Snail-expressing Cells Research Articles

Abstract 5390: Exploring signaling pathways of tumor-nerve interactions in prostate and breast cancer

Abstract A significant portion of prostate cancer (PCa) and breast cancer (BCa) patients develop bone metastatic disease. In some cases, cancer spreads through the nervous system, a process known as perineural invasion (PNI). Neurite outgrowth is believed to be a precursor to PNI. Snail is an important gene which regulates the epithelial-mesenchymal transition (EMT) process in which tumor cells at the invasive front undergo this transition to promote invasion, migration, and subsequent metastasis. We recently published that Snail promotes neurite outgrowth in PCa cells. We hypothesize that Snail expression will stimulate neurite outgrowth in both PCa and BCa cells through extracellular vesicles from cancer cells interacting with neurons. To test this hypothesis, we first collected conditioned media from PCa and BCa cells expressing Snail and isolated exosomes. Exosomal markers were detected using western blot to confirm the isolated exosomes. Additionally, we used Transmission Electron Microscopy (TEM) to observe the secretion of exosomes. Proteomics was performed to analyze proteins expressed within exosomes of LNCaP Snail overexpressing or C4-2 Snail knockdown PCa cells. Our results showed that Snail-expressing cells secrete exosomes containing Talin1, specifically, the proteolyzed C-terminal rod domain, while full-length Talin1 is found in whole cell lysates from Snail-expressing cells. Talin1 has previously been associated with neurite outgrowth. Neurite outgrowth assays were then performed using conditioned medium collected from C4-2 PCa cells with Snail knockdown and MCF-7 BCa cells with Snail overexpression. Increased neurite outgrowth is observed in PC-12 cells and NPC (Induced Pluripotent Stem Cells differentiated neuronal progenitor cells) when cultured with conditioned medium collected from PCa and BCa cells expressing high levels of Snail. AKT activation was observed in the neuronal cells in response to conditioned media from Snail-expressing cells. Furthermore, we found that mH4, a proprietary small molecule inhibitor of Talin1, reduces Snail-mediated neurite outgrowth and AKT activation (p-AKT Thr308 and Ser473). Overall, we have uncovered a pathway whereby Snail transcription factor promotes secretion of exosomes containing Talin1 which promotes AKT signaling in neuronal cells and neurite outgrowth. Talin1 small molecule inhibitor shows promise for therapeutic targeting of tumor-nerve interactions. Acknowledgements: The authors acknowledge the use of core facilities supported by the National Institute on Minority Health and Health Disparities through grant number 5U54MD013376 and 1U54GM128729-01-DaCCoTA Scholars Program (PI: Rezvani) National Institute of General Medical Sciences (NIGMS). Citation Format: Bor-Jang Hwang, Gabrielle J. Edwards, Cole Davis Knoblich, Khosrow Rezvani, Valerie Odero-Marah. Exploring signaling pathways of tumor-nerve interactions in prostate and breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5390.

Snail maintains the stem/progenitor state of skin epithelial cells and carcinomas through the autocrine effect of matricellular protein Mindin.

Preservation of a small population of cancer stem cells (CSCs) within a heterogeneous carcinoma serves as a paradigm to understand how select cells in a tissue maintain their undifferentiated status. In both embryogenesis and cancer, Snail has been correlated with stemness, but the molecular underpinning of this phenomenon remains largely ill-defined. In models of cutaneous squamous cell carcinoma (cSCC), we discovered a non-epithelial-mesenchymal transition function for the transcription factor Snail in maintaining the stemness of epidermal keratinocytes. Snail-expressing cells secrete the matricellular protein Mindin, which functions in an autocrine fashion to activate a Src-STAT3 pathway to reinforce their stem/progenitor phenotype. This pathway is activated by the engagement of Mindin with the leukocyte-specific integrin, CD11b (ITGAM), which is also unexpectedly expressed by epidermal keratinocytes. Interestingly, disruption of this signaling module in human cSCC attenuates tumorigenesis, suggesting that targeting Mindin would be a promising therapeutic approach to hinder cancer recurrence.

