6591

SNAI2

Although dysregulation of tight junction (TJ) proteins is observed in epithelial malignancy, their participation in epithelial transformation is poorly understood. Recently we demonstrated that expression of oncogenic Raf 1 in Pa4 epithelial cells disrupts TJs and induces an oncogenic phenotype by downregulating expression of the TJ protein, occludin. Here we report the mechanism by which Raf 1 regulates occludin expression. Raf 1 inhibited occludin transcription by repressing a minimal segment of the occludin promoter in concert with upregulation of the transcriptional repressor, Slug without influencing the well-documented transcriptional repressor, Snail. Overexpression of Slug in Pa4 cells recapitulated the effect of Raf 1 on occludin expression, and depletion of Slug by small interfering RNA abrogated the effect of Raf 1 on occludin. Finally, chromatin immunoprecipitation assays and site-directed mutagenesis demonstrated a direct interaction between Slug and an E-box within the minimal Raf 1-responsive segment of the occludin promoter. These findings support a role of Slug in mediating Raf 1-induced transcriptional repression of occludin and subsequent epithelial to mesenchymal transition.

Aberrant expression of Jagged1 and Notch1 are associated with poor outcome in breast cancer. However, the reason that Jagged1 and/or Notch overexpression portends a poor prognosis is unknown. We identify Slug, a transcriptional repressor, as a novel Notch target and show that elevated levels of Slug correlate with increased expression of Jagged1 in various human cancers. Slug was essential for Notch-mediated repression of E-cadherin, which resulted in beta-catenin activation and resistance to anoikis. Inhibition of ligand-induced Notch signaling in xenografted Slug-positive/E-cadherin-negative breast tumors promoted apoptosis and inhibited tumor growth and metastasis. This response was associated with down-regulated Slug expression, reexpression of E-cadherin, and suppression of active beta-catenin. Our findings suggest that ligand-induced Notch activation, through the induction of Slug, promotes tumor growth and metastasis characterized by epithelial-to-mesenchymal transition and inhibition of anoikis.

epithelial-mesenchymal transition (EMT) is a critical process occurring during embryonic development and in fibrosis and tumor progression. Dissociation of cell-cell contacts and remodeling of the actin cytoskeleton are major events of the EMT. Here, we show that myocardin-related transcription factors (MRTFs; also known as MAL and MKL) are critical mediators of transforming growth factor beta (TGF-beta) 1-induced EMT. In all epithelial cell lines examined here, TGF-beta1 triggers the nuclear translocation of MRTFs. Ectopic expression of constitutive-active MRTF-A induces EMT, whereas dominant-negative MRTF-A or knockdown of MRTF-A and -B prevents the TGF-beta1-induced EMT. MRTFs form complexes with Smad3. Via Smad3, the MRTF-Smad3 complexes bind to a newly identified cis-element GCCG-like motif in the promoter region of Canis familiaris and the human slug gene, which activates slug transcription and thereby dissociation of cell-cell contacts. MRTFs also increase the expression levels of actin cytoskeletal proteins via serum response factor, thereby triggering reorganization of the actin cytoskeleton. Thus, MRTFs are important mediators of TGF-beta1-induced EMT.

The short splice variant of the basic helix-loop-helix Per-Arnt-Sim transcription factor Singleminded-2, SIM2s, has been implicated in development and is frequently lost or reduced in primary breast tumors. Here, we show that loss of Sim2s causes aberrant mouse mammary gland ductal development with features suggestive of malignant transformation, including increased proliferation, loss of polarity, down-regulation of E-cadherin, and invasion of the surrounding stroma. Additionally, knockdown of SIM2s in MCF-7 breast cancer cells contributed to an epithelial-mesenchymal transition (EMT) and increased tumorigenesis. In both Sim2(-/-) mammary glands and SIM2s-depleted MCF7 cells, these changes were associated with increased SLUG and MMP2 levels. SIM2s protein was detectable on the SLUG promoter, and overexpression of SIM2s repressed expression from a SLUG-controlled reporter in a dose-dependent manner. To our knowledge, SIM2s is the first protein shown to bind and repress the SLUG promoter, providing a plausible explanation for the development role and breast tumor-suppressive activity of SIM2s. Together, our results suggest that SIM2s is a key regulator of mammary-ductal development and that loss of SIM2s expression is associated with an invasive, EMT-like phenotype.

