6615

SNAI1

expression of Snail transcriptional factor is a determinant in the acquisition of a mesenchymal phenotype by epithelial tumor cells. However, the regulation of the transcription of this gene is still unknown. We describe here the characterization of a human SNAIL promoter that contains the initiation of transcription and regulates the expression of this gene in tumor cells. This promoter was activated in cell lines in response to agents that induce Snail transcription and the mesenchymal phenotype, as addition of the phorbol ester PMA or overexpression of integrin-linked kinase (ILK) or oncogenes such as Ha-ras or v-Akt. Although other regions of the promoter were required for a complete stimulation by Akt or ILK, a minimal fragment (-78/+59) was sufficient to maintain the mesenchymal specificity. Activity of this minimal promoter and SNAIL RNA levels were dependent on ERK signaling pathway. NFkappaB/p65 also stimulated SNAIL transcription through a region located immediately upstream the minimal promoter, between -194 and -78. These results indicate that Snail transcription is driven by signaling pathways known to induce epithelial to mesenchymal transition, reinforcing the role of Snail in this process.

The phenotypic changes of increased motility and invasiveness of cancer cells are reminiscent of the epithelial-mesenchymal transition (EMT) that occurs during embryonic development. Snail, a zinc-finger transcription factor, triggers this process by repressing E-cadherin expression; however, the mechanisms that regulate Snail remain elusive. Here we find that Snail is highly unstable, with a short half-life about 25 min. We show that GSK-3beta binds to and phosphorylates Snail at two consensus motifs to dually regulate the function of this protein. Phosphorylation of the first motif regulates its beta-Trcp-mediated ubiquitination, whereas phosphorylation of the second motif controls its subcellular localization. A variant of Snail (Snail-6SA), which abolishes these phosphorylations, is much more stable and resides exclusively in the nucleus to induce EMT. Furthermore, inhibition of GSK-3beta results in the upregulation of Snail and downregulation of E-cadherin in vivo. Thus, Snail and GSK-3beta together function as a molecular switch for many signalling pathways that lead to EMT.

Down-regulation of E-cadherin marks the initiation of the epithelial-mesenchymal transition, a process exploited by invasive cancer cells. The zinc finger transcription factor, Snail, functions as a potent repressor of E-cadherin expression that can, acting alone or in concert with the Wnt/beta-catenin/T cell factor axis, induce an epithelial-mesenchymal transition. Although mechanisms that coordinate signaling events initiated by Snail and Wnt remain undefined, we demonstrate that Snail displays beta-catenin-like canonical motifs that support its GSK3beta-dependent phosphorylation, beta-TrCP-directed ubiquitination, and proteasomal degradation. Accordingly, Wnt signaling inhibits Snail phosphorylation and consequently increases Snail protein levels and activity while driving an in vivo epithelial-mesenchymal transition that is suppressed following Snail knockdown. These findings define a potential mechanism whereby Wnt signaling stabilizes Snail and beta-catenin proteins in tandem fashion so as to cooperatively engage transcriptional programs that control an epithelial-mesenchymal transition.

Mechanisms for breast cancer recurrence and metastases are poorly understood. New evidence from a transgenic mouse mammary tumor model suggests that the transcriptional repressor, Snail, may play a role in recurrence by downregulating E-cadherin and inducing an epithelial-to-mesenchymal transition. Preliminary information from expression microarray data sets from primary human breast cancers suggests that high levels of Snail are correlated with poor clinical outcome for women with early breast cancer. The identification of a molecular pathway involved in mammary tumor recurrence in a mouse model offers both opportunity and challenge to confirm, extend, and exploit these findings in the clinic.

Accumulating evidence indicates that hyperactive Wnt signalling occurs in association with the development and progression of human breast cancer. As a consequence of engaging the canonical Wnt pathway, a beta-catenin-T-cell factor (TCF) transcriptional complex is generated, which has been postulated to trigger the epithelial-mesenchymal transition (EMT) that characterizes the tissue-invasive phenotype. However, the molecular mechanisms by which the beta-catenin-TCF complex induces EMT-like programmes remain undefined. Here, we demonstrate that canonical Wnt signalling engages tumour cell dedifferentiation and tissue-invasive activity through an Axin2-dependent pathway that stabilizes the Snail1 zinc-transcription factor, a key regulator of normal and neoplastic EMT programmes. Axin2 regulates EMT by acting as a nucleocytoplasmic chaperone for GSK3beta, the dominant kinase responsible for controlling Snail1 protein turnover and activity. As dysregulated Wnt signalling marks a diverse array of cancerous tissue types, the identification of a beta-catenin-TCF-regulated Axin2-GSK3beta-Snail1 axis provides new mechanistic insights into cancer-associated EMT programmes.

PURPOSE: Inflammatory cytokines have been implicated in the progression of head and neck squamous cell carcinoma (HNSCC). Herein we investigate the mechanisms by which interleukin-1beta (IL-1beta) might contribute to epithelial-mesenchymal transition (EMT) in HNSCC. EXPERIMENTAL DESIGN: We evaluated the effect of IL-1beta 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: Cyclooxygenase-2 (COX-2)-dependent pathways contribute to the modulation of E-cadherin expression in HNSCC. An inverse relationship between COX-2 and E-cadherin was shown in situ by double immunohistochemical staining of human HNSCC tissue sections. Treatment of HNSCC cells with IL-1beta caused the downregulation of E-cadherin expression and upregulation of COX-2 expression. This effect was blocked in the presence of COX-2 small hairpin RNA. IL-1beta-treated HNSCC cell lines showed a significant decrease in E-cadherin mRNA and an increase in the mRNA expression of the transcriptional repressor Snail. IL-1beta exposure led to enhanced Snail binding at the chromatin level. Small hairpin RNA-mediated knockdown of Snail interrupted the capacity of IL-1beta to downregulate E-cadherin. In a SCID xenograft model, HNSCC Snail-overexpressing cells showed significantly increased primary and metastatic tumor burdens. CONCLUSIONS: IL-1beta 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.

SNAI1, ZEB1, E-cadherin (CDH1), and vitamin D receptor (VDR) genes regulate the epithelial-mesenchymal transition (EMT) that initiates the invasion process of many tumor cells. We hypothesized that this process could also affect the behavior of normal cells adjacent to the tumor. To verify this hypothesis, the expression level of these genes was determined by quantitative RT-PCR in tumor, normal adjacent, and normal distant tissues from 32 colorectal cancer (CC) patients. In addition, we extended the study to human HaCaT normal keratinocytes and SW480-ADH colon cancer cells co-cultured with SW480-ADH cells overexpressing the mouse Snai1 gene. Of 18 CC cases with SNAI1 expression in tumor tissue, five also had SNAI1 in normal adjacent tissue (NAT). expression of SNAI1 in tumor tissue correlated with downregulation of CDH1 and VDR genes in both tumor (P=0.047 and P=0.014, respectively) and NAT lacking SNAI1 expression (P=0.054 and P=0.003). ZEB1 expression was directly related to VDR expression in tumor tissue (r=0.39; P=0.027) and inversely to CDH1 in NAT (r=-0.46; P=0.010). CDH1 and VDR were also downregulated in SW480-ADH and MaCaT cells, respectively, when they were co-cultured with Snai1-expressing cells. Furthermore, cytokine analysis showed differences in the conditioned media obtained from the two cell types. These results indicate that histologically normal tissue adjacent to tumor tissue expressing the EMT-inducing gene SNAI1 shows alterations in the expression of epithelial differentiation genes such as CDH1 and VDR.

BACKGROUND AND OBJECTIVE: epithelial-mesenchymal transition (EMT) not only initiates invasion and metastasis of tumors, but also induces multidrug resistance in tumor cells. Our experiment analyzed the dependability between breast cancer resistant protein (BCRP) and EMT in breast cancer to explore the effect of EMT on BCRP-mediated multidrug resistance. METHODS: The expressions of BCRP and transcription inhibitor Snai1 (Snail) in breast cancer were detected by immunohistochemistry. The eukaryotic expression vector pCDNA3.1-Snail was constructed and then transfected into human breast cancer cell line MCF-7. Snail, epithelial marker gene E-cadherin, interstitial marker gene Vimentin, multidrug resistance protein BCRP, and relative drug resistance were measured by immunofluorescence, Western blot, real-time polymerase chain reaction (PCR), and MTT assay. RESULTS: Immunohistochemistry showed that Snail was highly correlated with BCRP in breast cancer. Immunofluorescence, Western blot, real-time PCR revealed that compared with parent cell MCF-7, after transfected with Snail, the expression of E-cadherin in MCF-7 decreased, but Snail, Vimentin, and BCRP increased. MTT displayed that the relative drug resistance increased to 9.93. CONCLUSION: After transfected with eukaryotic expression vector pCDNA3.1-Snail, breast cancer cells MCF-7 showed EMT with BCRP-mediated multidrug resistance.

Notch signalling pathway has been implicated as an important contributor to epithelial to myofibroblast transformation (EMT) in tumourigenesis. However, its role in kidney tubular cells undergoing EMT is not defined. This study assessed Notch signalling and the downstream effects on Snail in cultured proximal tubular epithelial cells. EMT was induced by exposure to transforming growth factor beta-1 (TGFbeta(1)) and angiotensin II (AngII). The expressions of Notch1, Snail, E-cadherin and alpha-smooth muscle actin (alpha-SMA) were determined by Western blot. Matrix Metalloproteinase (MMP)-2 and -9 production were determined by zymography. The specific roles of Notch1-ICD and Snail were determined by gene expression or siRNA technique respectively. TGFbeta(1) and AngII resulted in EMT as characterized by the expected decrease in E-cadherin expression, an increase in alpha-SMA, MMP-2 and MMP-9 expression and associated increase of Notch1 and Snail. Over-expression of Notch1-ICD similarly resulted in increased Snail expression, loss of E-cadherin and increase dalpha-SMA. Inhibiting Snail degradation by pre-treatment with lithium chloride (LiCl) led to a further decrease in E-cadherin expression in cells concurrently exposed to TGFbeta(1)+AngII, confirming that Snail is a repressor of E-cadherin. Silencing of Snail blocked TGFbeta(1)+AngII induced EMT. Inhibition of Notch activation, by concurrent exposure to DAPT during the induction of EMT attenuated the decrease in E-cadherin expression, limited the increase in alpha-SMA and MMP-2 and -9 expression and decreased Snail expression. These results suggest a direct role for Notch signalling via the Snail pathway in the development of EMT and renal fibrosis.CI - Copyright 2010 Elsevier Ltd. All rights reserved.

epithelial-mesenchymal transition is an important mechanism behind initiation of cancer invasion and metastasis. This study was performed to clarify the involvement of epithelial-mesenchymal transition in the progression of cholangiocarcinoma. Cholangiocarcinoma cell lines, CCKS-1 and TFK-1, were treated with transforming growth factor-beta1 (TGF-beta1), and the phenotypic changes and invasive activity were examined. Immunohistochemical analysis was performed using tissue sections of cholangiocarcinoma. In vitro, TGF-beta1 induced mesenchymal features in CCKS-1 and TFK-1 characterized by the reduction of E-cadherin and cytokeratin 19 expression and the induction of mesenchymal markers, such as vimentin and S100A4. TGF-beta1 also induced the nuclear expression of Snail, and the invasive activity was significantly increased in both cell lines. Studies using a mouse xenograft model showed that TGF-beta1 worsened the peritoneal dissemination of CCKS-1. All these changes by TGF-beta1 were inhibited by the simultaneous administration of soluble TGF-beta type II receptor. In vivo, six (16%) of 37 cholangiocarcinoma cases showed marked immunoreactivity of Snail in their nuclei. In these six cases, the immuno-expression of cytokeratin 19 was significantly reduced, and the expression of vimentin was significantly increased. The Snail expression significantly correlated with the lymph node metastasis and a poor survival rate of the patients. These results suggest that epithelial-mesenchymal transition induced by TGF-beta1/Snail activation is closely associated with the aggressive growth of cholangiocarcinoma, resulting in a poor prognosis.

BACKGROUND: Ultraviolet (UV) irradiation from the sun is the major cause of keratinocyte skin cancer. Transcription factor Snail plays an important role in epithelial-to-mesenchymal transition and epithelial tumor formation. OBJECTIVE: The aims of this study are to determine the regulation of Snail expression of ultraviolet (UV) irradiation on Snail expression in human skin in vivo, and the mechanisms by which UV irradiation induces Snail expression, in human keratinocytes. METHODS: Real-time RT-PCR was employed to measure Snail expression in human skin in vivo and cultured human keratinocytes. Luciferase assay and electrophoretic mobility shift assay (EMSA) were employed to investigate transcriptional regulation of the in Snail gene promoter. RESULTS: Ultraviolet (UV) irradiation transiently induces Snail expression in human skin in vivo and cultured human keratinocytes. Snail induction is significantly reduced by specific inhibitors of ERK, p38 or JNK, indicating each of the three major mitogen-activated protein kinase (MAPK) pathways participate in Snail regulation. AP-1 transcription factor complex, a downstream target of MAPK signaling, is required for Snail induction. Inhibition of AP-1 activity by over-expression of dominant-negative c-Jun substantially reduces Snail induction. Analyses of the Snail promoter, revealed the presence of an AP-1 binding site. EMSA assay demonstrated that UV irradiation specifically induced c-Jun binding to this AP-1 site. mutation of the AP-1-binding site completely blocked protein binding and inhibited UV irradiation-induced Snail promoter activity. CONCLUSION: UV irradiation induces Snail gene expression in human skin keratinocytes. This induction is mediated by MAPK-AP-1 dependent signaling pathway. Elevated expression of Snail in response to chronic UV irradiation in human skin may contribute to UV irradiation-induced skin tumor development.CI - Published by Elsevier Ireland Ltd.

Protein O-phosphorylation often occurs reciprocally with O-GlcNAc modification and represents a regulatory principle for proteins. O-phosphorylation of serine by glycogen synthase kinase-3beta on Snail1, a transcriptional repressor of E-cadherin and a key regulator of the epithelial-mesenchymal transition (EMT) programme, results in its proteasomal degradation. We show that by suppressing O-phosphorylation-mediated degradation, O-GlcNAc at serine112 stabilizes Snail1 and thus increases its repressor function, which in turn attenuates E-cadherin mRNA expression. Hyperglycaemic condition enhances O-GlcNAc modification and initiates EMT by transcriptional suppression of E-cadherin through Snail1. Thus, dynamic reciprocal O-phosphorylation and O-GlcNAc modification of Snail1 constitute a molecular link between cellular glucose metabolism and the control of EMT.

The transcription factor Snail is an important repressor of E-cadherin gene expression. It plays a key role in the induction of epithelial-mesenchymal transition, an essential process important not only in embryonic development and tumor progression but also in organ fibrogenesis. We studied the expression of Snail by immunohistochemistry, along with several epithelial phenotypic changes suggestive of epithelial-mesenchymal transition, in 14 patients with multiple myeloma cast nephropathy. This nephropathy is characterized by a rapid progression toward fibrosis. As controls, we used normal kidneys and kidneys from patients displaying an idiopathic nephrotic syndrome, a syndrome unassociated with renal fibrosis. We discovered that, in all patients with multiple myeloma nephropathy, a drastic accumulation of Snail is seen in the nuclei from tubular epithelial cells showing epithelial phenotypic changes. In contrast, normal and idiopathic nephrotic syndrome kidneys did not exhibit either of these markers. Snail, a major player in the process of epithelial-to-mesenchymal transition, is highly expressed by tubular epithelial cells during multiple myeloma nephropathy. It is, therefore, a potential target to prevent multiple myeloma kidneys from fibrosing. Intranuclear accumulation of Snail is a characteristic in phenotypically altered tubular cells from multiple myeloma kidneys. The epithelial-mesenchymal transition pathway could, therefore, be involved in the rapid renal fibrogenesis observed in this setting.CI - Copyright (c) 2011 Elsevier Inc. All rights reserved.

BACKGROUND: epithelial-mesenchymal transition (EMT) underlying cancer cell invasion and metastasis has been thoroughly studied in prostate cancer. Although EMT markers have been clinically observed in benign prostate hyperplasia, molecular events underlying the onset and progression of EMT in benign prostate cells have not been described. METHODS: EMT in BPH-1 cells was induced by TGF-beta1 treatment and the kinetics of expression of EMT markers, regulators, and selected miRNAs was assessed by western blotting and quantitative RT-PCR. RESULTS: EMT in BPH-1 cells was accompanied by rapid up-regulation of SNAI2/Slug and ZEB1 transcription factors, while changes in expression levels of ZEB2 and miR-200 family members were observed after extended time intervals. Invasive phenotype with EMT hallmarks, characterizing tumorigenic clones derived from BPH-1 cells, was associated with increased mRNA levels of SNAI2, ZEB1, and ZEB2, but was not associated with significant changes in basal levels of miR-200 family members. RNA interference revealed that SNAI2/Slug is crucial for TGF-beta1-induced vimentin up-regulation and migration of BPH-1 cells. CONCLUSIONS: This study suggests that in BPH-1 cells the transcription factor SNAI2/Slug is important for EMT initiation, while the ZEB family of transcription factors in cooperation with the miR-200 family may oppose the reversal of the EMT phenotype.CI - Copyright (c) 2011 Wiley-Liss, Inc.