Functional Alterations of Multidrug Resistance-Associated Proteins 2 and 5, and Breast Cancer Resistance Protein upon Snail-Induced Epithelial-Mesenchymal Transition in HCC827 Cells.

Our previous report indicated that Snail-induced epithelial-mesenchymal transition (EMT) enhanced P-glycoprotein (P-gp) function and drug resistance to P-gp substrate anticancer drug in a human non-small cell lung cancer (NSCLC) cell line, HCC827. Our objective is to evaluate the changes in the mRNA and protein expression levels and the functions of multidrug resistance-associated protein (MRP) 2, MRP5 and breast cancer resistance protein (BCRP). Snail-expressing HCC827 cells showed increased mRNA levels of Snail and a mesenchymal marker vimentin, and decreased mRNA levels of an epithelial marker E-cadherin after transduction, indicating that Snail had induced EMT consistent with our previous reports. The mRNA level of MRP2 was significantly decreased, while that of MRP5 remained unchanged, in Snail-expressing cells. The expression levels of MRP2 and MRP5 proteins in whole-cell homogenate were unchanged in Snail-expressing cells, but MRP5 protein showed significantly increased membrane localization. Snail-transduction increased the efflux transport of 5-(and-6)-carboxy-2',7'-dichlorofluorescein (CDCF), a substrate of MRP2, 3 and 5. This increase was blocked by MK571, which inhibits MRP1, 2, and 5. Toxicity of cisplatin, a substrate of MRP2 and 5, was significantly decreased in Snail-expressing cells. BCRP mRNA and protein levels were both decreased in Snail-expressing cells, which showed an increase in the intracellular accumulation of 7-ethyl-10-hydroxycamptothecin (SN-38), a BCRP substrate, resulting in reduced viability. These results suggested that MRP5 function appears to be increased via an increase in membrane localization, whereas the BCRP function is decreased via a decrease in the expression level in HCC827 cells with Snail-induced EMT.

Snail levels control the migration mechanism of mesenchymal tumor cells.

Cancer cells use two major types of movement: Mesenchymal, which is typical of cells of mesenchymal origin and depends on matrix metalloproteinase (MMP) activity, and amoeboid, which is characteristic of cells with a rounded shape and relies on the activity of Rho-associated kinase (ROCK). The present authors previously demonstrated that, during neoplastic transformation, telomerase-immortalized human fibroblasts (cen3tel cells) acquired a ROCK-dependent/MMP independent mechanism of invasion, mediated by the downregulation of the ROCK cellular inhibitor Round (Rnd)3/RhoE. In the present study, cen3tel transformation was also demonstrated to be paralleled by downregulation of Snail, a major determinant of the mesenchymal movement. To test whether Snail levels could determine the type of movement adopted by mesenchymal tumor cells, Snail was ectopically expressed in tumorigenic cells. It was observed that ectopic Snail did not increase the levels of typical mesenchymal markers, but induced cells to adopt an MMP-dependent mechanism of invasion. In cells expressing ectopic Snail, invasion became sensitive to the MMP inhibitor Ro 28-2653 and insensitive to the ROCK inhibitor Y27632, suggesting that, once induced by Snail, the mesenchymal movement prevails over the amoeboid one. Snail-expressing cells had a more aggressive behavior in vivo, and exhibited increased tumor growth rate and metastatic ability. These results confirm the high plasticity of cancer cells, which can adopt different types of movement in response to changes in the expression of specific genes. Furthermore, the present findings indicate that Rnd3 and Snail are possible regulators of the type of invasion mechanism adopted by mesenchymal tumor cells.

Incremental evolution of the neural crest, neural crest cells and neural crest-derived skeletal tissues.

