN-deficient U87 cells with high levels of both AKT activation and intracellular reactive oxygen species (ROS) underwent senescence, whereas PTEN-proficient LN18 cells entered apoptosis. ROS were indispensable for inducing senescence in PTEN-deficient cells, but not for apoptosis in PTEN-proficient cells. Furthermore, transfection with wild-type (wt) PTEN or AKT small interfering RNA induced a change from premature senescence to apoptosis and depletion of p53 or p21 prevented IR-induced premature senescence in U87 cells. Our data indicate that PTEN acts as a pivotal determinant of cell fate, regarding senescence and apoptosis in IR-exposed glioma cells. We conclude that premature senescence could have a compensatory role for apoptosis in the absence of the tumor suppressor PTEN through the AKT/ROS/p53/p21 signaling pathway.
PMCID: PMC3131905  PMID: 21072054
premature senescence; apoptosis; PTEN; glioma
25.  Snail1 Protein in the Stroma as a New Putative Prognosis Marker for Colon Tumours 
PLoS ONE  2009;4(5):e5595.
Over-expression of Snail1 gene transcriptional repressor promotes an epithelial-to-mesenchymal transition in epithelial tumour cell lines. Expression of Snail1 RNA has been associated to the pathogenesis of a number of malignancies; however, the lack of good monoclonal antibodies against this protein has precluded a definitive analysis of Snail1 protein. In this study, we aimed to determine the expression of this transcriptional factor in colorectal tumours. Using a Snail1 well-characterized monoclonal antibody developed in our laboratories we have analyzed by immunohistochemistry a cohort of 162 human colorectal tumours. Ninety tumours (56%) showed nuclear expression in the tumoral tissue and the adjacent stroma; in 34 (21%), Snail1 was detected just in the stroma, whereas in only 4 the expression of Snail1 was detected in the tumoral tissue and the stroma was negative. No correlation was found between the presence of Snail1 in the tumour and tumour stage; however, a trend (p = 0.054) was detected when the expression of this factor in the stroma was considered. Snail1 immunoreactivity in this compartment was associated with presence of distant metastasis (p = 0.006). Moreover, expression of Snail1 in the tumor stroma correlated with lower specific survival of cancer patients (p = 0.011). Interestingly, this correlation was also detected in stage I and II tumors. Therefore, our results indicate that the presence of nuclear Snail1 immunoreactive cells in the stroma may be an informative indicator of prognosis of colon tumours especially useful in those corresponding to lower stages and identify a new marker suitable to label activated stroma in colon tumours.
PMCID: PMC2680015  PMID: 19440385

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