PURPOSE: To investigate the transcriptional factors associated with epithelial-mesenchymal transition (EMT) in choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD). METHODS: Paraffin sections of CNV obtained from patients with AMD (n = 12) were stained for transcriptional factors related to EMT, i.e., Snail, Slug, SIP1, and Twist. As a control, postmortem sections of ocular normal tissue were used. Furthermore, using a human retinal pigment epithelial (RPE) cell line (ARPE-19), reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence microscopy were performed to explore the cellular localization and expression levels of EMT-associated transcriptional factors upon cytokine stimulation. RESULTS: Of 12 specimens, 11 CNV tissues (91.6%) showed staining for Snail localized in cellular nuclei, particularly in those of RPE cells. Snail was strongly co-localized with alpha-smooth muscle antigen (SMA) in RPE cells. In contrast, postmortem human retina showed no Snail staining in RPE cells. Other transcriptional factors, Slug, Twist and SIP1 were not detected in CNV or normal human retina. In ARPE-19 cells, RT-PCR and immunofluorescence microscopy showed that Snail mRNA was upregulated by transforming growth factor (TGF)-beta and VEGF stimulation. Furthermore, TGF-beta induced relocalization of Snail to the nucleus in RPE cells. CONCLUSIONS: The current data indicate that Snail is a major transcriptional factor for EMT changes of RPE cells in human CNV.

The progression of cancer from non-metastatic to metastatic is the critical transition in the course of the disease. The epithelial to mesenchymal transition (EMT) is a mechanism by which tumor cells acquire characteristics that improve metastatic efficiency. Targeting EMT processes in patients is therefore a potential strategy to block the transition to metastatic cancer and improve patient outcome. To develop models of EMT applicable to in vitro and in vivo settings, we engineered NCI-H358 non-small cell lung carcinoma cells to inducibly express three well-established drivers of EMT: activated transforming growth factor beta (aTGFbeta), Snail or Zeb1. We characterized the morphological, molecular and phenotypic changes induced by each of the drivers and compared the different end-states of EMT between the models. Both in vitro and in vivo, induction of the transgenes Snail and Zeb1 resulted in downregulation of epithelial markers and upregulation of mesenchymal markers, and reduced the ability of the cells to proliferate. Induced autocrine expression of aTGFbeta caused marker and phenotypic changes consistent with EMT, a modest effect on growth rate, and a shift to a more invasive phenotype. In vivo, this manifested as tumor cell infiltration of the surrounding mouse stromal tissue. Overall, Snail and Zeb1 were sufficient to induce EMT in the cells, but aTGFbeta induced a more complex EMT, in which changes in extracellular matrix remodeling components were pronounced.

The novel tight junction protein marvelD3 contains a conserved MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domain like occludin and tricellulin. However, little is yet known about the detailed role and regulation of marvelD3 in normal epithelial cells and cancer cells, including pancreatic cancer. In the present study, we investigated marvelD3 expression in well and poorly differentiated human pancreatic cancer cell lines and normal pancreatic duct epithelial cells in which the hTERT gene was introduced into human pancreatic duct epithelial cells in primary culture, and the changes of marvelD3 during Snail-induced epithelial-mesenchymal transition (EMT) under hypoxia, TGF-beta treatment and knockdown of FOXA2 in well differentiated pancreatic cancer HPAC cells. MarvelD3 was transcriptionally downregulated in poorly differentiated pancreatic cancer cells and during Snail-induced EMT of pancreatic cancer cells in which Snail was highly expressed and the fence function downregulated, whereas it was maintained in well differentiated human pancreatic cancer cells and normal pancreatic duct epithelial cells. Depletion of marvelD3 by siRNAs in HPAC cells resulted in downregulation of barrier functions indicated as a decrease in transepithelial electric resistance and an increase of permeability to fluorescent dextran tracers, whereas it did not affect fence function of tight junctions. In conclusion, marvelD3 is transcriptionally downregulated in Snail-induced EMT during the progression for the pancreatic cancer.CI - Copyright (c) 2011 Elsevier Inc. All rights reserved.

epithelial mesenchymal transition (EMT) plays an important role in the development of metastases. One of the hallmarks of EMT is loss of E-cadherin and gain of N-cadherin expression, which are regulated by transcription factors, such as SNAIL, SLUG, and TWIST. We examined the prognostic value of these factors as well as E-cadherin and N-cadherin, in a well-described large cohort of breast cancer patients treated with primary surgery. Analyses were stratified by estrogen receptor (ER) status, because of its crucial role in the regulation of these transcription factors. SNAIL, SLUG, and TWIST expression were examined on a TMA containing 575 breast tumors using immunohistochemistry. Nuclear expression was quantified using a weighted histoscore and classified as high versus low expression, based on the median histoscore. High expression of SNAIL, SLUG, and TWIST was seen in 54, 50, and 50% of tumors, respectively. The level of SNAIL (P = 0.014) and TWIST (P = 0.006) expression was associated with a worse patient relapse-free period, specifically in patients with ER-positive tumors (interaction Cox proportional hazards P = 0.039). Combining both factors resulted in an independent prognostic factor with high discriminative power (both low versus either high: HR 1.15; both low versus both high HR 1.84; P = 0.010). Co-expression of SNAIL-TWIST was associated with low-E-cadherin and high-N-cadherin expression, especially in ER-positive tumors (P = 0.009), suggesting that, through interactions with ER, SNAIL and TWIST may regulate E- and N-cadherin expression, thereby inducing EMT. Our results are indicative that SNAIL and TWIST play a crucial role in EMT through regulation of E- and N-cadherin expression, exclusively in ER-positive breast cancer patients.

BACKGROUND: The proliferation of retinal pigment epithelium (RPE) cells resulting from an epithelial-mesenchymal transition (EMT) plays a key role in proliferative vitreoretinopathy (PVR), which leads to complex retinal detachment and the loss of vision. Genes of Snail family encode the zinc finger transcription factors that have been reported to be essential in EMT during embryonic development and cancer metastasis. However, the function of Snail in RPE cells undergoing EMT is largely unknown. PRINCIPAL FINDINGS: Transforming growth factor beta(TGF-beta)-1 resulted in EMT in human RPE cells (ARPE-19), which was characterized by the expected decrease in E-cadherin and Zona occludin-1(ZO-1) expression, and the increase in fibronectin and alpha-smooth muscle actin (alpha-SMA) expression, as well as the associated increase of Snail expression at both mRNA and protein levels. Furthermore, TGF-beta1 treatment caused a significant change in ARPE-19 cells morphology, with transition from a typical epithelial morphology to mesenchymal spindle-shaped. More interestingly, Snail silencing significantly attenuated TGF-beta1-induced EMT in ARPE-19 cells by decreasing the mesenchymal markers fibronectin and a-SMA and increasing the epithelial marker E-cadherin and ZO-1. Snail knockdown could effectively suppress ARPE-19 cell migration. Finally, Snail was activated in epiretinal membranes from PVR patients. Taken together, Snail plays very important roles in TGF-beta-1-induced EMT in human RPE cells and may contribute to the development of PVR. SIGNIFICANCE: Snail transcription factor plays a critical role in TGF-beta1-induced EMT in human RPE cells, which provides deep insight into the pathogenesis of human PVR disease. The specific inhibition of Snail may provide a new approach to treat and prevent PVR.

Recently, the inhibition of epithelial-mesenchymal-transition (EMT) by p53 has been described as a new mode of tumor suppression which presumably prevents metastasis. Here we report that activation of p53 down-regulates the EMT-inducing transcription factor SNAIL via induction of the miR-34a/b/c genes. Suppression of miR-34a/b/c caused up-regulation of SNAIL and cells displayed EMT markers and related features, as enhanced migration and invasion. Ectopic miR-34a induced mesenchymal-epithelial-transition (MET) and down-regulation of SNAIL, which was mediated by a conserved miR-34a/b/c seed-matching sequence in the SNAIL 3'-UTR. miR-34a also down-regulated SLUG and ZEB1, as well as the stemness factors BMI1, CD44, CD133, OLFM4 and c-MYC. Conversely, the transcription factors SNAIL and ZEB1 bound to E-boxes in the miR-34a/b/c promoters, thereby repressing miR-34a and miR-34b/c expression. Since ectopic miR-34a prevented TGF-beta-induced EMT, the repression of miR-34 genes by SNAIL and related factors is part of the EMT program. In conclusion, the frequent inactivation of p53 and/or miR-34a/b/c found in cancer may shift the equilibrium of these reciprocal regulations towards the mesenchymal state and thereby lock cells in a metastatic state.

Ovarian cancer is the fifth most common cancer among women worldwide. Detection of metastasis of ovarian cancer is crucial for diagnosis and prolongs the life of patients. This study focused on whether SNAI1 overexpression relates to invasion of ovarian cancer in vitro and in vivo. Invasion, colony formation and wound healing assays and flow cytometric analysis were performed to test the invasion and proliferation of SKOV3 ovarian cancer cells after transfection. The effect of SNAI1 on ovarian cancer in vivo was validated using a murine xenograft model. In vitro, SNAI1 upregulation led to an increased percent of CD133+ SKOV3 cells and promoted SKOV3 cell invasion and proliferation. In vivo, the SNAI1 overexpression group showed the highest rate of tumor growth compared with SNAI2 and the control group (60 and 50%, respectively). Our results show that SNAI1 expression induces an increase in the number of CD133+ cells, a change important for the epithelial to mesenchymal transition and the proliferation in ovarian cancer. It is suggested that SNAI1 may serve as a novel target for ovarian cancer prediction and therapy.

Local invasiveness and distant metastasis are critical factors that contribute to oral squamous cell carcinoma-related deaths. Increasing evidence has shown that the epithelial to mesenchymal transition (EMT) is involved in cancer progression and is associated with the 'stemness' of cancer cells. Snail is a transcriptional factor that can induce EMT and preserve stem-cell function, which may induce resistance to radio- and chemotherapies in the cells. In the present study, SCC9 cells were transfected with an empty vector or a vector encoding human Snail (SCC9-S). Overexpression of Snail induced SCC9 cells to undergo EMT, in which the cells presented a fibroblast-like appearance, downregulated the epithelial markers E-cadherin and beta-catenin, upregulated the mesenchymal marker vimentin, and associated with highly invasive and metastatic properties. Furthermore, the induction of EMT promoted cancer stem cell (CSC)-like characteristics in the SCC9-S cells, such as low proliferation, self-renewal, and CSC-like markers expression. These results indicate that overexpression of Snail induces EMT and promotes CSC-like traits in the SCC9 cells. Further understanding the role of Snail in cancer progression may reveal new targets for the prevention or therapy of oral cancers.

Aberrant activation of canonical Wingless-type MMTV integration site family (Wnt) signaling is pathognomonic of colorectal cancers (CRC) harboring functional mutations in either adenomatous polyposis coli or beta-catenin. Coincident with Wnt cascade activation, CRCs also up-regulate the expression of Wnt pathway feedback inhibitors, particularly the putative tumor suppressor, Axin2. Because Axin2 serves as a negative regulator of canonical Wnt signaling in normal cells, recent attention has focused on the utility of increasing Axin2 levels in CRCs as a means to slow tumor progression. However, rather than functioning as a tumor suppressor, we demonstrate that Axin2 acts as a potent promoter of carcinoma behavior by up-regulating the activity of the transcriptional repressor, Snail1, inducing a functional epithelial-mesenchymal transition (EMT) program and driving metastatic activity. Silencing Axin2 expression decreases Snail1 activity, reverses EMT, and inhibits CRC invasive and metastatic activities in concert with global effects on the Wnt-regulated cancer cell transcriptome. The further identification of Axin2 and nuclear Snail1 proteins at the invasive front of human CRCs supports a revised model wherein Axin2 acts as a potent tumor promoter in vivo.

epithelial-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 was shown to be associated with the cancer stem cell (CSC) phenotype in breast cancer. Snail is a transcription factor that mediates EMT in a number of tumor types, including colorectal cancer (CRC). Our study was done to determine the role of Snail in mediating EMT and CSC function in CRC. Human CRC specimens were stained for Snail expression, and human CRC cell lines were transduced with a retroviral Snail construct or vector control. Cell proliferation and chemosensitivity to oxaliplatin of the infected cells were determined by the MTT (colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Migration and invasion were determined in vitro using modified Boyden chamber assays. EMT and putative CSC markers were analyzed using Western blotting. Intravenous injection of tumor cells was done to evaluate their metastatic potential in mice. Snail was overexpressed in human CRC surgical specimens. This overexpression induced EMT and a CSC-like phenotype in human CRC cells and enhanced cell migration and invasion (P < 0.002 vs. control). Snail overexpression also led to an increase in metastasis formation in vivo (P < 0.002 vs. control). Furthermore, the Snail-overexpressing CRC cells were more chemoresistant to oxaliplatin than control cells. Increased Snail expression induces EMT and the CSC-like phenotype in CRC cells, which enhance cancer cell invasion and chemoresistance. Thus, Snail is a potential therapeutic target in metastatic CRC.

BACKGROUND: Previous research has suggested a putative utility of pancreatic (pro)enzymes in cancer treatment. The aim of the present study was to investigate the in vitro effects of a mixture of two pancreatic pro-enzymes, i.e., Chymotrypsinogen and Trypsinogen, and the enzyme Amylase on three human cancer cell lines, i.e., OE33 (derived from an oesophageal carcinoma), Panc1 (derived from a pancreatic carcinoma) and Caco-2 (derived from a colon carcinoma). RESULTS: After treatment of the three cancer cell lines with different doses of the (pro)enzymes for up to 7 days, we observed (i) growth inhibition in a dose-dependent manner, (ii) enhanced expression of beta-catenin and E-cadherin and decreased expression of several epithelial-mesenchymal transition (EMT)-associated genes, such as Vimentin, Snail and Slug, (iii) differentiation of Caco-2 cells, including the appearance of cell-specific differentiated structures such as microvilli and tight junctions, the acquisition of a more regular polygonal morphology, and an increased expression of the intestinal differentiation markers alkaline phosphatase and cytokeratin 8, and (iv) differentiation of Panc1 cells, including the formation of cell aggregates, an increment on lamellar bodies and an increased expression of the pancreatic differentiation markers glucagon and insulin. CONCLUSIONS: Our results show that the treatment of three different human cancer cell lines with pancreatic (pro)enzymes results in an enhancement of cell adhesion, an attenuation of several EMT-associated markers, and an increase in the expression of several differentiation-associated markers, suggesting the acquisition of a less malignant phenotype and a decrease in proliferative capacity due to lineage-specific cellular differentiation.

epithelial mesenchymal transition (EMT) plays a major role in cancer metastasis. Several genes have been shown to play a role in EMT, and one of these is Amplified-in-breast cancer 1 (AIB1), which has oncogenic function and is known to be amplified in breast cancer. However, the role of AIB1 in EMT remains largely undefined at the molecular level. In this study, the effect of AIB1 overexpression on the EMT of the breast cancer cell line T47D was investigated. Overexpression of AIB1 disrupted the epithelial morphology of the cells. At the same time, the cells displayed a strong metastasis and reduced level of the epithelial marker E-cadherin. In contrast, knockdown of AIB1 in T47D cells increased cell-cell adhesion and produced weak metastasis, as well as a higher level of E-cadherin expression. We proposed that the regulation of EMT by AIB1 occurred through the action of the transcription factor SNAI1, and demonstrated that such interaction required the participation of ERalpha and the presence of ERalpha-binding site on SNAI1 promoter. The expression level of E-cadherin and the extent of cell migration and invasion in SNAI1-knocked down T47D cells that overexpressed AIB1 were similar to those of T47D cells that did not overexpress AIB1 and had no SNAI1 knockdown. Taken together, these results suggested that AIB1 exerted its effect on EMT through its interaction with ERalpha, which could directly bind to the ERalpha-binding site on the SNAI1 promoter, allowing the AIB1-ERalpha complex to promote the transcription of SNAI1 and eventually led to repression of E-cadherin expression, consistent with the loss of E-cadherin being a hallmark of EMT.

The glioma-associated oncogene homolog 1 (GLI1) family of zinc finger transcription factors is the nuclear mediator of the Hedgehog pathway that regulates genes essential for various stages of tumor development and progression. However, the role and mechanism by which high expression of GLI1 contributes to the invasion and metastasis of human esophageal squamous cell cancer (ESCC) has not been fully elucidated. In the present study, we demonstrated that GLI1 was over-expressed in human ESCC tissues, especially in ESCC tissues with deep invasion and lymph-node metastasis. Moreover, GLI1 was also over-expressed in ESCC cell lines and correlated with the aggressiveness of ESCC cell lines. In addition, GLI signaling pathway agonist purmorphamine could increase the invasion and metastasis ability of ESCC cells in vitro. There is increasing evidence for the contribution of epithelial-mesenchymal transition (EMT) to ESCC invasion and metastasis, therefore we investigated GLI1's role in EMT. Our results showed that high expression of GLI1 dampened expression of E-cadherin and enhanced the expression of Vimentin, and it also improved the expression of Snail, indicative of its role in EMT occurrence. Mechanistic studies showed that down-expression of Snail reversed GLI1 activation-regulated expression of EMT markers, suggesting the role of Snail in GLI1-mediated EMT. Taken together, our results had revealed that GLI1 could participate in the invasion and metastasis of ESCC through EMT. These studies indicated that in ESCC, GLI1 could be a useful target for cancer prevention and therapy.