Urochordates (ascidians) have recently supplanted cephalochordates (amphioxus) as the extant sister taxon of vertebrates. Given that urochordates possess migratory cells that have been classified as 'neural crest-like'- and that cephalochordates lack such cells--this phylogenetic hypothesis may have significant implications with respect to the origin of the neural crest and neural crest-derived skeletal tissues in vertebrates. We present an overview of the genes and gene regulatory network associated with specification of the neural crest in vertebrates. We then use these molecular data--alongside cell behaviour, cell fate and embryonic context--to assess putative antecedents (latent homologues) of the neural crest or neural crest cells in ascidians and cephalochordates. Ascidian migratory mesenchymal cells--non-pigment-forming trunk lateral line cells and pigment-forming 'neural crest-like cells' (NCLC)--are unlikely latent neural crest cell homologues. Rather, Snail-expressing cells at the neural plate of border of urochordates and cephalochordates likely represent the extent of neural crest elaboration in non-vertebrate chordates. We also review evidence for the evolutionary origin of two neural crest-derived skeletal tissues--cartilage and dentine. Dentine is a bona fide vertebrate novelty, and dentine-secreting odontoblasts represent a cell type that is exclusively derived from the neural crest. Cartilage, on the other hand, likely has a much deeper origin within the Metazoa. The mesodermally derived cellular cartilages of some protostome invertebrates are much more similar to vertebrate cartilage than is the acellular 'cartilage-like' tissue in cephalochordate pharyngeal arches. Cartilage, therefore, is not a vertebrate novelty, and a well-developed chondrogenic program was most likely co-opted from mesoderm to the neural crest along the vertebrate stem. We conclude that the neural crest is a vertebrate novelty, but that neural crest cells and their derivatives evolved and diversified in a step-wise fashion--first by elaboration of neural plate border cells, then by the innovation or co-option of new or ancient metazoan cell fates.

Interplay between β1-Integrin and Rho Signaling Regulates Differential Scattering and Motility of Pancreatic Cancer Cells by Snail and Slug Proteins

The Snail family of transcription factors has been implicated in pancreatic cancer progression. We recently showed that Snail (Snai1) promotes membrane-type 1 matrix metalloproteinase (MT1-MMP)- and ERK1/2-dependent scattering of pancreatic cancer cells in three-dimensional type I collagen. In this study, we examine the role of Slug (Snai2) in regulating pancreatic cancer cell scattering in three-dimensional type I collagen. Although Slug increased MT1-MMP expression and ERK1/2 activity, Slug-expressing cells failed to scatter in three-dimensional collagen. Moreover, in contrast to Snail-expressing cells, Slug-expressing cells did not demonstrate increased collagen I binding, collagen I-driven motility, or α2β1-integrin expression. Significantly, inhibiting β1-integrin function decreased migration and scattering of Snail-expressing cells in three-dimensional collagen. As Rho GTPases have been implicated in invasion and migration, we also analyzed the contribution of Rac1 and Rho signaling to the differential migration and scattering of pancreatic cancer cells. Snail-induced migration and scattering were attenuated by Rac1 inhibition. In contrast, inhibiting Rho-associated kinase ROCK1/2 increased migration and scattering of Slug-expressing cells in three-dimensional collagen and thus phenocopied the effects of Snail in pancreatic cancer cells. Additionally, the increased migration and scattering in three-dimensional collagen of Slug-expressing cells following ROCK1/2 inhibition was dependent on β1-integrin function. Overall, these results demonstrate differential effects of Snail and Slug in pancreatic cancer and identify the interplay between Rho signaling and β1-integrin that functions to regulate the differential scattering and migration of Snail- and Slug-expressing pancreatic cancer cells.

Reciprocal Repression between Sox3 and Snail Transcription Factors Defines Embryonic Territories at Gastrulation

SummaryIn developing amniote embryos, the first epithelial-to-mesenchymal transition (EMT) occurs at gastrulation, when a subset of epiblast cells moves to the primitive streak and undergoes EMT to internalize and generate the mesoderm and the endoderm. We show that in the chick embryo this decision to internalize is mediated by reciprocal transcriptional repression of Snail2 and Sox3 factors. We also show that the relationship between Sox3 and Snail is conserved in the mouse embryo and in human cancer cells. In the embryo, Snail-expressing cells ingress at the primitive streak, whereas Sox3-positive cells, which are unable to ingress, ensure the formation of ectodermal derivatives. Thus, the subdivision of the early embryo into the two main territories, ectodermal and mesendodermal, is regulated by changes in cell behavior mediated by the antagonistic relationship between Sox3 and Snail transcription factors.