BACKGROUND: Acquired features of cells under epithelial-mesenchymal transition (EMT) have not yet been fully identified. The current study was conducted to assess alterations in both the proliferative potential and the responsiveness to extracellular matrices (ECMs) in EMT. METHODS: MDCK cells and SLUG-transfected MDCK clones (SLUG-MDCK) were used in this study. The cell cycle was analyzed by using flow cytometry and Western blotting. ECM-stimulated cell proliferation was examined by using the following ECMs, type I collagen, type IV collagen, fibronectin, and laminin. Protein phosphorylation was detected by immunoprecipitation-Western by using the 4G10 antibody. RESULTS: Both G1 and G2/M arrest were found in the SLUG-MDCK cells, and the responsible molecules for the cell-cycle arrests were, at least in part, p21WAF1/Cip1 and Wee1. Once in contact with type I collagen, the SLUG-MDCK cells, showing the Wee1 degradation, dramatically started to proliferate up to 6-fold in cell number at Day 5, in contrast to only a 2-fold increase in the control. The analysis of the collagen receptors in the SLUG-MDCK cells disclosed a striking increase in the discoid domain receptor (DDR) 2 expression and a clear decrease in the DDR1 expression. The immunoprecipitated DDR2 protein extracted from SLUG-MDCK cells, which were cultured on collagen for 30 minutes, was tyrosine-phosphorylated, indicating valid functionality of the up-regulated receptor. The altered expression from DDR1 to DDR2 was also found in the naturally dedifferentiated sister cell lines of human liver cancer. CONCLUSIONS: Collectively, SLUG-induced EMT may alter the expression profile of receptor tyrosine kinases, including DDRs.

Here, we report that lipocalin 2 (Lcn2) promotes breast cancer progression, and we identify the mechanisms underlying this function. We first found that Lcn2 levels were consistently associated with invasive breast cancer in human tissue and urine samples. To investigate the function of Lcn2 in breast cancer progression, Lcn2 was overexpressed in human breast cancer cells and was found to up-regulate mesenchymal markers, including vimentin and fibronectin, down-regulate the epithelial marker E-cadherin, and significantly increase cell motility and invasiveness. These changes in marker expression and cell motility are hallmarks of an epithelial to mesenchymal transition (EMT). In contrast, Lcn2 silencing in aggressive breast cancer cells inhibited cell migration and the mesenchymal phenotype. Furthermore, reduced expression of estrogen receptor (ER) alpha and increased expression of the key EMT transcription factor Slug were observed with Lcn2 expression. Overexpression of ERalpha in Lcn2-expressing cells reversed the EMT and reduced Slug expression, suggesting that ERalpha negatively regulates Lcn2-induced EMT. Finally, orthotopic studies demonstrated that Lcn2-expressing breast tumors displayed a poorly differentiated phenotype and showed increased local tumor invasion and lymph node metastasis. Taken together, these in vitro, in vivo, and human studies demonstrate that Lcn2 promotes breast cancer progression by inducing EMT through the ERalpha/Slug axis and may be a useful biomarker of breast cancer.