Although epithelial-mesenchymal transition (EMT) and the subsequent development of peritoneal fibrosis are key processes leading to the peritoneal failure related to peritoneal dialysis (PD), mechanisms underlying these processes remain largely unclear. In the present study, we found that miR-30a was significantly down-regulated in peritoneal tissues, with progressive fibrosis in patients with continuous ambulatory peritoneal dialysis and in a rat model of PD. In vitro, transforming growth factor (TGF)-beta1-induced EMT, identified by de novo expression of alpha-smooth muscle actin and a loss of E-cadherin in both human and rat peritoneal mesothelial cells, was associated with down-regulation of miR-30a but up-regulation of Snai1, suggesting a close link between miR-30a and Snai1 in TGF-beta1-induced peritoneal fibrosis. It was further demonstrated in vitro that miR-30a was able to bind the 3' untranslated region of Snai1 and overexpression of miR-30a blocked TGF-beta1-induced up-regulation of Snai1 and, therefore, inhibited EMT and collagen expression. To determine the functional role of miR-30a, we overexpressed miR-30a in the peritoneal tissue in a rat model of PD and found that overexpression of miR-30a blocked both Snai1 and EMT and inhibited peritoneal fibrosis, with improvement of peritoneal dysfunction. In conclusion, miR-30a negatively regulates Snai1-mediated EMT during peritoneal fibrosis in vitro and in vivo. Blockade of peritoneal fibrosis by overexpressing miR-30a in a rat model of PD reveals a therapeutic potential of miR-30a for peritoneal fibrosis associated with PD.CI - Copyright (c) 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Chemokine (C-C motif) ligand 18 (CCL18), which is derived from tumour-associated macrophages (TAMs), plays a critical role in promoting breast cancer metastasis via its receptor, PYK2 N-terminal domain interacting receptor 1 (Nir1). However, the molecular mechanism by which Nir1 promotes breast cancer metastasis by binding to CCL18 remains elusive. In this study, Nir1 expression was associated with lymph node and distant metastasis in patients with invasive ductal carcinoma. For the first time, we report that Nir1 binding to CCL18 promotes the phosphorylation of Akt, LIN-11, Isl1 and MEC-3 protein domain kinase (LIMK), and cofilin, which is a critical step in cofilin recycling and actin polymerisation. Interestingly, Nir1 binding to CCL18 can enhance cell mesenchymal properties and induce epithelial-mesenchymal transition (EMT). Mechanistically, Nir1 binding to CCL18 stabilises Snail via the Akt/GSK3beta signalling pathway. In support of these observations, Nir1 binding to CCL18 promoted lung metastasis and LY294002 could inhibit it in vivo. In summary, our in vitro and in vivo results indicate that Nir1 binding to CCL18 plays an important role in breast cancer invasion/metastasis. This study identified both Nir1 and CCL18 as potential anti-invasion targets for therapeutic intervention in breast cancer.CI - Copyright (c) 2013 Elsevier Ltd. All rights reserved.

Notch has recently been shown to promote epithelial-to-mesenchymal transition (EMT) by involving in the EMT process that occurs during tumor progression and converts polarized epithelial cells into motile, invasive cells. However, it is still unclear whether the Notch signaling pathway is associated with the regulation of EMT in esophageal carcinoma. The present study explored Notch-1-mediated esophageal carcinoma EC-9706 cell invasion and metastasis by inducing epithelial-mesenchymal transition through Snail. The results demonstrated that the inhibition of Notch-1 expression in the esophageal carcinoma cell line EC-9706 could suppress the occurrence of EMT and at the same time could decrease the invasion and metastasis ability of the EC-9706 cells, indicative of its role in EMT. Snail is a transcriptional repressor of E-cadherin. We found that with the inhibition of Notch-1 expression in the esophageal carcinoma cell line EC-9706, the expression of Snail also decreased. Mechanistic studies showed that the up-expression of Snail in the EC-9706 cells restored the suppression of EMT regulated by Notch-1 inhibition, suggesting the role of Snail in Notch-1-mediated EMT. At the same time, the up-expression of Snail in the EC-9706 cells could also rescue the invasion and metastasis ability inhibited by Notch-1 siRNA. Taken together, our results had revealed that Notch-1 could participate in the invasion and metastasis of esophageal carcinoma through EMT via Snail. This study indicated that Notch-1 might be a useful target for esophageal carcinoma prevention and therapy.

Airway remodeling is characterized by airway wall thickening, subepithelial fibrosis, increased smooth muscle mass, angiogenesis and an increase in mucous glands, which may lead to a chronic and obstinate asthma with pulmonary function depression. In the present study, we observed substantially thickened lung tissue with extensive fibrosis in ovalbumin-sensitized mice, which was interrelated with transforming growth factor-beta1 (TGF-beta1) expression in bronchoalveolar lavage fluid. In vitro experiments further demonstrated that TGF-beta1 resulted in epithelial-mesenchymal transition (EMT) in bronchial epithelial cells, which was characterized by the expected decrease in E-cadherin expression and the increase in vimentin and alpha-smooth muscle actin expression, as well as the associated increase in Snail expression at mRNA and protein levels. Furthermore, the downregulation of Snail by small interfering RNA (siRNA) attenuated the TGF-beta1induced EMT-like phenotype. Of note, a significantly increased synthesis of fibronectin was observed following TGF-beta1 treatment, which further supported the hypothesis that EMT is a pivotal factor in peribronchial fibrosis. In combination, the results indicated that myofibroblasts deriving from bronchial epithelial cells via EMT may contribute to peribronchial fibrosis and that Snail may be an important factor in this phenomenon.

To research the effects of silencing transcription factor SNAI1 on the in vitro biological phenotypes of breast cancer cell line MCF-7, based on the gene sequence of SNAI1, we linked shRNA with the green fluorescent protein-expressing eukaryotic expression vector pGCsilencer U6/Neo/GFP, and transfected it into MCF-7 cells. The SNAI1 gene-silencing effect was authenticated by RT-PCR and immunofluorescence. We then examined the effect of gene silencing on the expression of epithelial and mesenchymal markers and on their biological phenotypes of the target cells. Finally, we explained that SNAI1 was bound to E-cadherin in MCF-7 cells by ChIP. Silencing SNAI1 upregulated the expression of epithelial markers claudin-4, claudin-7, and E-cadherin, while expression of the mesenchymal marker matrix metalloproteinase-2 was downregulated. The capacity for proliferation, migration, and invasion was diminished. SNAI1 binds to the E-cadherin gene promoter and inhibits its transcription. We can conclude that silencing gene SNAI1 inhibits expression of properties that are associated with the malignant phenotype of MCF-7 cells and reverses the epithelial-mesenchymal transition process by regulating relevant target gene E-cadherin.

Cisplatin is a first-line chemotherapeutic agent in the treatment of non-small cell lung cancer (NSCLC), but the therapeutic effect is disappointing, partly due to drug resistance. Emerging evidence showed that chemoresistance associates with acquisition of epithelial-mesenchymal transition (EMT) phenotype and cancer stem cell-like properties. However, the underlying mechanism is not entirely clear. In this study, we showed that cisplatin-resistant A549 cells (A549/CDDP) acquire EMT phenotype associated with migratory and invasive capability. A549/CDDP cells also displayed enhanced cancer stem cell-like properties. Increased expression of transcription factor Snail, but not ZEB1, Slug and Twist, was observed in A549/CDDP cells. Knockdown of Snail reversed EMT and significantly attenuated migration, invasion and cancer stem cell-like properties of A549/CDDP cells. Conversely, overexpressed Snail in A549 cells induced EMT and cancer stem cell-like properties. Finally, we demonstrated that activated AKT signal leads to increased beta-catenin expression and subsequently up-regulates Snail in A549/CDDP cells. Taken together, these results revealed that AKT/beta-catenin/Snail signaling pathway is mechanistically associated with cancer stem cell-like properties and EMT features of A549/CDDP cells, and thus, this pathway could be a novel target for the treatment of NSCLC.CI - (c) 2013 Published by Elsevier B.V.

By fostering cell commitment to the epithelial-to-mesenchymal transition (EMT), SNAIL proteins endow cells with motility, thereby favoring the metastatic spread of tumor cells. Whether the phenotypic change additionally facilitates tumor initiation has never been addressed. Here we demonstrate that when a SNAIL protein is ectopically produced in non-transformed mammary epithelial cells, the cells are protected from anoikis and proliferate under low-adherence conditions: a hallmark of cancer cells. The three SNAIL proteins show unequal oncogenic potential, strictly correlating with their ability to promote EMT. SNAIL3 especially behaves as a poor EMT-inducer comforting the concept that the transcription factor functionally diverges from its two related proteins.

BACKGROUND: Intraductal papillary mucinous neoplasm (IPMN) is a well-established entity among pancreatic neoplasms that ranges from low-grade dysplasia to invasive carcinoma. epithelial-mesenchymal transition (EMT) contributes to tumor progression in various cancers. Moreover, Notch signaling is one of the important upstream effectors of EMT promotion. Currently, it is unclear whether EMT causes pathological progression of IPMN. AIM: We evaluated the expression of EMT-promoting transcription factors Twist and B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi1) in IPMN. METHODS: Patients who underwent resections at our institute and its affiliated hospital were enrolled in this study (n = 35). Protein expression of EMT markers Twist, Bmi1, Jagged1, and E-cadherin in resected specimens was investigated by immunohistochemistry. expression of these proteins was compared with the clinicopathological factors and patient survival. RESULTS: Positive expression of Twist and Bmi1 was observed in 40.0% and 42.9% of IPMNs, respectively. Twist and Bmi1 expression was significantly higher in IPMNs with high-grade dysplasia (P < 0.05) and invasive carcinoma (P < 0.05) than that in IPMNs with low-grade dysplasia. High expression of Twist was correlated with Jagged1 expression and inversely correlated with expression of E-cadherin (P = 0.06 and P < 0.05, respectively). In survival analyses, the recurrence rate was significantly higher in the group that showed simultaneous high expression of Twist and Bmi1 (P < 0.05). CONCLUSIONS: expression of Twist and Bmi1 is associated with aggressiveness and poor prognoses of IPMN through EMT promotion that might be induced by Notch signaling.CI - (c) 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

MicroRNA miR-199a is clustered with miR-214 on chromosome 1 and its expression is up-regulated by various factors that are associated with epithelial-to-mesenchymal transition (EMT), such as a transcriptional repressor Twist1 and transforming growth factor (TGF)-beta. miR-199a is either up-regulated or down-regulated in a variety of cancers, although EMT is associated with the progression of cancer. We found here that miR-199a suppressed the translation of SNAI1, a transcriptional repressor that plays a role in EMT, by targeting the sequence within the 3'UTR of the SNAI1 mRNA, and reduced the protein level of SNAI1. miR-199a increased the protein level of claudin-1 in both the TGF-beta1-treated and -untreated cells at least partly by decreasing the protein level of SNAI1, a transcriptional repressor for claudin-1. In addition, miR-199a targeted the sequence within the 3'UTR of the N-cadherin mRNA and suppressed the TGF-beta1-induced increase in the protein level of N-cadherin in a manner independent of SNAI1. These results indicate that miR-199a suppresses the TGF-beta1-induced protein expression of SNAI1 and N-cadherin.CI - (c) 2014 The Authors Genes to Cells (c) 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

BACKGROUND: epithelial-mesenchymal transition (EMT) plays a pivotal role in the development of metastatic cancers. Basic fibroblast growth factor (bFGF) is significantly elevated in metastatic prostate cancers, which has been mentioned mainly to induce EMT in normal cells. However, there is no description about bFGF induced EMT and its underlying mechanism in prostate cancer cells. METHODS: Western blotting, immunofluorescence and qRT-PCR assays were used to study protein or mRNA expression profiles of the EMT. Wound healing scratch, migration and invasion assays were used to test the motility of cells undergoing EMT. More methods were used to explore the underlying mechanisms. RESULTS: We demonstrated that bFGF promoted EMT and motility of human prostate cancer PC-3 cells. Both protein and mRNA expression of Snail were rapidly increased after bFGF treatment. Ectopic expression of Snail triggered EMT and enhanced cell motility in PC-3 cells, and knockdown of Snail almost abolished bFGF induced EMT, suggesting the critical role of Snail. Mechanistic study demonstrated that bFGF promoted the stability, nuclear localization and transcription of Snail by inhibiting the activity of glycogen synthase kinase 3 beta (GSK-3beta) through phosphatidylinositide 3 kinases (PI3K)/protein kinase B (AKT) signaling pathway. CONCLUSIONS: It is concluded that bFGF can promote EMT and motility of PC-3 cells, and AKT/GSK-3beta signaling pathway controls the stability, localization and transcription of Snail which is crucial for this bFGF induced EMT. GENERAL SIGNIFICANCE: To our knowledge, this is the first study to demonstrate that bFGF can induce EMT via AKT/GSK-3beta/Snail signaling pathway in prostate cancer cells.CI - Copyright (c) 2014 Elsevier B.V. All rights reserved.

Snail transcription factor is up-regulated in several cancers and associated with increased tumor migration and invasion via induction of epithelial-to-mesenchymal transition (EMT). MAPK (ERK1/2) signaling regulates cellular processes including cell motility, adhesion, and invasion. We investigated the regulation of ERK1/2 by Snail in breast cancer cells. ERK1/2 activity (p-ERK) was higher in breast cancer patient tissue as compared to normal tissue. Snail and p-ERK were increased in several breast cancer cell lines as compared to normal mammary epithelial cells. Snail knockdown in MDA-MB-231 and T47-D breast cancer cells decreased or re-localized p-ERK from the nuclear compartment to the cytoplasm. Snail overexpression in MCF-7 breast cancer cells induced EMT, increased cell migration, decreased cell adhesion and also increased tumorigenicity. Snail induced nuclear translocation of p-ERK, and the activation of its subcellular downstream effector, Elk-1. Inhibiting MAPK activity with UO126 or knockdown of ERK2 isoform with siRNA in MCF-7 Snail cells reverted EMT induced by Snail as shown by decreased Snail and vimentin expression, decreased cell migration and increased cell adhesion. Overall, our data suggest that ERK2 isoform activation by Snail in aggressive breast cancer cells leads to EMT associated with increased cell migration and decreased cell adhesion. This regulation is enhanced by positive feedback regulation of Snail by ERK2. Therefore, therapeutic targeting of ERK2 isoform may be beneficial for breast cancer.

cancer metastasis is considered a major challenge in cancer therapy. Recently, epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR) signaling has been shown to induce epithelial-mesenchymal transition (EMT) and thereby to promote cancer metastasis. However, the underlying mechanism has not been fully elucidated. We demonstrate that EGF can induce EMT in human prostate and lung cancer cells and thus promote invasion and migration. EGF-induced EMT has been characterized by the cells acquiring mesenchymal spindle-like morphology and increasing their expression of N-cadherin and fibronectin, with a concomitant decrease of E-cadherin. Both protein and mRNA expression of transcription factor Snail rapidly increases after EGF treatment. The knockdown of Snail significantly attenuates EGF-induced EMT, suggesting that Snail is crucial for this process. To determine the way that Snail is accumulated, we demonstrate (1) that EGF promotes the stability of Snail via inhibiting the activity of glycogen synthase kinase 3 beta (GSK-3beta), (2) that protein kinase C (PKC) rather than the phosphatidylinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is responsible for GSK-3beta inhibition and (3) that GSK-3beta inhibition promotes the transcription of Snail. Taken together, these results reveal that the PKC/GSK-3beta signaling pathway controls both the stability and transcription of Snail, which is crucial for EMT induced by EGF in PC-3 and A549 cells. Our study suggests a novel signaling pathway for Snail regulation and provides a better understanding of growth-factor-induced tumor EMT and metastasis.

Metastatic dissemination is often initiated by the reactivation of an embryonic development program referred to as epithelial-mesenchymal transition (EMT). The transcription factor SNAIL promotes EMT and elicits associated pathological characteristics such as invasion, metastasis, and stemness. To better understand the posttranslational regulation of SNAIL, we performed a luciferase-based, genome-wide E3 ligase siRNA library screen and identified SCF-FBXO11 as an important E3 that targets SNAIL for ubiquitylation and degradation. Furthermore, we discovered that SNAIL degradation by FBXO11 is dependent on Ser-11 phosphorylation of SNAIL by protein kinase D1 (PKD1). FBXO11 blocks SNAIL-induced EMT, tumor initiation, and metastasis in multiple breast cancer models. These findings establish the PKD1-FBXO11-SNAIL axis as a mechanism of posttranslational regulation of EMT and cancer metastasis.CI - Copyright (c) 2014 Elsevier Inc. All rights reserved.