Pancreatic Cancer Cells Respond to Type I Collagen by Inducing Snail Expression to Promote Membrane Type 1 Matrix Metalloproteinase-dependent Collagen Invasion

Pancreatic ductal adenocarcinoma (PDAC) is characterized by pronounced fibrotic reaction composed primarily of type I collagen. Although type I collagen functions as a barrier to invasion, pancreatic cancer cells have been shown to respond to type I collagen by becoming more motile and invasive. Because epithelial-mesenchymal transition is also associated with cancer invasion, we examined the extent to which collagen modulated the expression of Snail, a well known regulator of epithelial-mesenchymal transition. Relative to cells grown on tissue culture plastic, PDAC cells grown in three-dimensional collagen gels induced Snail. Inhibiting the activity or expression of the TGF-β type I receptor abrogated collagen-induced Snail. Downstream of the receptor, we showed that Smad3 and Smad4 were critical for the induction of Snail by collagen. In contrast, Smad2 or ERK1/2 was not involved in collagen-mediated Snail expression. Overexpression of Snail in PDAC cells resulted in a robust membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14)-dependent invasion through collagen-coated transwell chambers. Snail-expressing PDAC cells also demonstrated MT1-MMP-dependent scattering in three-dimensional collagen gels. Mechanistically, Snail increased the expression of MT1-MMP through activation of ERK-MAPK signaling, and inhibiting ERK signaling in Snail-expressing cells blocked two-dimensional collagen invasion and attenuated scattering in three-dimensional collagen. To provide in vivo support for our findings that Snail can regulate MT1-MMP, we examined the expression of Snail and MT1-MMP in human PDAC tumors and found a statistically significant positive correlation between MT1-MMP and Snail in these tumors. Overall, our data demonstrate that pancreatic cancer cells increase Snail on encountering collagen-rich milieu and suggest that the desmoplastic reaction actively contributes to PDAC progression.

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 aldehyde oxidase I, in response to Snail. The ROS scavenger, N-acetyl cysteine partially reversed Snail-mediated EMT after 7days characterized by increased E-cadherin levels and decreased ERK activity, while treatment with the MEK inhibitor, UO126, resulted in a more marked effect by 3days, 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.

Abstract 2300: Overexpression of Snail induces epithelial-mesenchymal transition and cancer stem cell-like phenotypes in CRC cells

Abstract Background: Epithelial-to-mesenchymal transition (EMT) is a critical process providing tumor cells with the ability to migrate and escape from the primary tumor and metastasize to distant sites. Recently, EMT has been shown to be associated with the cancer stem cell (CSC) phenotype in breast cancer. Snail is a transcriptional factor that mediates EMT in a number of tumor types. Its aberrant expression has been found in various cancers including colorectal cancer (CRC). The role of Snail in mediating EMT and CSC function in CRC has not yet been fully elucidated. Methods: The human CRC cell lines HT29 and KM12L4 were transduced with a retrovirus containing the pBabe-puro-Snail expression construct or the empty vector. The Snail expressing cells and controls were selected with the medium containing 2µg/ml puromycin. Changes in cell proliferation and chemo-sensitivity were determined by MTT assay. Migration and invasion were determined using in vitro wound healing assay and modified Boyden Chamber assays, respectively. EMT makers (E-cadherin, vimentin) and the putative CSC marker CD133 was analyzed by Western blotting. ALDH1 enzyme activity was analyzed by the Aldefluor assay. Results: Overexpression of Snail in CRC cells induced an EMT phenotype with elevated expression of vimentin and reduced expression of E-cadherin. Snail-induced EMT did not alter cell proliferation but showed enhanced cell motility, in which Snail-expressing cells were 5- to 6-fold more migratory than the control cells. Snail also promoted the CSC-like phenotype demonstrated by increased expression of the CSC markers CD133. The aldefluor positive cell population in the Snail-expressing cells was increased ∼4 fold. Moreover, the Snail-expressing CRC cells showed increased chemoresistance to 5FU by 50%. Conclusion. High level of Snail expression confers the EMT and CSC phenotype in CRC cells, which enhanced cancer cell invasion and chemoresistance. Our data suggest that Snail may be a potential therapeutic target in metastatic colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2300.