epithelial-mesenchymal transition (EMT) induced by transforming growth factor-beta (TGF-beta) is implicated in hepatocarcinogenesis and hepatocellular carcinoma (HCC) metastasis. HAb18G/CD147, which belongs to the CD147 family, is an HCC-associated antigen that has a crucial role in tumor invasion and metastasis. The goal of this study was to investigate the role of HAb18G/CD147 during EMT in hepatocarcinogenesis. Human normal hepatic cell lines QZG and L02, primary mouse hepatocytes and nude mouse models were used to determine the role of HAb18G/CD147 in EMT, and the involvement of the TGF-beta-driven pathway. A dual-luciferase reporter assay and ChIP were used to investigate the transcriptional regulation of the CD147 gene. Samples from patients with liver disease were assessed to determine the relationship between HAb18G/CD147 and typical markers for EMT. Our results show that upregulation of HAb18G/CD147 is induced by TGF-beta coupled with downregulation of E-cadherin and upregulation of N-cadherin and vimentin. The expression of HAb18G/CD147 is controlled by the cell survival PI3K/Akt/GSK3beta signaling pathway, and is directly regulated by the transcription factor Slug. Transfection of CD147 also induces an elevated expression of TGF-beta. CD147-transfected hepatocytes have mesenchymal phenotypes that accelerate tumor formation and tumor metastasis in vivo. Immunohistochemistry analysis shows a negative correlation between HAb18G/CD147 and E-cadherin expression (r(s)=-0.3622, P=0.0105), and a positive correlation between HAb18G/CD147 and Slug expression (r(s)=0.3064, P=0.0323) in human HCC tissues. Our study uncovers a novel role of HAb18G/CD147 in mediating EMT in the process of HCC progression and showed that CD147 is a Slug target gene in the signaling cascade TGF-beta-->PI3K/Akt-->GSK3beta-->Snail-->Slug-->CD147. Our results suggest that CD147 may be a potential target for the treatment and prevention of HCC.

The epithelial-mesenchymal transition (EMT) is a complex process that occurs during organogenesis and in cancer metastasis. Despite recent progress, the molecular pathways connecting the physiological and pathological functions of EMT need to be better defined. Here we show that the transcription factor Elf5, a key regulator of mammary gland alveologenesis, controls EMT in both mammary gland development and metastasis. We uncovered this role for Elf5 through analyses of Elf5 conditional knockout animals, various in vitro and in vivo models of EMT and metastasis, an MMTV-neu transgenic model of mammary tumour progression and clinical breast cancer samples. Furthermore, we demonstrate that Elf5 suppresses EMT by directly repressing the transcription of Snail2, a master regulator of mammary stem cells and a known inducer of EMT. These findings establish Elf5 not only as a key cell lineage regulator during normal mammary gland development, but also as a suppressor of EMT and metastasis in breast cancer.

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.

The ETS family of transcription factors is involved in several physiological and pathological processes including tumor progression. The ETS transcription factors are divided into subfamilies based on the sequence and location of the ETS domain. ETV1 (Ets variant gene 1; also known as ER81), is a member of the PEA3 subfamily, which has been found to promote metastatic progression in several types of human cancer. Previous findings demonstrated that ETV1 expression is upregulated in gastric adenocarcinomas; however, the underlying mechanisms of ETV1-induced metastatic progression in gastric cancer remain elusive. In the present study, we found that the overexpression of ETV1 in normal gastric epithelial cells resulted in epithelial to mesenchymal transition (EMT) and increased invasiveness. Conversely, knockdown of ETV1 resulted in decreased aggressiveness of the invasive gastric cancer cells. Mechanistically, ETV1 transcriptionally upregulates Snail expression. Of note, ETV1 expression is significantly correlated with Snail expression in human gastric tumor samples. In summary, we present data that ETV1 promotes Snail expression to induce EMT-like metastatic progression in gastric cancer.

BACKGROUND/AIMS: MicroRNAs (miRNAs) play critical roles during carcinogenesis and cancer progression. Down-regulation of miR-204 has been frequently observed in various cancers. In this study, we investigated the roles and mechanisms of miR-204 in human intrahepatic cholangiocarcinoma (ICC). METHODS: The relative expression of miR-204 in ICC tissues and cell lines was monitored by qRT-PCR. Effects of miR-204 were studied in human ICC cell lines HuH28 and HuCCT1, and cells were analyzed for proliferation, migration and invasion. expression levels of miR-204 target gene Slug and EMT markers (E-cadherin and vimentin) in ICC cell lines and tissues were measured by qRT-PCR, western blotting and immunofluorescence. RESULTS: miR-204 was frequently downregulated in human ICC, and the low-level expression of miR-204 was significantly associated with lymph node metastasis. Overexpression of miR-204 dramatically suppressed ICC cell migration and invasion, as well as the epithelial-mesenchymal transition process (EMT). Slug was identified as a direct target of miR-204, and its downregulation by miR-204 in HuH28 cells reversed EMT, as shown by the increased expression of the epithelial marker E-cadherin and decreased expression of the mesenchymal marker vimentin. CONCLUSION: These findings suggest that miR-204 plays negative roles in the invasive and/or metastatic potential of ICC, and that its suppressive effects are mediated by repressing Slug expression.CI - (c) 2013 S. Karger AG, Basel.