EPHB3 is a critical cellular guidance factor in the intestinal epithelium and an important tumor suppressor in colorectal cancer (CRC) whose expression is frequently lost at the adenoma-carcinoma transition when tumor cells become invasive. The molecular mechanisms underlying EPHB3 silencing are incompletely understood. Here we show that EPHB3 expression is anti-correlated with inducers of epithelial-mesenchymal transition (EMT) in primary tumors and CRC cells. In vitro, SNAIL1 and SNAIL2, but not ZEB1, repress EPHB3 reporter constructs and compete with the stem cell factor ASCL2 for binding to an E-box motif. At the endogenous EPHB3 locus, SNAIL1 triggers the displacement of ASCL2, p300 and the Wnt pathway effector TCF7L2 and engages corepressor complexes containing HDACs and the histone demethylase LSD1 to collapse active chromatin structure, resulting in rapid downregulation of EPHB3. Beyond its impact on EPHB3, SNAIL1 deregulates markers of intestinal identity and stemness and in vitro forces CRC cells to undergo EMT with altered morphology, increased motility and invasiveness. In xenotransplants, SNAIL1 expression abrogated tumor cell palisading and led to focal loss of tumor encapsulation and the appearance of areas with tumor cells displaying a migratory phenotype. These changes were accompanied by loss of EPHB3 and CDH1 expression. Intriguingly, SNAIL1-induced phenotypic changes of CRC cells are significantly impaired by sustained EPHB3 expression both in vitro and in vivo. Altogether, our results identify EPHB3 as a novel target of SNAIL1 and suggest that disabling EPHB3 signaling is an important aspect to eliminate a roadblock at the onset of EMT processes.CI - Copyright (c) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Breast cancer is highly metastatic disease and the most lethal of the gynecologic malignancies. epithelial-to-mesenchymal is a crucial process for the invasion of epithelial tumors. Recent studies revealed that breast cancer cells that have undergone EMT acquire aggressive malignant properties, but the molecular mechanisms underlying this transition are not well-understood. In this study, we report findings that human X-box binding protein1 (XBP1) acts as a novel regulator of EMT. We found that increased expression of XBP1 was associated with the progression of breast cancer and that XBP1 protein was significantly over-expressed in matched metastatic tumor. High XBP1 protein also predicts shorter overall survival of breast cancer patients. RNA interference-mediated knockdown of XBP1 expression restored E-cadherin expression and cell-cell junction formation in breast cancer cells, suppressing cell invasion, and tumor formation. In contrast, overexpression of XBP1 decreased the expression of the epithelial marker E-cadherin but increased the mesenchymal markers in breast cancer cells. Our finding demonstrates the upregulated expression of the key EMT regulator Snail and that it mediated EMT activation and cell invasion by XBP1.CI - Copyright (c) 2014 Elsevier Inc. All rights reserved.

Accumulating evidence has indicated a large-scale regulatory network generated by 3'untranslated regions (3'UTRs) in cancer. The 3'UTRs act not only in cis but, most likely even more importantly, as trans regulators of gene expression, consequently leading to phenotypic alterations. Here, we found that ectopic expression of SNAI1 3'UTR induced migration and invasion of ovarian cancer cell line RMUG-L without significantly affecting cell viability. Additionally, SNAI1 3'UTR overexpression regulated key epithelial-to-mesenchymal transition (EMT) markers, including SNAI1, Vimentin, and E-cadherin, and functioned as a sponge for multiple migration-/invasion-related microRNAs (miRNAs) in RMUG-L cells. These findings revealed the noncoding function of SNAI1 for the first time.

Macrophages are a major component of the leukocyte infiltrate of tumors and play a pivotal role in the progression of hepatocellular carcinoma (HCC). However, the molecular mechanisms by which macrophages promote HCC invasion are poorly understood. The present study was undertaken to investigate the relationship between macrophages and epithelial-mesenchymal transition (EMT) of HCC. Double-staining immunohistochemistry was used to observe the association between macrophages and EMT markers in clinical HCC samples and it showed that EMT primarily occurred at the edge of the tumor nest, in which infiltrating macrophages were always observed. This indicated that CD68 which is a marker of macrophages, was correlated with EMT marker levels. In addition, after being cultured with macrophages for 24 h, the ability of HCC cells to migrate and invade increased, Snail and N-Cadherin expression was upregulated, and E-Cadherin was downregulated. An antibody array assay was applied to analyze the supernatant of these cultures and it demonstrated IL-8 increased significantly in the macrophage co-culture system. Finally, the role of macrophage-derived IL-8 in the invasion of HCC cells was assayed, and downstream signaling pathways were also investigated. We found that IL-8: i) may induce EMT and promote HCC cell migration and invasion and ii) is associated with the JAK2/STAT3/Snail signaling pathway. Taking together, these findings revealed that macrophages that have infiltrated tumors may induce epithelial-mesenchymal transition of HCC cells via the IL-8 activated JAK2/STAT3/Snail pathway. Thus, this may offer a potential target for developing new HCC therapies.

Here we showed that Osthole, 7-methoxy-8-(3-methyl-2-butenyl) coumarin, a bioactive coumarin derivative extracted from medicinal plants, inhibited migration, invasion, epithelial to mesenchymal transition (EMT) in androgen-independent prostate cancer (AIPC) cells in vitro and metastasis of AIPC in vivo. In patients, high Snail levels were correlated with a higher histological Gleason sum and poor survival rates. Osthole inhibited the TGF-beta/Akt/MAPK pathways, reduced Snail-DNA-binding activity and induced E-cadherin. We found that osthole decreased miR-23a-3p. Ectopic miR-23a-3p suppressed E-cadherin 3' untranslated region reporter activity and E-cadherin expression, and relieved the motility suppression caused by osthole treatment.

epithelial-mesenchymal transition (EMT) is a key process involved in the invasion and metastasis of cancer cells. Furthermore, EMT can induce a cancer stem cell (CSC)-like phenotype in a number of tumor types. We demonstrated that Snail is one of the master regulators that promotes EMT and mediates cancer cell migration and invasion in many types of malignancies including head and neck squamous cell carcinoma (HNSCC). In the present study, we investigated the role of Snail in inducing and maintaining CSC-like properties through EMT in HNSCC. We established HNSCC cell lines transfected with Snail. Stem cell markers were evaluated with real-time RT-PCR and western blot analysis. CSC properties were assessed using sphere formation and WST-8 assays as well as chemosensitivity and chick chorioallantoic membrane in vivo invasion assays. Introduction of Snail induced EMT properties in HNSCC cells. Moreover, Snail-induced EMT maintained the CSC-like phenotype, and enhanced sphere formation capability, chemoresistance and invasive ability. These data suggest that Snail could be one of the critical molecular targets for the development of therapeutic strategies for HNSCC.

Downregulation of E-cadherin by the transcriptional repressor Snail is associated with acquisition of metastatic potential. Although hepatitis C virus (HCV) core protein has been implicated in hepatocarcinogenesis, it is unclear whether Snail is involved in HCV core-induced dysregulation of E-cadherin. Herein, we investigated the mechanism by which HCV core induces E-cadherin repression and the role of Snail in HCV core-mediated invasiveness and metastasis. We found that HCV infection, especially HCV core expression, effectively induced the epithelial-mesenchymal transition (EMT) in hepatoma cells by repressing E-cadherin. HCV core interacted with Snail and enhanced its binding to the E-box in the promoter region of E-cadherin, leading to decreased E-cadherin promoter activity. We found that HCV core, Snail, and the histone deacetylases HDAC1/HDAC2 formed a co-repressor complex at the E-cadherin promoter. Moreover, HCV core was shown to stabilize Snail through activation of the PI3K/Akt/GSK3beta pathway. Silencing Snail expression restored E-cadherin expression and inhibited HCV core-promoted tumor growth and distant lung metastasis in vivo. Collectively, these results demonstrated that HCV core induced EMT by interacting with the transcriptional repressor complex Snail/HDACs at the E-cadherin promoter, which led to E-cadherin repression and increased invasiveness of hepatoma cells. These findings increase understanding of factors regulating metastasis in hepatoma and may ultimately lead to the development of novel treatment strategies for HCV-associated hepatocellular carcinoma.

Gastric cancer is one of the most common causes of digestive tract tumor. Despite of recent advances in surgical techniques and development of adjuvant therapy, the underlying mechanisms of gastric cancer remain poorly understood and relevant insight into novel treatment strategies using gene target remains incomplete. Recently, several studies report that epithelial to mesenchymal transition (EMT) is a crucial process for the invasion and metastasis of epithelial tumors; however, the molecular mechanisms underlying this transition are unknown. As a cis-Golgi matrix protein, GM130 plays an important role in cell cycle progression and transport of protein in the secretory pathway. In this study, we found that GM130 expression has a positive correlation with the pathological differentiation and tumor node metastasis (TNM) stage of gastric cancer. High GM130 expression levels also predict shorter overall survival of gastric cancer patients. RNA interference-mediated knockdown of GM130 expression increased epithelial marker (E-cadherin) and decreased mesenchymal marker (N-cadherin and vimentin) expression in gastric cancer cells, suppressing cell invasion, and tumor formation. Furthermore, we found that GM130 upregulated expression of the key EMT regulator Snail (SNAI1), which mediated EMT activation and cell invasion by GM130. Taken together, our study indicates GM130 may be a promising therapeutic biomarker for gastric cancer.

epithelial to mesenchymal transition (EMT)(1) occurs naturally during embryogenesis, tissue repair, cancer progression, and metastasis. EMT induces cellular and microenvironmental changes resulting in loss of epithelial and acquisition of mesenchymal phenotypes, which promotes cellular invasive and migratory capabilities. EMT can be triggered by extracellular factors, including TGF-beta, HGF, and EGF. Overexpression of transcription factors, such as SNAIL, SLUG, ZEB1/2, and TWIST1, also induces EMT and is correlated to cancer aggressiveness. Here, the breast adenocarcinoma cell line MCF7 was transduced with SNAIL to identify specific mechanisms controlled by this transcription factor during EMT. Overexpression of SNAIL led to EMT, which was thoroughly validated by molecular, morphological, and functional experiments. Subcellular proteome enrichment followed by GEL-LC-MS/MS was performed to provide extensive protein fractionation and in-depth proteomic analysis. Quantitative analysis relied on a SILAC strategy, using the invasive breast cancer cell line MDA-MB-231 as a reference for quantitation. Subsets of proteins enriched in each subcellular compartment led to a complementary list of 4289 proteins identified with high confidence. A subset of differentially expressed proteins was validated by Western blot, including regulation in specific cellular compartments, potentially caused by protein translocation. Protein network analysis highlighted complexes involved in cell cycle control and epigenetic regulation. Flow cytometry analysis indicated that SNAIL overexpression led to cell cycle arrest in G0/G1 phases. Furthermore, down-regulation of HDAC1 was observed, supporting the involvement of epigenetic processes in SNAIL-induced EMT. When HDAC1 activity was inhibited, MCF7 not only apparently initiated EMT but also up-regulated SNAIL, indicating the cross-talk between these two proteins. Both HDAC1 inhibition and SNAIL overexpression activated the AKT pathway. These molecular mechanisms appear to be essential to EMT and therefore for cancer metastasis. Specific control of such epigenetic processes might then represent effective approaches for clinical management of metastatic cancer.CI - (c) 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Most cancer cells depend on enhanced glucose and glutamine (Gln) metabolism for growth and survival. Oncogenic metabolism provides biosynthetic precursors for nucleotides, lipids, and amino acids; however, its specific roles in tumor progression are largely unknown. We previously showed that distal-less homeobox-2 (Dlx-2), a homeodomain transcription factor involved in embryonic and tumor development, induces glycolytic switch and epithelial-mesenchymal transition (EMT) by inducing Snail expression. Here we show that Dlx-2 also induces the expression of the crucial Gln metabolism enzyme glutaminase (GLS1), which converts Gln to glutamate. TGF-beta and Wnt induced GLS1 expression in a Dlx-2-dependent manner. GLS1 shRNA (shGLS1) suppressed in vivo tumor metastasis and growth. Inhibition of Gln metabolism by shGLS1, Gln deprivation, and Gln metabolism inhibitors (DON, 968 and BPTES) prevented Dlx-2-, TGF-beta-, Wnt-, and Snail-induced EMT and glycolytic switch. Finally, shDlx-2 and Gln metabolism inhibition decreased Snail mRNA levels through p53-dependent upregulation of Snail-targeting microRNAs. These results demonstrate that the Dlx-2/GLS1/Gln metabolism axis is an important regulator of TGF-beta/Wnt-induced, Snail-dependent EMT, metastasis, and glycolytic switch.

Inhibition of epithelial-mesenchymal transition (EMT)-inducing transcription factors Twist and Snail prevents tumor metastasis but enhances metastatic growth. Here, we report an unexpected role of a tumor suppressor tristetraprolin (TTP) in inhibiting Twist and Snail without enhancing cellular proliferation. TTP bound to the AU-rich element (ARE) within the mRNA 3'UTRs of Twist1 and Snail1, enhanced the decay of their mRNAs and inhibited the EMT of cancer cells. The ectopic expression of Twist1 or Snail1 without their 3'UTRs blocked the inhibitory effects of TTP on the EMT. We also observed that TTP overexpression suppressed the growth of cancer cells. Our data propose a new model whereby TTP down-regulates Twist1 and Snail1 and inhibits both the EMT and the proliferation of cancer cells.

epithelial to mesenchymal transition (EMT) has received considerable attention as a conceptual paradigm for explaining the invasive and metastatic behavior of cells during cancer progression. Our previous study showed that loss of expression of TSSC3 is positively associated with osteosarcoma malignancy and progression. However, whether TSSC3 mediates EMT in osteosarcoma is poorly understood. In the present study, we determined that TSSC3 downregulation induced cell migration and invasion ability and promoted mesenchymal transition of osteosarcoma cells by upregulating mesenchymal markers and inhibiting the epithelial markers. Furthermore, TSSC3 downregulation elicited a signaling cascade that included increased levels of Wnt3a and LRP5, inactivation of GSK-3beta, accumulation of nuclear beta-catenin and Snail, the augmented binding of beta-catenin to TCF-4, and accordingly increased the expression of Wnt target genes (CD44, MMP7). The gene knockdown of these signaling proteins could inhibit TSSC3 downregulation-promoted EMT, migration, and invasion in osteosarcoma. Finally, TSSC3 overexpression obviously inhibited cell migration, invasion, and repressed mesenchymal phenotypes, reducing lung metastasis through GSK-3beta activation. Collectively, TSSC3 downregulation promotes the EMT of osteosarcoma cells by regulating EMT markers via a signal transduction pathway that involves Snail, Wnt-beta-catenin/TCF, and GSK-3beta.CI - Copyright (c) 2016 Elsevier Ireland Ltd. All rights reserved.

BACKGROUND: epithelial-to-mesenchymal transition (EMT) has been associated with the acquisition of metastatic potential and the resistance of cancer cells to therapeutic treatments. MCF-7 breast cancer cells engineered to constitutively express the zinc-finger transcriptional repressor gene Snail (MCF-7-Snail cells) have been previously shown to display morphological and molecular changes characteristic of EMT. We report here the results of a comprehensive systems level molecular analysis of changes in global patterns of gene expression and levels of glutathione and reactive oxygen species (ROS) in MCF-7-Snail cells and the consequence of these changes on the sensitivity of cells to radiation treatment and therapeutic drugs. METHODS: Snail-induced changes in global patterns of gene expression were identified by microarray profiling using the Affymetrix platform (U133 Plus 2.0). The resulting data were processed and analyzed by a variety of system level analytical methods. Levels of ROS and glutathione (GSH) were determined by fluorescent and luminescence assays, and nuclear levels of NF-kappaB protein were determined by an ELISA based method. The sensitivity of cells to ionizing radiation and anticancer drugs was determined using a resazurin-based cell cytotoxicity assay. RESULTS: Constitutive ectopic expression of Snail in epithelial-like, luminal A-type MCF-7 cells induced significant changes in the expression of >7600 genes including gene and miRNA regulators of EMT. mesenchymal-like MCF-7-Snail cells acquired molecular profiles characteristic of triple-negative, claudin-low breast cancer cells, and displayed increased sensitivity to radiation treatment, and increased, decreased or no change in sensitivity to a variety of anticancer drugs. Elevated ROS levels in MCF-7-Snail cells were unexpectedly not positively correlated with NF-kappaB activity. CONCLUSIONS: Ectopic expression of Snail in MCF-7 cells resulted in morphological and molecular changes previously associated with EMT. The results underscore the complexity and cell-type dependent nature of the EMT process and indicate that EMT is not necessarily predictive of decreased resistance to radiation and drug-based therapies.