High frequency of SNAIL-expressing cells confirms and predicts metastatic potential of phaeochromocytoma

Phaeochromocytomas are uncommon tumours of adrenal or extra-adrenal chromaffin tissue. About 2-26% of these have been reported to metastasize, but, on histological criteria, it is virtually impossible to predict malignant behaviour of the tumour. Using immunohistochemistry, we analysed the protein expression of SNAIL, a zinc-finger transcription factor, in a series of 50 phaeochromocytoma specimens from 42 patients. We found that SNAIL-expressing cells are frequent in metastatic primary tumours and their metastases, whereas in tumours without metastases, SNAIL expression is commonly absent. We conclude that the expression of SNAIL may be of use in predicting the metastatic potential of phaeochromocytoma.

Yin Yang 1 Regulates the Expression of Snail through a Distal Enhancer

Expression of the Snail gene is required for the epithelial-mesenchymal transitions that accompany mammalian gastrulation, neural crest migration, and organ formation. Pathologic expression of Snail contributes to the migratory capacity of invasive tumors, including melanomas. To investigate the mechanism of Snail up-regulation in human melanoma cells, a conserved enhancer located 3' of the Snail gene was analyzed. An overlapping Ets and yin yang 1 (YY1) consensus sequence, in addition to a SOX consensus sequence, was required for full enhancer activity. Proteins specifically binding these sequences were detected by electrophoretic mobility shift assay. The Ets/YY1 binding activity was purified by DNA-affinity chromatography and identified as YY1. Although ubiquitously expressed, YY1 was bound at the Snail 3' enhancer in vivo in Snail-expressing cells but not in cells that did not express Snail. Knockdown of YY1 in A375 cells led to decreased Snail expression. These results identify a role for YY1 in regulating transcription of Snail in melanoma cells through binding to the Snail 3' enhancer.

Snail Up-regulates Proinflammatory Mediators and Inhibits Differentiation in Oral Keratinocytes

The transcriptional repressor Snail2 is overexpressed in head and neck squamous cell carcinomas (HNSCC) relative to nonmalignant head and neck mucosal epithelium, and in locally recurrent relative to nonrecurrent HNSCCs. We investigated the mechanisms by which Snails might contribute to the pathogenesis of HNSCCs using cell biological and molecular analyses. Oral keratinocytes that expressed Snails acquired an enhanced ability to attract monocytes and to invade a dense interstitial collagen matrix. They were also found to up-regulate production of proinflammatory cytokines and cyclooxygenase-2 (COX2), which have previously been shown to correlate with malignancy. Induction of nuclear factor-kappaB transcriptional activity by Snails was weak and not sufficient to account for the elevated levels of COX2, interleukin (IL)-6, IL8, or CXCL1. In addition, expression of Snails in oral keratinocytes impaired desquamation in vitro and strongly repressed expression of both ELF3 and matriptase-1, which play important roles in the terminal differentiation of keratinocytes. Reexpression of matriptase-1 in Snail-expressing cells partially rescued desquamation. This implicates Snails as contributing to malignancy both at the early stages, by impeding terminal differentiation, and at later stages, when invasion and inflammation are important.