Bone morphogenetic proteins (BMPs) initiate differentiation in human embryonic stem cells (hESCs) but the exact mechanisms have not been fully elucidated. We demonstrate here that SLUG and MSX2, transcription factors involved in epithelial-mesenchymal transitions, essential features of gastrulation in development and tumor progression, are important mediators of BMP4-induced differentiation in hESCs. Phosphorylated Smad1/5/8 colocalized with the SLUG protein at the edges of hESC colonies where differentiation takes place. The upregulation of the BMP target SLUG was direct as shown by the binding of phosphorylated Smad1/5/8 to its promoter, which interrupted the formation of adhesion proteins, resulting in migration. Knockdown of SLUG by short hairpin RNA blocked these changes, confirming an important role for SLUG in BMP-mediated mesodermal differentiation. Furthermore, BMP4-induced MSX2 expression leads to mesoderm formation and then preferential differentiation toward the cardiovascular lineage.CI - (c) 2013 AlphaMed Press.

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.

Androgen receptor (AR) has been indicated to be involved in bladder cancer progression. We showed androgen induced epithelial-mesenchymal transition (EMT) in AR-positive bladder cancer cells and promoted tumor metastasis in xenograft models. We subsequently identified that Slug was the mediator of EMT induced by androgen. Furthermore, upregulation of Slug was due to activation of Wnt/beta-catenin signaling in response to androgen. Finally, expression of AR showed strong correlation with loss of E-cadherin, higher expression of Slug and nuclear accumulation of beta-catenin in bladder tumor tissues. Taken together, our results suggest AR signaling promotes bladder cancer metastasis through Slug mediated EMT.CI - Copyright (c) 2014 Elsevier Ireland Ltd. All rights reserved.

The transcriptional repressor Slug is best known to control epithelial-mesenchymal transition (EMT) and promote cancer invasion/metastasis. In this study, we demonstrate that Slug is temporally regulated during cell cycle progression. At G1/S transition, cyclin E-cyclin-dependent kinase 2 mediates the phosphorylation of Slug at Ser-54 and Ser-104, resulting in its ubiquitylation and degradation. Non-phosphorylatable Slug is markedly stabilized at G1/S transition compared with wild-type Slug and greatly leads to downregulation of DNA synthesis and checkpoint-related proteins, including TOP1, DNA Ligase IV and Rad17, reduces cell proliferation, delays S-phase progression and contributes to genome instability. Our results indicate that Slug has multifaceted roles in cancer progression by controlling both EMT and genome stability.

In recent decades, the CXC chemokine receptor 7 (CCR7) [corrected] and its ligand CCL21 have been extensively reported to be associated with tumorigenesis. Meanwhile, Slug signaling induces the epithelial-mesenchymal transition (EMT) process in chondrosarcoma development. In the present study, we explored the functions of CCL21/CCR7 [corrected] in Slug-mediated EMT in the chondrosarcoma. We analyzed protein expression of CCR7 [corrected] and Slug in human chondrosarcoma samples. Effects of CCR7 [corrected] on chondrosarcoma cells were assessed by in vitro assays. Additionally, CCR7 [corrected] pathways were further investigated by pharmacological and genetic approaches. We found that the altered CCR7 [corrected] (81.7 %) and Slug (85.0 %) expression in human chondrosarcoma tissues were significantly associated with grade, recurrence, and 5-year overall survival. According to in vitro assays, CCL21 stimulation induced the expression of phosph-ERK, phosph-AKT, Slug and N-cadherin in SW1353 cells, while the expression of E-cadherin was down-regulated. Furthermore, Slug signaling modulated E- to N-cadherin switch, which was influenced by the kinase inhibitor PD98059 and LY294002. In addition, the genetic silencing of Slug inhibited the capacity of migration and invasion of SW1353 cells. In conclusion, CCL21/CCR7 [corrected] pathway activates ERK and PI3K/AKT signallings to up-regulate Slug pathway, leading to the occurrence of EMT process in human chondrosarcoma. This study lays a new foundation for molecule-targeted therapy of human chondrosarcoma.