Fibrosis is an important feature of inflammatory bowel diseases (IBD), particularly Crohn's disease (CD), but its pathogenesis is poorly understood. To determine the postulated involvement of epithelial-mesenchymal transition (EMT) in the development of fibrosis in IBD, we analysed the expression profiles of the miR-200 family which has been shown to induce EMT in experimental models and various human diseases. We also analysed the expression of Snail and Slug, postulated targets of the investigated microRNAs. Ten patients with ulcerative colitis (UC) and 10 patients with CD who underwent colon resection were included. From each, two tissue samples were chosen (one with the most severely and one with the least affected or normal mucosa) for analysis of microRNAs expression using real-time polymerase chain reaction, and Snail and Slug expression using immunohistochemistry. We found significant down-regulation of all investigated microRNAs in CD, and of three investigated microRNAs in UC, in comparison to the normal or the least affected mucosa. Comparing UC and CD, four microRNAs were significantly more down-regulated in CD than in UC. Snail and Slug were expressed in the injured epithelium and occasionally in mesothelial cells and submesothelial fibroblasts. Our finding of down-regulation of the miR-200 family and up-regulation of transcription repressors Snail and Slug supports the postulated role of EMT in the pathogenesis of fibrosis in IBD. The described expression patterns are consistent with the notion that fibrosis does not occur only in CD but also in UC, being much more severe in CD.CI - (c) 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

The oncogene, mouse double minute 2 (MDM2), has been implicated in the pathogenesis of numerous cancers. In this study, we investigated the role of MDM2 in epithelial-to-mesenchymal transition (EMT) and the underlying mechanisms in breast cancer cells in vitro and in vivo. The results showed that up-regulation of MDM2 in MCF-7 cells altered the cell morphology to a mesenchymal phenotype. Knockdown of MDM2 in MDA-MB-231 cells altered the cell morphology to the epithelial phenotype. In addition, overexpression of MDM2 increased the expression of N-cadherin and Vimentin and decreased the expression of E-cadherin, at both the mRNA and protein levels, in vitro and in vivo. Conversely, down-regulation of MDM2 decreased the expression of N-cadherin and Vimentin, and increased the expression of E-cadherin in vitro. Furthermore, MDM2 up-regulated both the mRNA and protein expression of Snail in vitro and in vivo. Knockdown of Snail almost abolished MDM2 induced EMT in vitro. Finally, we found that MDM2 expression correlated with EMT markers and Snail: Snail expression was inversely associated with E-cadherin in human breast cancer samples. Our findings demonstrated that MDM2 induces EMT by enhancing Snail expression in vitro and in vivo. Thus, MDM2 may be a potential target for therapy against human metastatic breast cancer.

Polychlorinated biphenyls (PCBs) are ubiquitous legacy persistent pollutants and epidemiological data showed that PCB burdens were associated with failed implantation in human. However, the mechanism how PCB exposure affects the embryo implantation is not clear. Using an in vitro model for human embryo implantation employing the human choriocarcinoma cell line JAR and the human endometrial cell line Ishikawa, we have shown that PCB mixture Aroclor 1254 at environmental-relevant concentrations (2.5, 12.5, and 62.5muM) dose-dependently impaired the endometrial receptivity by reducing the adhesion of JAR spheroid attachment and increasing the spheroid outgrowth. The receptive-up-regulated micro-RNA, mir-30d was also down-regulated in endometrial cells by the exposure. Following transient transfection of mir-30d mimic, the disrupted attachment and outgrowth of JAR spheroids was partially restored in the model. By measurement of cadherin switch and vimentin expression, the PCB exposure also activated epithelial mesenchymal transition (EMT) in endometrial cells. In accordance, mir-30d mimic suppressed the EMT markers induced by PCBs. Luciferase reporter assay confirmed that the EMT regulator Snai1 was targeted by mir-30d, and the expression of Snai1 was dose-dependently up-regulated by PCB exposure. Taken together, our study revealed that PCBs may affect the receptivity of endometrial cells by impairing the interaction between receptivity-up-regulated microRNA and EMT process.CI - Copyright (c) 2016 Elsevier Ireland Ltd. All rights reserved.

Snail1 transcriptional factor is essential for triggering epithelial-to-mesenchymal transition (EMT) and inducing tumor cell invasion. We report here an EMT-independent action of Snail1 on tumor invasion, as it is required for the activation of cancer-associated fibroblasts (CAF). Snail1 expression in fibroblasts requires signals derived from tumor cells, such as TGFbeta; reciprocally, in fibroblasts, Snail1 organizes a complex program that stimulates invasion of epithelial cells independent of the expression of Snail1 in these cells. epithelial cell invasion is stimulated by the secretion by fibroblast of diffusible signaling molecules, such as prostaglandin E2 The capability of human or murine CAFs to promote tumor invasion is dependent on Snail1 expression. Inducible Snail1 depletion in mice decreases the invasion of breast tumors; moreover, epithelial tumor cells coxenografted with Snail1-depleted fibroblasts originated tumors with lower invasion than those transplanted with control fibroblasts. Therefore, these results demonstrate that the role of Snail1 in tumor invasion is not limited to EMT, but it is also dependent on its activity in stromal fibroblasts, where it orchestrates the cross-talk with epithelial tumor cells. cancer Res; 76(21); 6205-17. (c)2016 AACR.CI - (c)2016 American Association for cancer Research.

Transcriptional silencing is a major cause for the inactivation of tumor suppressor genes, however, the underlying mechanisms are only poorly understood. The EPHB2 gene encodes a receptor tyrosine kinase that controls epithelial cell migration and allocation in intestinal crypts. Through its ability to restrict cell spreading, EPHB2 functions as a tumor suppressor in colorectal cancer whose expression is frequently lost as tumors progress to the carcinoma stage. Previously we reported that EPHB2 expression depends on a transcriptional enhancer whose activity is diminished in EPHB2 non-expressing cells. Here we investigated the mechanisms that lead to EPHB2 enhancer inactivation. We show that expression of EPHB2 and SNAIL1 - an inducer of epithelial-mesenchymal transition (EMT) - is anti-correlated in colorectal cancer cell lines and tumors. In a cellular model of Snail1-induced EMT, we observe that features of active chromatin at the EPHB2 enhancer are diminished upon expression of murine Snail1. We identify the transcription factors FOXA1, MYB, CDX2 and TCF7L2 as EPHB2 enhancer factors and demonstrate that Snail1 indirectly inactivates the EPHB2 enhancer by downregulation of FOXA1 and MYB. In addition, Snail1 induces the expression of Lymphoid enhancer factor 1 (LEF1) which competitively displaces TCF7L2 from the EPHB2 enhancer. In contrast to TCF7L2, however, LEF1 appears to repress the EPHB2 enhancer. Our findings underscore the importance of transcriptional enhancers for gene regulation under physiological and pathological conditions and show that SNAIL1 employs a combinatorial mechanism to inactivate the EPHB2 enhancer based on activator deprivation and competitive displacement of transcription factors.CI - Copyright (c) 2016 Elsevier B.V. All rights reserved.

EMT (epithelial-mesenchymal transition) occurs in a wide range of tumor types, and has been shown to be crucial for metastasis. Epigenetic modifications of histones contribute to chromatin structure and result in the alterations in gene expression. Tri-methylation of histone H3 lysine 4 (H3K4me3) is associated with the promoters of actively transcribed genes and can serve as a transcriptional on/off switch. RbBP5 is a component of the COMPASS/ -like complex, which catalyzes H3K4me3 formation. In this study, we found that in the process of TGF-Beta1 induced EMT in the prostate cancer cell line DU145, H3K4me3 enrichment and RbBP5 binding increased in the vicinity of Snail (SNAI1) transcription start site. Knocking-down of RbBP5 notably decreased Snail expression and EMT. Recruitment of RbBP5 and formation of H3K4me3 at Snail TSS during EMT depend on binding of SMAD2/3 and CBP at Snail TSS. This study links the SMAD2/3 signal with Snail transcription via a histone modification - H3K4me3. Furthermore, our research also demonstrates that RbBP5 and even WRAD may be a promising therapeutic candidates in treating prostate cancer metastasis, and that DU145 cells maintain their incomplete mesenchymal state in an auto/ paracrine manner.

Several members of the Poly(ADP-ribose) polymerase (PARP) family are essential regulators of genome integrity, actively prospected as drug targets for cancer therapy. Among them, PARP3 is well characterized for its functions in double-strand break repair and mitotis. Here we report that PARP3 also plays an integral role in TGFbeta and reactive oxygen species (ROS) dependent epithelial-to-mesenchymal transition (EMT) and stem-like cell properties in human mammary epithelial and breast cancer cells. PARP3 expression is higher in breast cancer cells of the mesenchymal phenotype and correlates with the expression of the mesenchymal marker Vimentin while being in inverse correlation with the epithelial marker E-cadherin. Furthermore, PARP3 expression is significantly upregulated during TGFbeta-induced EMT in various human epithelial cells. In line with this observation, PARP3 depletion alters TGFbeta-dependent EMT of mammary epithelial cells by preventing the induction of the Snail-E-cadherin axis, the dissolution of cell junctions, the acquisition of cell motility and chemoresistance. PARP3 responds to TGFbeta-induced ROS to promote a TG2-Snail-E-cadherin axis during EMT. Considering the link between EMT and cancer stem cells, we show that PARP3 promotes stem-like cell properties in mammary epithelial and breast cancer cells by inducing the expression of the stem cell markers SOX2 and OCT4, by increasing the proportion of tumor initiating CD44high/CD24low population and the formation of tumor spheroid bodies, and by promoting stem cell self-renewal. These findings point to a novel role of PARP3 in the control of TGFbeta-induced EMT and acquisition of stem-like cell features and further motivate efforts to identify PARP3 specific inhibitors.

Biological changes in Snail-overexpressed SGC7901 cells were studied by establishing a pEGFP-C1-Snail carrier. The significance of Snail in epithelial-mesenchymal transition (EMT) as well as the invasion and metastatic capacity of gastric cancer cells was also discussed; moreover, we attempted to verify the probable cancer stem cell characteristics of Snail-overexpressed cells. A pEGFP-C1-Snail eukaryotic expression plasmid was constructed and pEGFP-C1(-) and pEGFP-C1-Snail plasmids were extracted and transfected into SGC7901 cells using Lipofectamine 2000. Stably expressed SGC7901-N [control group containing pEGFP-C1(-)] and SGC7901-S (test group containing pEGFP-C1-Snail) cells were screened using a G418 resistance medium. Snail, E-cadherin, b-catenin, vimentin, and fibronectin gene and protein expressions were detected by real-time quantitative PCR, western blot, and immunofluorescence. Cell invasion and metastasis were tested by scratch test, invasion assay, and an adhesion experiment. The positive rate of aldehyde dehydrogenase-1 (ALDH-1) expression was analyzed by flow cytometry. The results indicated the occurrence of EMT, accompanied by morphological changes in the cells and a weakening of the cell adhesion capacity. We also observed a decrease in the expression of epithelial markers E-cadherin and b-catenin and an increase in mesenchymal (Snail and vimentin) marker expression. Moreover, the cells showed increased invasiveness and metastatic capacity, and decreased proliferative ability. Moreover, the Snail-treated SGC7901 cells moved towards the scratch and produced fewer clones compared to the control cells. Owing to its capacity for self-renewal, SGC7901-S cells produced new clones and expressed ALDH-1. Therefore, we concluded that Snail overexpression induced EMT and endowed cells with tumor stem cell characteristics.

OBJECTIVE: Reprogramming energy metabolism has been defined as the ninth hallmark of cancer; glucose deprivation might be a novel, feasible and effective approach for cancer treatment. However, the comprehensive illustration of behavior alteration of hepatocellular carcinoma (HCC) cells induced by glucose restriction is lacking and associated molecular mechanism is still unclear. METHODS: Three human HCC cell lines were cultured with normal control (25.0 mM D-glucose) and low glucose (5.5 mM D-glucose) Dulbecco's modified Eagle's medium. Cell migration ability was assessed by both transwell migration assay and wound healing assay. Levels of heat shock factor 1 (HSF1) and epithelial-mesenchymal transition (EMT) markers, E-cadherin and N-cadherin, were estimated by real-time polymerase chain reaction and western blotting. Moreover, low glucose-induced inhibition of EMT-associated migration via the HSF1 was demonstrated in HCC cells by knocking down HSF1 with the HSF1 siRNA technique; in terms of molecular mechanism, direct regulation of HSF1 on snail1 expression was identified by chromatin immunoprecipitation followed by Luciferase reporter assay. RESULTS: Herein, we showed that low glucose culture hampered typical EMT-like morphological change, "cadherin switching," and cell migration of HCC cells and further demonstrated that persistent down-regulation of HSF1 induced by glucose restriction, resulting in direct inhibition of snail1 expression and promoting the up-regulation of E-cadherin expression, and HSF1 was required for EMT-associated migration of HCC cells when cultured with low glucose media. CONCLUSION: All these results illustrate that glucose restriction can repress the EMT and migration of HCC cells, and demonstrate the importance of HSF1 to our understanding on HCC treatment as a novel and effective therapeutic target or adjuvant strategy.

epithelial-mesenchymal transition (EMT) can contribute to tumor invasion, metastasis, and resistance to chemotherapy or hormone therapy. EMT may be induced by a variety of growth factors, such as epidermal growth factor (EGF). Most studies regarding EMT have focused on TGF-beta-Smads signaling. The mechanism of EGF-induced EMT via activation of the Smad2/3 in breast cancer cells, MCF-7 and MDA-MB-231, remains unclear. The expression levels of Snail, vimentin, and fibronectin were increased by EGF treatment in a time-dependent manner, while the expression level of E-cadherin was decreased. EGF-induced nuclear co-localization of phospho-Smad2/3 and Snail and cancer cell migration were inhibited by pretreatment with an ERK1/2 inhibitor, PD98059 and a phospho-Smad2 inhibitor, SB203580. Knockdown of Smad2/3 expression suppressed EGF-induced expressions of Snail, vimentin, fibronectin, and cancer cell invasion, suggesting an acquisition of the mesenchymal and migratory phenotype in less aggressive MCF-7 cells. Moreover, MDA-MB-231 cells were shown that EGF-induced EMT, and cell invasion through ERK1/2-phospho-Smad2/3-Snail signaling pathway. We have discovered that EGF-stimulated activation of Smad2/3 upregulated several key EMT markers, inhibited E-cadherin expression, promoted EMT, enhanced migration and invasion in MCF-7 and MDA-MB-231 breast cancer cells. Identification of this molecular mechanism may provide new molecular targets for the development of therapies for metastatic breast cancer.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in multiple cellular functions including metabolism and gene transcription. Our previous study showed that GAPDH expression was elevated in colon cancer and further upregulated in liver metastatic tissues, suggesting a possilbe role of GAPDH in promoting cancer metastasis. The present study was designed to investigate the underlying mechanism, using multiple experimental approaches including genetic silencing of GAPDH expression by short hairpin RNA (shRNA) and biochemcial/molecular analyses of the key events involved in glycolytic metabolism and epithelial-mesenchymal transition (EMT). We showed that silencing of GAPDH expression resulted in a significant reduction of glycolysis in colon cancer cell lines, accompanied by a decrease in cell proliferation and an apparent change in cell morphology associated with alterations in actin expression and phalloidine staining patterns. Furthermore, GAPDH suppression also caused a downregulation of gene expression involved in cancer stem-like cells and EMT. CHIP assay and co-immunoprecipitation revealed that GAPDH physically interacted with the transcriptional factor Sp1 and enhance the expression of SNAIL, a major regulator of EMT. Suppression of GAPDH expression resulted in a signficant decrease in SNAIL expression, leading to inhibition of EMT and attenuation of colon cancer cell migration in vitro and reduced metastasis in vivo. Overall, the present study suggests that GAPDH plays an important role in cancer metastasis by affecting EMT through regulation of Sp1-mediated SNAIL expression.

epithelial mesenchymal transition (EMT), the transition of epithelial cells into motile mesenchymal cells, plays an important role in embryogenesis, cancer invasion, and metastasis. Ameloblastomas are common epithelial odontogenic tumors, occurring exclusively in the mandible with locally invasive growth. Thirty-seven ameloblastoma cases were evaluated for the involvement of EMT by immunohistochemical staining and western blotting using antibodies against Slug, Snail, Twist, TGF-beta, and E-cadherin. Double immunostaining was also performed. Slug and TGF-beta were expressed in the nuclei of peripheral and stellate reticulum cells of ameloblastoma nests. Twenty cases of Snail, 36 of Slug, 8 of Twist, and 19 of TGF-beta showed strong expression in tumor cells in follicular and plexiform patterns. expression of Slug and TGF-beta increased in regions where the expression of E-cadherin was reduced. EMT was found to be associated with the local invasive growth of ameloblastoma. These data suggest that reduced expression of E-cadherin and over-expression of Slug, Snail, and TGF-beta induce EMT. Given that ameloblastomas are characterized by local invasiveness, EMT might be related to their development. Thus, strong expression of Slug and TGF-beta and reduced expression of E-cadherin might be related to the local invasiveness of ameloblastoma.