Snail and Sonic Hedgehog activation in neuroendocrine tumors of the ileum

The transcription factor Snail represses E-cadherin and induces epithelial-mesenchymal transition, a process also exploited by invasive cancer cells. Aberrant Hedgehog (Hh) signaling was recently observed in a variety of epithelial cancers and it has been shown that the Hh target gene Gli1 induces expression of Snail. In this study, we examined whether Snail and Sonic Hedgehog (SHH) are expressed in neuroendocrine tumors (NETs) of the ileum. Using immunohistochemistry, we found expression of Snail in 22 out of 37 (59%) of evaluated NET samples, but not in adjacent normal tissues. Snail expression was mostly restricted to the invasive front of the tumors. Six of seven liver metastases analyzed were positive for Snail. Intratumoral expression of SHH was detected in 27 out of 37 (73%) tumors. As opposed to Snail, cells expressing SHH were found to be distributed more randomly throughout the tumors. Out of 30 primary NETs, 16 (53%) showed both Snail and SHH expression. Furthermore, we found downregulation of E-cadherin in Snail-expressing cells by immunofluorescence. Real-time RT-PCR revealed conservation of the Hh target genes Gli1, Gli2, and Ptch in the pancreatic carcinoid cell line BON-1, which were downregulated upon Hh inhibition with cyclopamine. Moreover, Hh inhibition attenuated in vitro cell growth in a dose-dependent manner. In conclusion, we describe for the first time that Snail and SHH are overexpressed in a large subset of NETs of the ileum. Aberrant activation of these pathways might be involved in invasion and metastatic spread in NETs.

A 3′ Enhancer Controls Snail Expression in Melanoma Cells

The snail gene encodes a transcriptional repressor that functions during animal development and in cancer progression to promote epithelial-mesenchymal transitions. Strict spatial and temporal boundaries of Snail expression in development imply precise transcriptional control, which becomes inappropriately activated in many cancer subtypes. To gain insight into the molecular mechanism(s) governing transcriptional control of Snail, we analyze chromatin structural changes associated with Snail transcription in melanoma cells. Regardless of transcriptional status, the Snail promoter displays three constitutive DNase hypersensitive sites (HS) and a moderate level of histone H3 Lys(4) dimethylation. A robust HS is found in the 3' region of A375 melanoma cells, in which Snail is highly expressed, but is absent in cells not expressing Snail. This element is conserved throughout the mammalian lineage and strongly activates expression of a reporter in A375 and Colo829 melanoma cells, but not in keratinocytes or primary melanocytes. Activity of this enhancer is associated with enrichment of H3 Lys(4) dimethylation and H3 acetylation at both the enhancer and the promoter. Additionally, enhancer activity is associated with H3 Lys(4) trimethylation at the promoter. A physical interaction between the 3' enhancer and promoter was observed in Snail-expressing cells, demonstrating a direct role for the enhancer in Snail expression. These results suggest a model in which the Snail promoter is constitutively packaged in a poised chromatin structure that can be activated in melanoma cells by a tissue-specific enhancer, which physically contacts the promoter.

Id-1 is induced in MDCK epithelial cells by activated Erk/MAPK pathway in response to expression of the Snail and E47 transcription factors

Id-1, a member of the helix–loop–helix transcription factor family has been shown to be involved in cell proliferation, angiogenesis and invasion of many types of human cancers. We have previously shown that stable expression of E47 and Snail repressors of the E-cadherin promoter in MDCK epithelial cell line triggers epithelial mesenchymal transition (EMT) concomitantly with changes in gene expression. We show here that both factors activate the Id-1 gene promoter and induce Id-1 mRNA and protein. The upregulation of the Id-1 gene occurs through the transactivation of the promoter by the Erk/MAPK signaling pathway. Moreover, oncogenic Ras is also able to activate Id-1 promoter in MDCK cells in the absence of both E47 and Snail transcription factors. Several transcriptionally active regulatory elements have been identified in the proximal promoter, including AP-1, Sp1 and four putative E-boxes. By EMSA, we only detected an increased binding to Sp1 and AP-1 elements in E47- and Snail-expressing cells. Binding is affected by the treatment of cells with PD 98059 MEK inhibitor, suggesting that MAPK/Erk contributes to the recruitment or assembly of proteins to Id-1 promoter. Small interfering RNA directed against Sp1 reduced Id-1 expression and the upregulation of the promoter, indicating that Sp1 is required for Id-1 induction in E47- and Snail-expressing cells. Our results provide new insights into how some target genes are activated during and/or as a consequence of the EMT triggered by both E47 and Snail transcription factors.

Upregulation of MMP-9 in MDCK epithelial cell line in response to expression of the Snail transcription factor

Overexpression of the transcription factor Snail in epithelial MDCK cells promotes the epithelial-mesenchymal transition (EMT) and the acquisition of an invasive phenotype. We report here that the expression of Snail is associated with an increase in the promoter activity and expression of the matrix metalloproteinase MMP-9. The effect of Snail silencing on MMP-9 expression corroborates this finding. Induced transcription of MMP-9 by Snail is driven by a mechanism dependent on the MAPK and phosphoinositide 3-kinase (PI3K) signalling pathways. Although other regions of the promoter were required for a complete stimulation by Snail, a minimal fragment (nucleotides -97 to +114) produces a response following an increased phosphorylation of Sp-1 and either Sp-1 or Ets-1 binding to the GC-box elements contained in this region. The expression of a dominant negative form of MEK decreased these complexes. A moderate increase in the binding of the nuclear factor kappaB (NFkappaB) to the upstream region (nucleotide -562) of the MMP-9 promoter was also observed in Snail-expressing cells. Interestingly, oncogenic H-Ras (RasV12) synergistically co-operates with Snail in the induction of MMP-9 transcription and expression. Altogether, these results indicate that MMP-9 transcription is activated in response to Snail expression and that it might explain, at least in part, the invasive properties of the Snail-expressing cells.

Analysis of forkhead and snail expression reveals epithelial–mesenchymal transitions during embryonic and larval development of Nematostella vectensis

The winged helix transcription factor Forkhead and the zinc finger transcription factor Snail are crucially involved in germ layer formation in Bilateria. Here, we isolated and characterized a homolog of forkhead/ HNF3 (FoxA/group 1) and of snail from a diploblast, the sea anemone Nematostella vectensis. We show that Nematostella forkhead expression starts during late Blastula stage in a ring of cells that demarcate the blastopore margin during early gastrulation, thereby marking the boundary between ectodermal and endodermal tissue. snail, by contrast, is expressed in a complementary pattern in the center of forkhead-expressing cells marking the presumptive endodermal cells fated to ingress during gastrulation. In a significant portion of early gastrulating embryos, forkhead is expressed asymmetrically around the blastopore. While snail-expressing cells form the endodermal cell mass, forkhead marks the pharynx anlage throughout embryonic and larval development. In the primary polyp, forkhead remains expressed in the pharynx. The detailed analysis of forkhead and snail expression during Nematostella embryonic and larval development further suggests that endoderm formation results from epithelial invagination, mesenchymal immigration, and reorganization of the endodermal epithelial layer, that is, by epithelial–mesenchymal transitions (EMT) in combination with extensive morphogenetic movements. snail also governs EMT at different processes during embryonic development in Bilateria. Our data indicate that the function of snail in Diploblasts is to regulate motility and cell adhesion, supporting that the triggering of changes in cell behavior is the ancestral role of snail in Metazoa.

Snail mediates E-cadherin repression by the recruitment of the Sin3A/histone deacetylase 1 (HDAC1)/HDAC2 complex.

The transcription factor Snail has been described as a direct repressor of E-cadherin expression during development and carcinogenesis; however, the specific mechanisms involved in this process remain largely unknown. Here we show that mammalian Snail requires histone deacetylase (HDAC) activity to repress E-cadherin promoter and that treatment with trichostatin A (TSA) is sufficient to block the repressor effect of Snail. Moreover, overexpression of Snail is correlated with deacetylation of histones H3 and H4 at the E-cadherin promoter, and TSA treatment in Snail-expressing cells reverses the acetylation status of histones. Additionally, we demonstrate that Snail interacts in vivo with the E-cadherin promoter and recruits HDAC activity. Most importantly, we demonstrate an interaction between Snail, histone deacetylase 1 (HDAC1) and HDAC2, and the corepressor mSin3A. This interaction is dependent on the SNAG domain of Snail, indicating that the Snail transcription factor mediates the repression by recruitment of chromatin-modifying activities, forming a multimolecular complex to repress E-cadherin expression. Our results establish a direct causal relationship between Snail-dependent repression of E-cadherin and the modification of chromatin at its promoter.