BACKGROUND: The E-cadherin, N-cadherin, and Snail genes are epithelial-mesenchymal transition (EMT)-inducible genes. Previous studies demonstrated that the expression of EMT markers in the primary tumor sites of gastric cancer correlates with tumor progression and prognosis. However, the clinical significance of the expression of these EMT markers in metastatic lymph nodes remains unclear. In the present study, we investigated the expression of these EMT markers in the primary tumor sites and metastatic lymph nodes. METHODS: Immunohistochemistry was used to investigate the expression of E-cadherin, N-cadherin, and Snail in 89 primary tumors and 511 metastatic lymph nodes obtained from patients with gastric cancer. RESULTS: The weak expression of E-cadherin in tumors and lymph nodes increased with more lymph node metastasis and in more undifferentiated tumors. The strong expression of N-cadherin in lymph nodes correlated with more lymph nodes metastasis, an advanced stage, and poor prognosis. The weak expression of Snail in tumors correlated with lymphatic invasion. The strong expression of Snail in lymph nodes correlated with more lymph node metastasis and an advanced stage. The strong expression of Snail in tumors and its weak expression in lymph nodes correlated with more lymph node metastasis, an advanced stage, and poor prognosis. CONCLUSIONS: The expression of N-cadherin in metastatic lymph nodes is useful for predicting the prognosis of patients with gastric cancer. The Snail switch-namely, the positive-to-negative conversion of the Snail status-between primary tumors and lymph node metastasis may be important for confirming EMT and mesenchymal-epithelial transition.

epithelial-to-mesenchymal transition (EMT) is essential for tumor invasion and metastasis. Snail has been proven to be a key regulator of EMT. Several studies have shown compelling evidence that Snail is also an important regulator of tumor growth and aggression; however, the role of Snail in the cell cycle has not been clarified. We decreased Snail expression by siRNA transfection and lentiviralmediated RNAi, to explore the effect of silencing Snail on the tumorigenicity and migration of lung carcinoma (lung cancer) cells. The results showed that silencing Snail conferred significant anti-proliferative activity and inhibited cell migration, tumor growth and metastasis both in vitro and in vivo. To understand the mechanism of these effects, we further investigated correlations among Snail expression, EMT and cell cycle. Significantly, Snail knockdown reversed EMT processes in lung cancer cells. Furthermore, the cyclin-dependent kinase inhibitor P21 was upregulated after silencing Snail. P21 upregulation manifested its tumor suppressor effects and arrested cells in the G2/M phase, not the G1/S phase following Snail depletion in lung cancer cells. These data suggest that silencing Snail decreases the malignant behaviors of lung cancer cells by reversing EMT processes and causing cell cycle defects.

JPJD was an ideal alternative traditional Chinese medicine compound in the prevention and treatment of CRC, but its underlying mechanisms has not been fully elucidated. In this study, we demonstrated in vitro that TGF-beta-induced EMT promoted the invasion and metastasis of CRC cells, reduced the expression of E-cadherin, and elevated the expression of Vimentin. However, JPJD could inhibit the invasive and migratory ability of TGF-beta-stimulated CRC cells in a concentration-dependent manner through increasing the expression of E-cadherin and repressing the expression of Vimentin, as well as the inhibition of TGF-beta/Smad signaling pathway. Meanwhile, JPJD reduced the transcriptional activities of EMT-associated factors Snail and E-cadherin during the initiation of TGF-beta-induced EMT. In vivo, the results demonstrated that JPJD can significantly inhibit the liver and lung metastasis of orthotopic CRC tumor in nude mice, as well as significantly prolonging the survival time of tumor-bearing in a dose-dependent manner. Additionally, JPJD can upregulate the expression of E-cadherin and Smad2/3 in the cytoplasm and downregulate the expression of Vimentin, p-Smad2/3, and Snail in the orthotopic CRC tumor tissues. In conclusions, our new findings provided evidence that JPJD could inhibit TGF-beta-induced EMT in CRC through TGF-beta/Smad mediated Snail/E-cadherin expression.

BACKGROUND: The transcription factor sex-determining region Y-box protein 3 (SOX3) plays important roles in various types of cancer. However, its expression and function have not yet been elucidated in osteosarcoma (OS). METHODS: The expression levels of SOX3 in OS tissues and OS cell lines were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. The effects of SOX3 expression on OS cell biological traits were investigated by overexpressing and downregulating SOX3 protein. The expression of epithelial-mesenchymal transition (EMT) markers and transcription factors associated with EMT (EMT-TFs), were detected simultaneously. The mechanism underlying SOX3-mediated Snail1 expression was further investigated. RESULTS: SOX3 was upregulated in human OS tissues. SOX3 overexpression promoted the EMT, migration and invasion in OS cells. The downregulation of SOX3 resulted in opposing effects. Furthermore, SOX3 upregulation enhanced the expression of the transcriptional repressor Snail1 by binding to its promoter region. Additionally, a positive correlation among the expression of SOX3, Snail1, and E-cadherin was demonstrated in human OS tissues. CONCLUSIONS: SOX3 promotes migration, invasiveness, and EMT in OS cells via transcriptional activation of Snail1 expression, suggesting that SOX3 is a novel regulator of EMT in OS and may serve as a therapeutic target for the treatment of OS metastasis.

Despite an overall good prognosis, a significant proportion of patients with hormone receptor positive human epidermal growth factor receptor 2 negative breast cancers develop distant metastases. The metastatic potential of epithelial cells is known to be regulated by tumor-stromal interaction and mediated by epithelial-to-mesenchymal transition. Hormone receptor positive human epidermal growth factor receptor 2 negative tumors were used to estimate markers of epithelial-to-mesenchymal transition, and the luminal breast cancer cell line MCF-7 was used to examine the interactions between integrins and growth factor receptors in causation of epithelial-to-mesenchymal transition. A total of 140 primary tumors were sub-divided into groups enriched for the markers of epithelial-to-mesenchymal transition (snail family transcriptional repressor 2 and integrin beta6) versus those with low levels. Within the epithelial-to-mesenchymal transition+ tumors, there was a positive correlation between the transcripts of integrin beta6 and growth factor receptors-human epidermal growth factor receptor 2 and epidermal growth factor receptor. In tumors enriched for epithelial-to-mesenchymal transition markers, patients with tumors with the highest quartile of growth factor receptor transcripts had a shorter disease-free survival compared to patients with low growth factor receptor expression by Kaplan-Meier analysis (log rank, p = 0.03). epithelial-to-mesenchymal transition was induced in MCF-7 cells by treatment with transforming growth factor beta 1 and confirmed by upregulation of SNAI1 and SNAI2 transcripts, increase of vimentin and integrin beta6 protein, and repression of E-cadherin. Treatment of these cells with the dual-specificity tyrosine-kinase inhibitor lapatinib led to downregulation of epithelial-to-mesenchymal transition as indicated by lower levels of SNAI1 and SNAI2 transcripts, integrin alphavbeta6, and matrix metalloproteinase 9 protein. The results suggest that synergistic interactions between growth factor receptors and integrin beta6 could mediate epithelial-to-mesenchymal transition and migration in a subset of luminal breast cancers and lapatinib might be effective in disrupting this interaction.

miR-128 is expressed in various tumors, but its expression and function in gastric cancer have not been defined. Thus, the goal of this study was to characterize miR-128 in gastric cancer. We found first that miR-128 is down-regulated in gastric cancer cell lines and tissues, and this dysregulation is correlated with DNA methylation and the transcription factor SNAIL. Using prediction tools, western blotting, and luciferase reporter assays, we found that Bmi-1 was the direct target of miR-128. Additionally, overexpression of miR-128 inhibited gastric cancer cell migration, invasion, and proliferation by targeting Bmi-1 in vitro and in vivo. We also documented, with receiver operating characteristic curves and Kaplan-Meier survival analysis, that miR-128 and Bmi-1 may be useful markers for diagnosing and estimating the prognosis of gastric cancer patients. As the epithelial-to-mesenchymal transition is an important mechanism associated with cancer invasion and metastasis, we inferred that miR-128 could regulate this mechanism in gastric cancer. In fact, we found that miR-128 could reverse epithelial-to-mesenchymal transition induced by Bmi-1 via the PI3K/AKT pathway. Because SNAIL also acts as a mesenchymal marker, our findings identified a novel positive feedback loop in which the transcription factor SNAIL curbs the expression of miR-128, and then down-regulated miR-128 promotes the expression of Bmi-1; finally, overexpression of Bmi-1 drives the epithelial-to-mesenchymal transition process via the PI3K/AKT pathway, and the expression of SNAIL is up-regulated.

BACKGROUND: Chronic hepatitis C virus (HCV) infection is an important cause of hepatocellular carcinoma (HCC). epithelial to mesenchymal transition (EMT) is a key process associated with tumor metastasis and poor prognosis. HCV infection, HCV core and NS5A protein could induce EMT process, but the role of NS4B on EMT remains poorly understood. METHODS: We overexpressed HCV NS4B protein in HepG2 cells or Huh7.5.1 cells infected by HCVcc, the E-cadherin expression, N-cadherin expression and the EMT-associated transcriptional factor Snail were determined. The migration and invasion capabilities of the transfected cells were evaluated using wound-healing assay. Additionally, we used Snail siRNA interference to confirm the relation of HCV NS4B and Snail on EMT promotion. RESULTS: HCV NS4B increased the expression of EMT related markers and promoted cell migration and invasion. Snail knock-down almost completely eliminated the function of NS4B protein in EMT changes and reversed cell migration capacity to lower level. HCV NS4B protein could reduce the expression of Scribble and Hippo signal pathway were subsequently inactivated, resulting in the activation of PI3K/AKT pathway, which may be the reason for the up-regulation of Snail. CONCLUSIONS: This study demonstrates that HCV NS4B protein induces EMT progression via the upregulation of Snail in HCC, which may be a novel underlying mechanism for HCV-associated HCC development, invasion and metastasis.

epithelial-mesenchymal transition (EMT) is a highly conserved and fundamental process in development, fibrosis, and metastasis. During the process, epithelial cells lose their morphology and transcriptional program, and transdifferentiate to mesenchymal cells. It has been reported that lens epithelial cells undergo EMT during cataract formation, and regulation of microRNAs on genes is associated with lens development. However, the molecular mechanisms of this regulation in diabetic cataract still need to be investigated. In the present study, the expression of E-cadherin was downregulated, while the expression of alpha-SMA and vimentin was upregulated in diabetic cataract tissues and the in vitro model, suggesting the involvement of EMT in diabetic cataract formation. Results of miRNA profiling demonstrated that miR-30a was markedly downregulated in diabetic cataract tissues. Overexpression of miR-30a-5p decreased SNAI1, a known modulator of EMT, and the expression of vimentin and alpha-SMA in our diabetic cataract model in vitro. It is concluded that EMT is involved in human diabetic cataract, and upregulation of miR-30a can repress EMT through its targeting of SNAI1 in lens epithelial cells, which make miR-30a a novel target of therapeutic intervention for human diabetic cataract.

Hepatocellular carcinoma (HCC) is one of the common malignances in the world and is associated with high mortality and poor prognosis, partly due to early invasion and metastasis. Cx32 has been indicated to be involved in the progression of many cancers including HCC, but its relationship with tumor invasion and metastasis is still controversial. In the present study, the downregulated Cx32 in HCC tissue was found negatively correlated with histological grade and lymph node metastasis. Cx32 regulated HCC migration and invasion in vitro and inhibited tumor metastasis in xenograft models in vivo. We subsequently identified that Cx32 mediated epithelial-mesenchymal transition (EMT) by regulating Snail expression, and the enhanced Snail was due to activation of Wnt/beta-catenin signaling in response to Cx32 inhibition. Finally, decreased expression of Cx32 showed strong correlation with loss/reduction of E-cadherin, higher expression of Snail, and nuclear accumulation of beta-catenin in HCC tissues. Taken together, our results suggest that Cx32 inhibits HCC invasion and metastasis through Snail-mediated EMT, Cx32 and this signaling pathway molecules may offer potential targets for HCC cancer therapy.

We previously performed long non-coding RNA (lncRNA) expression microarray analyses to identify novel indicators for gastric cancer (GC) metastasis and prognosis in which we identified lncRNA XLOC_010235 (XLOC) as a candidate RNA. However, XLOC_010235 molecular mechanism of action remains unclear. Gain and loss of function approaches were used to investigate the biological role of XLOC in vitro. The effects of XLOC on cell viability were assessed by CCK-8 proliferation assays. Real-time PCR, western-blot and immunofluorescence were used to evaluate the mRNA and protein expression of Snail and multiple EMT related molecules. The positive XLOC/Snail1 interaction was identified and verified by immunohistochemistry assay and bivariate correlation analysis. Ectopic expression of XLOC facilitate cell viability, migration and invasion, leading to the acceleration of metastasis, while depletion of XLOC expression hindered cell migration and invasion. Moreover, over-expression of XLOC was found to play a important role in epithelial-to-mesenchymal transition (EMT) through the regulation of E-cadherin, N-cadherin and Vimentin expression, in which transcriptional factor Snail1 was involved. These results advance our understanding of the role of lncRNA XLOC_010235 as a active regulator of EMT by associating with Snail1, which may help in the development of new therapeutics.

Mithramycin exhibits certain anticancer effects in glioma, metastatic cerebral carcinoma, malignant lymphoma, chorionic carcinoma and breast cancer. However, its effects on salivary adenoid cystic carcinoma remain unclear. Here, we report that mithramycin significantly inhibited epithelial-to-mesenchymal transition and invasion in human salivary adenoid cystic carcinoma cell lines. The underlying mechanism for this activity was further demonstrated to involve decreasing the expression of the transcription factors specificity protein 1 and SNAI1. Specificity protein 1 is a pro-tumourigenic transcription factor that is overexpressed in SACC-LM and SACC-83 cells, and its expression is inhibited by mithramycin. Moreover, chromatin immunoprecipitation assays showed that specificity protein 1 induced SNAI1 transcription through direct binding to the SNAI1 promoter. In summary, this study uncovered the mechanism through which mithramycin inhibits epithelial-to-mesenchymal transition and invasion in salivary adenoid cystic carcinoma cell lines, namely, via downregulating specificity protein 1 and SNAI1 expression, which suggests mithramycin may be a promising therapeutic option for salivary adenoid cystic carcinoma.

Substantial evidence has shown that epithelial-mesenchymal transition (EMT) plays critical roles in colorectal cancer (CRC) development and prognosis. To uncover the pivotal regulators that function in the cooperative interactions between cancer cells and their microenvironment and consequently affect the EMT process, we carried out a systematic analysis and evaluated prognosis in CRC specimens. tumor buds and their surrounding stroma were captured using laser microdissection. We used gene expression profiling, bioinformatics analysis and regulatory network construction for molecular selection. The clinical significance of potential biomarkers was investigated. We identified potential EMT biomarkers, including BGN, MMP1, LGALS1, SERPINB5, and TM4SF4, all of which participated in the integrated pathway of TGFbeta/Snail with TNFalpha/NFkappaB. We also found that BGN, MMP1, LGALS1, SERPINB5 and TM4SF4 were related to CRC patient prognosis. Patients with higher expression of these individual potential biomarkers had poorer prognosis. Among the identified biomarkers, BGN and TM4SF4 are reported, for the first time, to probably be involved in the EMT process and to predict CRC prognosis. Our results strongly suggest that the integrated pathway of TGFbeta/Snail with TNFalpha/NFkappaB may be the principal axis that links cancer cells to their microenvironment during the EMT process and results in poor prognosis in CRC patients.

Filamin A (FLNA) is actin filament cross-linking protein involved in cancer progression. Its importance in regulating cell motility is directly related to the epithelial to mesenchymal transition (EMT) of tumor cells. However, little is known about the mechanism of action of FLNA at this early stage of cancer invasion. Using immunochemical methods, we evaluated the levels and localization of FLNA, pFLNA[Ser2152], beta1 integrin, pbeta1 integrin[Thr788/9], FAK, pFAK[Y379], and talin in stably transfected HT29 adenocarcinoma cells overexpressing Snail and looked for the effect of Snail in adhesion and migration assays on fibronectin-coated surfaces before and after FLNA silencing. Our findings indicate that FLNA upregulation correlates with Snail-induced EMT in colorectal carcinoma. FLNA localizes in the cytoplasm and at the sites of focal adhesion (FA) of invasive cells. Silencing of FLNA inhibits Snail-induced cell adhesion, reduces the size of FA sites, induces the relocalization of talin from the cytoplasm to the membrane area and augments cell migratory properties. Our findings suggest that FLNA may not act as a classic integrin inhibitor in invasive carcinoma cells, but is involved in other pro-invasive pathways. FLNA upregulation, which correlates with cell metastatic properties, maybe an additional target for combination therapy in colorectal carcinoma tumor progression.CI - Copyright (c) 2017 Elsevier Inc. All rights reserved.

It has been well documented that Snail plays a decisive role in various tumors. However, the direct effect of Snail on laryngeal squamous cell carcinoma (LSCC) has not been elaborated. In this study, we firstly detected the expression of Snail in 14 samples of patients with LSCC and found that its content was high in cancer tissues compared with adjacent tissues. Then we established LSCC Hep-2 cells with Snail silencing and validated the knockdown efficiency by Western blotting and real-time PCR. Results showed that silencing of Snail significantly inhibited the ability of adhesion, migration, and invasion of Hep-2 cells. Further study revealed that knockdown of Snail suppressed the epithelial-mesenchymal transition (EMT) process of Hep-2 cells, as evidenced by downregulation of matrix metallopeptidase (MMP)-2, MMP-9, integrin subunit beta 1 (ITGbeta1), beta-catenin, vimentin, N-cadherin, and fibronectin and upregulation of vitamin D receptor (VDR) and E-cadherin. Additionally, transfection with the small interfering RNA of VDR reversed the effect induced by Snail silencing in Hep-2 cells. Taken together, these results demonstrate that knockdown of Snail can inhibit the EMT process of LSCC cells through the VDR signaling pathway in vitro.

OBJECTIVE: Snail is an important factor in regulating epithelial mesenchymal transition (EMT). Its elevation is related to the enhancement of lung cancer invasion. MicroRNA-22 (MiR-22) plays a role in regulating lung cancer cell invasion. Bioinformatics analysis showed the complementary binding site between miR-22 and Snail. This study aimed to investigate the role of miR-22 in regulating Snail and affecting lung cancer cell invasion and metastasis. MATERIALS AND METHODS: Dual luciferase assay confirmed the targeted relationship between miR-22 and Snail. MiR-22 and Snail expressions were compared in MRC-5, Anip973, and AGZY83-a cells. Cell colony formation and invasion were tested in Anip973 and AGYZ83-a cells. Anip973 and AGYZ83-a cells were treated by 5 ng/ml transforming growth factor beta1 (TGF-beta1) to detect miR-22, Snail, E-cadherin, and N-cadherin expressions. Anip973 cells were cultured in vitro and divided into five groups, including miR-Normal control (miR-NC), miR-22 mimic, small interfering RNA-Normal control (si-NC), si-Snail, and miR-22 mimic + si-Snail groups. RESULTS: MiR-22 targeted inhibited Snail expression. MiR-22 significantly down-regulated, while Snail obviously elevated in Anip973 and AGYZ83-a cells compared with that in MRC-5 cells. Anip973 exhibited markedly stronger invasive and colony formation abilities than AGYZ83-a. TGFbeta1 apparently reduced miR-22 and E-cadherin, whereas increased Snail and N-cadherin stronger in Anip973 than that in AGYZ83-a. MiR-22 mimic and/or si-Snail transfection significantly reduced Snail and N-cadherin levels, up-regulated E-cadherin expression, and attenuated cell colony formation and invasion. CONCLUSIONS: Down-regulation of miR-22 plays a role in facilitating lung cancer cell EMT and invasion by elevating Snail. MiR-22 over-expression attenuated lung cancer cell EMT and invasion via targeted inhibiting Snail.

Phenotypic conversion of tumor cells through epithelial-mesenchymal transition (EMT) requires massive gene expression changes. How these are brought about is not clear. Here we examined the impact of the EMT master regulator SNAIL1 on the FOXA family of transcription factors which are distinguished by their particular competence to induce chromatin reorganization for the activation of transcriptional enhancer elements. We show that the expression of SNAIL1 and FOXA genes is anticorrelated in transcriptomes of colorectal tumors and cell lines. In cellular EMT models, ectopically expressed Snail1 directly represses FOXA1 and triggers downregulation of all FOXA family members, suggesting that loss of FOXA expression promotes EMT. Indeed, cells with CRISPR/Cas9-induced FOXA-deficiency acquire mesenchymal characteristics. Furthermore, ChIP-seq data analysis of FOXA chromosomal distribution in relation to chromatin structural features which characterize distinct states of transcriptional activity, revealed preferential localization of FOXA factors to transcriptional enhancers at signature genes that distinguish epithelial from mesenchymal colon tumors. To validate the significance of this association, we investigated the impact of FOXA factors on structure and function of enhancers at the CDH1, CDX2 and EPHB3 genes. FOXA-deficiency and expression of dominant negative FOXA2 led to chromatin condensation at these enhancer elements. Site-directed mutagenesis of FOXA binding sites in reporter gene constructs and by genome-editing in situ impaired enhancer activity and completely abolished the active chromatin state of the EPHB3 enhancer. Conversely, expression of FOXA factors in cells with inactive CDX2 and EPHB3 enhancers led to chromatin opening and de novo deposition of the H3K4me1 and H3K27ac marks. These findings establish the pioneer function of FOXA factors at enhancer regions of epithelial genes and demonstrate their essential role in maintaining enhancer structure and function. Thus, by repressing FOXA family members, SNAIL1 targets transcription factors at strategically important positions in gene-regulatory hierarchies, which may facilitate transcriptional reprogramming during EMT.

During epithelial-mesenchymal transition (EMT), reprogramming of gene expression is accompanied by histone modifications. Whether EMT-promoting signaling directs functional changes in histone methylation has not been established. We show here that the histone lysine methyltransferase SETDB1 represses EMT and that, during TGF-beta-induced EMT, cells attenuate SETDB1 expression to relieve this inhibition. SETDB1 also controls stem cell generation, cancer cell motility, invasion, metastatic dissemination, as well as sensitivity to certain cancer drugs. These functions may explain the correlation of breast cancer patient survival with SETDB1 expression. At the molecular level, TGF-beta induces SETDB1 recruitment by Smad3, to repress Smad3/4-activated transcription of SNAI1, encoding the EMT "master" transcription factor SNAIL1. Suppression of SNAIL1-mediated gene reprogramming by SETDB1 occurs through H3K9 methylation at the SNAI1 gene that represses its H3K9 acetylation imposed by activated Smad3/4 complexes. SETDB1 therefore defines a TGF-beta-regulated balance between histone methylation and acetylation that controls EMT.CI - (c) 2017 The Authors.

BACKGROUND: Aberrant expression of miRNAs was involved in tumor initiation, progression and metastasis in multiple cancers. Many kinds of microRNAs in esophageal squamous cell carcinoma (ESCC) have been researched, whereas miR-30c has not been included. METHODS: Firstly, we explored the expression of miR-30c in ESCC tissue and serum samples and its relations to the survival. To further investigate its effects on ESCC cells, we completed a series of experiments. We detected the effects of ectopic miR-30c expression on the proliferation, migration and invasion of ESCC cells in vitro. We identified the target role of SNAI1 in ESCC using Dual-luciferase reporter assay and western blot assay. RESULTS: The results showed miR-30c was significant down-regulated in ESCC tissues and cell lines. Clinically, we found lower miR-30c expression was significantly correlated with worse ESCC progression and survival. Also we clarified that miR-30c suppressed cell proliferation, invasion and epithelial to mesenchymal transition (EMT) of ESCC cell lines. What's more, we figured out that miR-30c inhibits ESCC biological behaviors and EMT progress by directly binding to the 3'-UTR of SNAI1. CONCLUSION: This study provides new insight into the mechanism responsible for the development of human ESCC. Therefore, miR-30c could be a promising biomarker and a therapeutic target for ESCC in the future.CI - Copyright (c) 2017 Elsevier Masson SAS. All rights reserved.

The E3 ubiquitin ligase F-box and WD repeat domain containing 7 (FBW7alpha) functions as a putative tumor suppressor in non-small cell lung cancer (NSCLC) due to its regulation of a set of oncogenic proteins associated with cell proliferation and mitosis. Increasing efforts have been focused on the understanding of FBW7 in determining cell cycle progression and apoptosis induction, however, the correlation between FBW7 and tumor metastasis is not fully understood. In this study, we reported a potential anti-metastatic effect of FBW7 in non-small cell lung cancer (NSCLC). In this model, FBW7 inhibited cancer cell metastasis primarily by inducing ubiquitination and proteolysis of the transcriptional factor Snail, which suppressed E-cadherin cell tight junction protein expression. Loss of FBW7 would stabilize the Snail protein, thus, inhibit E-cadherin expression and promote metastasis in vitro and in vivo. Moreover, Snail ubiquitination and degradation were also achieved by pharmacological approach, in which the FBW7 agonist oridonin treatment led to Snail proteolysis. Furthermore, FBW7 silencing stabilized Snail protein and induced epithelial-to mesenchymal transition (EMT), and acquisition of migration and invasion properties in NSCLC. Overall, our study provides new insights into the FBW7-Snail axis in regulating cell migration and invasion, and suggests that targeting FBW7 may be a potent approach to inhibit metastasis in NSCLC.CI - Copyright (c) 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

OBJECTIVE: Orofacial development is a complex process subjected to failure impairing. Indeed, the cleft of the lip and/or of the palate is among the most frequent inborn malformations. The JARID2 gene has been suggested to be involved in non-syndromic cleft lip with or without cleft palate (nsCL/P) etiology. JARID2 interacts with the polycomb repressive complex 2 (PRC2) in regulating the expression patterns of developmental genes by modifying the chromatin state. MATERIALS AND METHODS: Genes coding for the PRC2 components, as well as other genes active in cell differentiation and embryonic development, were selected for a family-based association study to verify their involvement in nsCL/P. A total of 632 families from Italy and Asia participated to the study. RESULTS: Evidence of allelic association was found with polymorphisms of SNAI1; in particular, the rs16995010-G allele was undertransmitted to the nsCL/P cases [P = 0.004, odds ratio = 0.69 (95% C.I. 0.54-0.89)]. However, the adjusted significance value corrected for all the performed tests was P = 0.051. CONCLUSIONS: The findings emerging by the present study suggest for the first time an involvement of SNAI1 in the nsCL/P onset. CLINICAL RELEVANCE: Interestingly, SNAI1 is known to promote epithelial to mesenchymal transition by repressing E-cadherin expression, but it needs an intact PRC2 to act this function. Alterations of this process could contribute to the complex etiology of nsCL/P.

Cyclindependent kinase 10 (CDK10) has been indicated to be a candidate tumor suppressor in multiple cancer types. However, to the best of the authors' knowledge, its biological and regulatory functions in glioma have not been previously reported. In the present study, it was demonstrated that overexpression of CDK10 inhibited glioma cell proliferation and metastasis. By contrast, knockdown of CDK10 expression promoted these malignant phenotypes. It was additionally indicated that dysregulated CDK10 expression was associated with epithelialmesenchymal transition (EMT) and that it regulated the expression of zinc finger protein SNAI1 (Snail). Furthermore, silencing Snail expression rescued EMT phenotypes induced by CDK10 knockdown, suggesting that Snail may be involved in the mechanistic association between CDK10 and EMT. The present study illustrated that downregulation of CDK10 expression activated Snaildriven EMT and consequently promoted glioma metastasis, suggesting that CDK10 may serve as a potential molecular target for glioma therapy.

Lung cancer patients with human immunodeficiency virus (HIV) have a poorer prognosis than do patients without HIV infection. HIV1 Tat is a secreted viral protein that penetrates the plasma membrane and interacts with a number of proteins in non-HIV-infected cells. The loss of function of Tat-interacting protein 30 (TIP30) has been linked to metastasis in non-small cell lung cancer (NSCLC). However, it is unknown how the interaction of HIV1 Tat with TIP30 regulates the metastasis of NSCLC cells. In this study, the overexpression of TIP30 decreased tumor growth factor-beta-induced epithelial-to-mesenchymal transition (EMT) and invasion of NSCLC cells, whereas the knockdown of TIP30 promoted EMT, invasion and stemness. Exposure to recombinant HIV1 Tat proteins promoted EMT and invasion. A mechanistic study showed that the interaction of HIV1 Tat with TIP30 blocked the binding of TIP30 to importin-beta, which is required for the nuclear translocation of Snail. Indeed, the loss of TIP30 promoted the nuclear translocation of Snail. In vivo studies demonstrated that the overexpression of TIP30 inhibited the metastasis of NSCLC cells. In contrast, the coexpression of HIV1 Tat and TIP30 diminished the inhibitory effect of TIP30 on metastasis. Immunohistochemistry confirmed that TIP30 overexpression reduced the nuclear localization of Snail, whereas the coexpression of HIV1 Tat and TIP30 increased nuclear Snail in metastatic tumors. In conclusion, the binding of HIV1 Tat to TIP30 enhanced EMT and metastasis by regulating the nuclear translocation of Snail. Targeting Tat-interacting proteins may be a potential therapeutic strategy to prevent metastasis in NSCLC patients with HIV infection.CI - (c) 2018 The Authors. cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese cancer Association.

Forkhead Box F1 (FOXF1) has been recently implicated in cancer progression and metastasis of lung cancer and breast cancer. However, the biological functions and underlying mechanisms of FOXF1 in the regulation of the progression of colorectal cancer (CRC) are largely unknown. We showed that FOXF1 was up-regulated in 93 paraffin-embedded archived human CRC tissue, and both high expression and nuclear location of FOXF1 were significantly associated with the aggressive characteristics and poorer survival of CRC patients. The GSEA analysis showed that the higher level of FOXF1 was positively associated with an enrichment of EMT gene signatures, and exogenous overexpression of FOXF1 induced EMT by transcriptionally activating SNAI1. Exogenous overexpression FOXF1 functionally promoted invasion and metastasis features of CRC cells, and inhibition of SNAI1 attenuates the invasive phenotype and metastatic potential of FOXF1-overexpressing CRC cells. Furthermore, the results of the tissue chip showed that the expression of FOXF1 was positively correlated with SNAI1 in CRC tissues chip. These results suggested that FOXF1 plays a critical role in CRC metastasis by inducing EMT via transcriptional activation of SNAI1, highlighting a potential new therapeutic strategy for CRC.CI - Copyright (c) 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Migration and invasion of cancer cells are greatly increased during epithelial to mesenchymal transition (EMT). Depletion of TRIM21 promotes the migration and invasion of MCF7 and T47D cells, and changes the expression of genes that regulate EMT. TRIM21 interacts with Snail, a master regulator of EMT. Overexpression of TRIM21 leads to increased ubiquitination and proteosomal degradation of Snail, while depletion of TRIM21 decreases the ubiquitination of Snail. Importantly, depletion of Snail suppresses the increased migration and invasion of MCF7 and T47D cells promoted by depletion of TRIM21. High-level expression of TRIM21 is associated with longer overall survival in breast cancer. Together, our study demonstrates that TRIM21 modulates EMT by mediating the stability of Snail in breast cancer cells.CI - Copyright (c) 2018 Elsevier B.V. All rights reserved.

Ubiquitin specific peptidase 11 (USP11) is a deubiquitinating enzyme (DUB) and has been observed to promote mammary tumor initiation and progression. Several reports have shown that USP11 plays an important role in cancer development. Previous research has shown that USP11 is upregulated in breast cancer, but the function of USP11 in ovarian cancer has not been determined to date. In the present study, we observed that USP11 was upregulated in ovarian cancer tissues and cell lines. To determine the role of USP11 in ovarian cancer, we performed several functional experiments, including Transwell migration and invasion analysis, in vitro and in vivo deubiquitination assays, colony formation assays, and CCK8 assays. Notably, we observed that USP11 facilitated the epithelialtomesenchymal transition (EMT), which is an important process in cancer cell invasion and metastasis. Furthermore, we observed that USP11 regulated EMT through the regulation of Snail. Our results revealed that USP11 promoted EMT in ovarian cancer by deubiquitinating Snail, and USP11 may be a novel therapeutic target for ovarian cancer treatment.

Members of the Snail gene superfamily, which encode zinc finger transcriptional repressors, play critical roles in the establishment of the vertebrate body plan. The Snail1 (Snai1) gene promotes epithelial-mesenchymal transitions during development and disease progression, and Snai1 null mouse embryos exhibit defects in gastrulation. However, the early embryonic lethality of Snai1 null embryos precludes the study of Snai1 function in other developmental contexts or diseases. To overcome this restriction, we generated a Snai1 conditional null allele by flanking the promoter and first two exons of the Snai1 gene with loxP sites. Cre-mediated deletion of the Snai1(flox) allele generates the Snai1(del2) allele, which behaves genetically as a Snai1 null allele. This conditional null allele will enable investigation of Snai1 function in a variety of developmental and pathological contexts.CI - (c) 2006 Wiley-Liss, Inc.

epithelial-mesenchymal transition (EMT) plays important roles in various physiological and pathological processes, and is regulated by signaling pathways mediated by cytokines, including transforming growth factor beta (TGFbeta). Embryonic endothelial cells also undergo differentiation into mesenchymal cells during heart valve formation and aortic maturation. However, the molecular mechanisms that regulate such endothelial-mesenchymal transition (EndMT) remain to be elucidated. Here we show that TGFbeta plays important roles during mural differentiation of mouse embryonic stem cell-derived endothelial cells (MESECs). TGFbeta2 induced the differentiation of MESECs into mural cells, with a decrease in the expression of the endothelial marker claudin 5, and an increase in expression of the mural markers smooth muscle alpha-actin, SM22alpha and calponin, whereas a TGFbeta type I receptor kinase inhibitor inhibited EndMT. Among the transcription factors involved in EMT, Snail was induced by TGFbeta2 in MESECs. Tetracycline-regulated expression of Snail induced the differentiation of MESECs into mural cells, whereas knockdown of Snail expression abrogated TGFbeta2-induced mural differentiation of MESECs. These results indicate that Snail mediates the actions of endogenous TGFbeta signals that induce EndMT.

epithelial-mesenchymal transition (EMT) is required for mesodermal differentiation during development. The zinc-finger transcription factor, Snail1, can trigger EMT and is sufficient to transcriptionally reprogram epithelial cells toward a mesenchymal phenotype during neoplasia and fibrosis. Whether Snail1 also regulates the behavior of terminally differentiated mesenchymal cells remains unexplored. Using a Snai1 conditional knockout model, we now identify Snail1 as a regulator of normal mesenchymal cell function. Snail1 expression in normal fibroblasts can be induced by agonists known to promote proliferation and invasion in vivo. When challenged within a tissue-like, three-dimensional extracellular matrix, Snail1-deficient fibroblasts exhibit global alterations in gene expression, which include defects in membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invasive activity. Snail1-deficient fibroblasts explanted atop the live chick chorioallantoic membrane lack tissue-invasive potential and fail to induce angiogenesis. These findings establish key functions for the EMT regulator Snail1 after terminal differentiation of mesenchymal cells.

Fibronectins are cell-secreted glycoproteins that modulate cell attachment, spreading, migration, morphology, differentiation and oncogenic transformation. Fibronectin expression is activated during EMT (epithelial-mesenchymal transition) and is a hallmark of mesenchymal cells. It is shown in the present study that a transcription factor previously unrelated with EMT, TFCP2c/LSF/LBP-1c, was translocated to the nucleus and bound to the fibronectin promoter upon EMT induction by Snail1. Consequently, the interference of TFCP2c/LSF/LBP-1c's activity prevented fibronectin expression. Moreover, TFCP2c/LSF/LBP-1c was detected in nuclei of embryonic dermal mesenchymal cells adjacent to the hair bud, a cell population that expresses endogenous nuclear Snail1 and fibronectin. Therefore we indicate a new molecular role for TFCP2c/LSF/LBP-1c in fibronectin expression.

Several E-box-binding transcription factors regulate individual and collective cell migration and enhance the motility of epithelial cells by promoting epithelial-mesenchymal transition (EMT). Here, we characterized the role of a subset of these transcription factors and the EMT proteome in branching morphogenesis of mammary epithelial tissues using a three-dimensional organotypic culture model of the mammary duct. We found that the transcription factors Snail1, Snail2, and E47 were transiently upregulated at branch sites; decreasing the expression of these transcription factors inhibited branching. Conversely, ectopic expression of Snail1, Snail2, and E47 induced branching in the absence of exogenous stimuli. These changes correlated with the expression of mesenchymal markers and repression of E-cadherin, which was essential for branching. Snail1 and Snail2 also promoted cell survival at branch sites, but this was not sufficient to induce branching. These findings indicate that Snail1, Snail2, and E47 can promote collective migration during branching morphogenesis of mammary epithelial tissues through key regulators of EMT.

Mouse embryonic cells isolated from focal adhesion kinase (FAK)-null animals at embryonic day 7.5 display multiple defects in focal adhesion remodeling, microtubule dynamics, mechanotransduction, proliferation, directional motility, and invasion. To date, the ability of FAK to modulate cell function has been ascribed largely to its control of posttranscriptional signaling cascades in this embryonic cell population. In this paper, we demonstrate that FAK unexpectedly exerts control over an epithelial-mesenchymal transition (EMT) program that commits embryonic FAK-null cells to an epithelial status highlighted by the expression of E-cadherin, desmoplakin, and cytokeratins. FAK rescue reestablished the mesenchymal characteristics of FAK-null embryonic cells to generate committed mouse embryonic fibroblasts via an extracellular signal-related kinase- and Akt-dependent signaling cascade that triggered Snail1 gene expression and Snail1 protein stabilization. These findings indentify FAK as a novel regulator of Snail1-dependent EMT in embryonic cells and suggest that multiple defects in FAK(-/-) cell behavior can be attributed to an inappropriate commitment of these cells to an epithelial, rather than fibroblastic, phenotype.

epithelial-mesenchymal transition (EMT) is a crucial step for the acquisition of invasive properties of carcinoma cells during tumor progression. Epidermal growth factor (EGF)-treatment of squamous cell carcinoma (SCC) cells provokes changes in the expression of lineage markers, morphological changes, and a higher invasive and metastatic potential. Here we show that chronic stimulation with EGF induces EMT in skin-derived SCC cell lines along with the down-regulation of the epithelial marker E-cadherin, and of the putative tumor suppressor VILIP-1 (visinin-like protein 1). In esophageal squamous cell carcinoma and non-small cell lung carcinoma the loss of VILIP-1 correlates with clinicopathological features related to enhanced invasiveness. VILIP-1 has previously been shown to suppress tumor cell invasion via enhancing cAMP-signaling in a murine SCC model. In mouse skin SCC cell lines the VILIP-1-negative tumor cells have low cAMP levels, whereas VILIP-1-positive SCCs possess high cAMP levels, but low invasive properties. We show that in VILIP-1-negative SCCs, Snail1, a transcriptional repressor involved in EMT, is up-regulated. Snail1 expression is reduced by ectopic VILIP-1-expression in VILIP-1-negative SCC cells, and application of the general adenylyl cyclase inhibitor 2',3'-dideoxyadenosine attenuated this effect. Conversely, EGF-stimulation of VILIP-1-positive SCC cells leads to the down-regulation of VILIP-1 and the induction of Snail1 expression. The induction of Snail is inhibited by elevated cAMP levels. The role of cAMP in EMT was further highlighted by its suppressive effect on the EGF-induced enhancement of migration in VILIP-1-positive SCC cells. These findings indicate that VILIP-1 is involved in EMT of SCC by regulating the transcription factor Snail1 in a cAMP-dependent manner.

The Snail genes are implicated in processes that involve cell movement, both during embryonic development and tumour progression. In teleosts, the vertebrate Snail1 gene is represented by two distinct genes, snail1a and snail1b (previously snail1 and snail2). These genes are expressed in complementary mesodermal domains and their combined expression matches that of their mammalian counterpart. By analysing their loss and gain of function, we found that the most-anterior axial mesendodermal cells, the precursors of the polster, move in a cohesive manner directed by the activity of snail1a- and snail1b-expressing cells surrounding these precursors. The cell-autonomous function of Snail1 proteins regulates cell motility and influences the behaviour of Snail-negative neighbouring cells. Snail1a is required by the prechordal plate for it to reach its normal position, whereas Snail1b controls the acquisition of its normal shape. These non-redundant functions of Snail1a and Snail1b in controlling axial mesendoderm migration comply with the duplication-degeneration-complementation model, and indicate that Snail genes not only act as inducers of epithelial-to-mesenchymal transition, but also as more general regulators of cell adhesion and movement.

Snail1 (Snail) and Snail2 (Slug) are transcription factors that share a similar DNA binding structure of four and five C2H2 zinc finger motifs (ZF), respectively. Both factors bind specifically to a subset of E-box motifs (E2-box: CAGGTG/CACCTG) in target promoters like the E-cadherin promoter and are key mediators of epithelial-to-mesenchymal transition (EMT). However, there are differences in the biological actions, in binding affinities to E-cadherin promoter, and in the target genes of Snail1 and Snail2, although the molecular bases are presently unknown. In particular, the role of each Snail1 and Snail2 ZF in the binding to E-boxes and in EMT induction has not been previously explored. We have approached this question by modeling Snail1 and Snail2 protein-DNA interactions and through mutational and functional assays of different ZFs. Results show that Snail1 efficient repression and binding to human and mouse E-cadherin promoter as well as EMT-inducing ability require intact ZF1 and ZF2, while for Snail2, either ZF3 or ZF4 is essential for those functions. Furthermore, the differential distribution of E2-boxes in mouse and human E-cadherin promoters also contributes to the differential Snail factor activity. These data indicate a non-equivalent role of Snail1 and Snail2 ZFs in gene repression, contributing to the elucidation of the molecular differences between these important EMT regulators.

epithelial-mesenchymal transition (EMT) is characterized by loss of cell-cell junctions, polarity and epithelial markers, and in turn, acquisition of mesenchymal features and motility. Changes associated with this developmental process have been extensively implicated in breast cancer progression and metastasis. Matrix metalloproteinases (MMPs) have been identified as specific inducers of EMT in mammary epithelial cells. MMP-3 induces EMT associated with malignant transformation via a pathway dependent upon production of reactive oxygen species (ROS). While the process by which exposure to MMP-3 leads to induction of ROS has been extensively studied, exactly how the MMP-3-induced ROS stimulate EMT remains unknown. Here, we used profiling methods to identify MMP-3-induced transcriptional alterations in mouse mammary epithelial cells, finding common overlap with changes mediated by nuclear factor-kappaB (NF-kappaB) and found in advanced breast cancer. In cultured cells, we found that Snail, an ROS-dependent key mediator of MMP-3-induced changes, is regulated by NF-kappaB in response to MMP-3. More specifically, we found MMP-3 to cause binding of p65 and cRel NF-kappaB subunits to the Snail promoter, leading to its transcription. Our results identify a specific pathway by which MMPs induce EMT and malignant characteristics, and provide insight into potential therapeutic approaches to target MMP-associated breast cancers.

Reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) entails a mesenchymal to epithelial transition (MET). While attempting to dissect the mechanism of MET during reprogramming, we observed that knockdown (KD) of the epithelial-to-mesenchymal transition (EMT) factor SNAI1 (SNAIL) paradoxically reduced, while overexpression enhanced, reprogramming efficiency in human cells and in mouse cells, depending on strain. We observed nuclear localization of SNAI1 at an early stage of fibroblast reprogramming and using mouse fibroblasts expressing a knockin SNAI1-YFP reporter found cells expressing SNAI1 reprogrammed at higher efficiency. We further demonstrated that SNAI1 binds the let-7 promoter, which may play a role in reduced expression of let-7 microRNAs, enforced expression of which, early in the reprogramming process, compromises efficiency. Our data reveal an unexpected role for the EMT factor SNAI1 in reprogramming somatic cells to pluripotency.CI - Copyright (c) 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Peritoneal dialysis (PD) is a form of renal replacement therapy whose repeated use can alter dialytic function through induction of epithelial-mesenchymal transition (EMT) and fibrosis, eventually leading to PD discontinuation. The peritoneum from Cav1-/- mice showed increased EMT, thickness, and fibrosis. Exposure of Cav1-/- mice to PD fluids further increased peritoneal membrane thickness, altered permeability, and increased the number of FSP-1/cytokeratin-positive cells invading the sub-mesothelial stroma. High-throughput quantitative proteomics revealed increased abundance of collagens, FN, and laminin, as well as proteins related to TGF-beta activity in matrices derived from Cav1-/- cells. Lack of Cav1 was associated with hyperactivation of a MEK-ERK1/2-Snail-1 pathway that regulated the Smad2-3/Smad1-5-8 balance. Pharmacological blockade of MEK rescued E-cadherin and ZO-1 inter-cellular junction localization, reduced fibrosis, and restored peritoneal function in Cav1-/- mice. Moreover, treatment of human PD-patient-derived MCs with drugs increasing Cav1 levels, as well as ectopic Cav1 expression, induced re-acquisition of epithelial features. This study demonstrates a pivotal role of Cav1 in the balance of epithelial versus mesenchymal state and suggests targets for the prevention of fibrosis during PD.CI - (c) 2014 The Authors. Published under the terms of the CC BY 4.0 license.

The miR-200 family promotes the epithelial state by suppressing the Zeb1/Zeb2 epithelial gene transcriptional repressors. To identify other miR-200-regulated genes, we isolated mRNAs bound to transfected biotinylated miR-200c in mouse breast cancer cells. In all, 520 mRNAs were significantly enriched in miR-200c binding at least twofold. Putative miR-200-regulated genes included Zeb2, enriched 3.5-fold in the pull down. However, Zeb2 knockdown does not fully recapitulate miR-200c overexpression, suggesting that regulating other miR-200 targets contributes to miR-200's enhancement of epithelial gene expression. Candidate genes were highly enriched for miR-200c seed pairing in their 3'UTR and coding sequence and for genes that were downregulated by miR-200c overexpression. Epidermal growth factor receptor and downstream MAPK signaling pathways were the most enriched pathways. Genes whose products mediate transforming growth factor (TGF)-beta signaling were also significantly overrepresented, and miR-200 counteracted the suppressive effects of TGF-beta and bone morphogenic protein 2 (BMP-2) on epithelial gene expression. miR-200c regulated the 3'UTRs of 12 of 14 putative miR-200c-binding mRNAs tested. The extent of mRNA binding to miR-200c strongly correlated with gene suppression. Twelve targets of miR-200c (Crtap, Fhod1, Smad2, Map3k1, Tob1, Ywhag/14-3-3gamma, Ywhab/14-3-3beta, Smad5, Zfp36, Xbp1, Mapk12, Snail1) were experimentally validated by identifying their 3'UTR miR-200 recognition elements. Smad2 and Smad5 form a complex with Zeb2 and Ywhab/14-3-3beta and Ywhag/14-3-3gamma form a complex with Snail1. These complexes that repress transcription assemble on epithelial gene promoters. miR-200 overexpression induced RNA polymerase II localization and reduced Zeb2 and Snail1 binding to epithelial gene promoters. expression of miR-200-resistant Smad5 modestly, but significantly, reduced epithelial gene induction by miR-200. miR-200 expression and Zeb2 knockdown are known to inhibit cell invasion in in vitro assays. Knockdown of each of three novel miR-200 target genes identified here, Smad5, Ywhag and Crtap, also profoundly suppressed cell invasion. Thus, miR-200 suppresses TGF-beta/BMP signaling, promotes epithelial gene expression and suppresses cell invasion by regulating a network of genes.

Loss of apical-basal polarity and activation of epithelial-mesenchymal transition (EMT) both contribute to carcinoma progression and metastasis. Here, we report that apical-basal polarity inhibits EMT to suppress metastatic dissemination. Using mouse and human epithelial three-dimensional organoid cultures, we show that the PAR-atypical protein kinase C (aPKC) polarity complex inhibits EMT and invasion by promoting degradation of the SNAIL family protein SNAI1. Under intact apical-basal polarity, aPKC kinases phosphorylate S249 of SNAI1, which leads to protein degradation. Loss of apical-basal polarity prevents aPKC-mediated SNAI1 phosphorylation and stabilizes the SNAI1 protein to promote EMT and invasion. In human breast tumour xenografts, inhibition of the PAR-complex-mediated SNAI1 degradation mechanism promotes tumour invasion and metastasis. Analyses of human breast tissue samples reveal negative correlations between PAR3 and SNAI1 protein levels. Our results demonstrate that apical-basal polarity functions as a critical checkpoint of EMT to precisely control epithelial-mesenchymal plasticity during tumour metastasis.

Paraquat (PQ) poisoning-induced pulmonary fibrosis is one of the primary causes of death in patients with PQ poisoning. Hypoxia-inducible factor-1alpha (HIF-1alpha) and epithelial-mesenchymal transition (EMT) are involved in the progression of pulmonary fibrosis. Snail and beta-catenin are two other factors involved in promoting EMT. However, the relationship among HIF-1alpha, Snail and beta-catenin in PQ poisoning-induced pulmonary fibrosis is not clear. Our research aimed to determine whether the regulation of HIF-1alpha in EMT occurs via the Snail and beta-catenin pathways in PQ poisoning-induced pulmonary fibrosis. Sixty-six Sprague-Dawley rats were randomly and evenly divided into a control group and a PQ group. The PQ group was treated with an intragastric infusion of a 20% PQ solution (50 mg/kg) for 2, 6, 12, 24, 48 and 72 hrs. A549 and RLE-6TN cell lines were transfected with HIF-1alpha siRNA for 48 hrs before being exposed to PQ. Western blotting, real-time quantitative PCR, immunofluorescence, immunohistochemistry and other assays were used in our research. In vivo, the protein levels of HIF-1alpha and alpha-SMA were increased at 2 hrs and the level of ZO-1 (Zonula Occluden-1) was reduced at 12 hrs. In vitro, the transient transfection of HIF-1alpha siRNA resulted in a decrease in the degree of EMT. The expression levels of Snail and beta-catenin were significantly reduced when HIF-alpha was silenced. These data demonstrate that EMT may be involved in PQ poisoning-induced pulmonary fibrosis and regulated by HIF-1alpha via the Snail and beta-catenin pathways. Hypoxia-inducible factor-1alpha may be a therapeutic target for the treatment of PQ poisoning-induced pulmonary fibrosis.CI - (c) 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.