BACKGROUND: The epithelial-mesenchymal transition (EMT) is crucial for the invasion and metastasis of breast cancer. However, how Notch signaling regulates the EMT process and invasion in breast cancer remains largely unknown. METHODS: The impact of Notch1 silencing by specific shRNAs on the EMT and invasion of human breast cancer MCF-7 and MDA-MB-231 cells as well as xenografts was tested by western blot, real-time polymerase chain reaction (RT-PCR), immunofluorescence, transwell, and immunohistochemistry assays. The effect of Slug silencing or upregulation on the EMT and invasion of breast cancer cells was analyzed, and the effect of Notch1 signaling on Slug expression was determined by the luciferase reporter assay. RESULTS: The Notch1 intracellular domain (N1ICD) and Jagged1 were expressed in breast cancer cells. Notch1 silencing reversed the spontaneous EMT process and inhibited the migration and invasion of breast cancer cells and the growth of xenograft breast cancers. The expression of N1ICD was upregulated significantly by Jagged1-mediated Notch signaling activation. Moreover, Jagged1-mediated Notch signaling promoted the EMT process, migration, and invasion of breast cancer cells, which were abrogated by Notch silencing. Furthermore, the N1ICD positively regulated the Slug expression by inducing Slug promoter activation. Importantly, the knockdown of Slug weakened the invasion ability of breast cancer cells and reversed the Jagged1-induced EMT process with significantly decreased expression of vimentin and increased expression of E-cadherin. In addition, Slug overexpression restored the Notch1 knockdown-suppressed EMT process. CONCLUSIONS: Our novel data indicate that Notch signaling positively regulates the EMT, invasion, and growth of breast cancer cells by inducing Slug expression. The Notch1-Slug signaling axis may represent a potential therapeutic target for breast cancer therapy.

BACKGROUND: Metastasis is the primary cause of prostate cancer (PCa) lethality and poses a huge clinical obstacle. Lipocalin 2 (LCN2), a member of the lipocalin family, is aberrantly expressed in some human cancers and has been implicated in the progression of some tumors. However, the role of LCN2 in the metastatic capacity of prostate cancer (PCa) is poorly understood. METHODS: LCN2 expression was examined by RT-qPCR and/or immunoblotting in human prostate tissue specimens and prostate cancer cell lines LNCaP, C4-2, 22RV1, PC3, DU-145, and PC3MM2. LCN2 protein level in human serum samples was determined by ELISA. Lentiviruses-mediated over-expression of LCN2 and knockdown of LCN2 was conducted to evaluate the role of LCN2 in cell migratory and invasive capacities of prostate cancer cells. Cell migration and invasion was examined by transwell chamber assay. Knockdown of SLUG by lentivirus was performed to investigate its role in LCN2-promoted cell migration and invasion in vitro (22RV1 cell line) and metastasis in vivo (tail vein metastasis assay in nude mice). Role of ERK signaling in LCN2-mediated up-regulation of SLUG was assayed by using ERK inhibitor U0126. RESULTS: We confirmed that LCN2 levels were correlated positively with invasive prostate cancer in human tissue and serum samples, and were also consistently associated with the invasive capacity of prostate cancer cell lines. The over-expression of LCN2 in 22RV1 cells (not highly invasive) promoted the epithelial-mesenchymal transition (EMT), increasing cell motility and invasiveness, while the knockdown of LCN2 in PC3 cells (highly invasive) inhibited EMT, decreasing cell motility and invasiveness. Among the multiple EMT transcription factors, LCN2 specifically induces the expression of SLUG, which was shown here to be required for the LCN2-induced increase in the invasive capacity of prostate cancer cells both in vitro and in vivo. Mechanistically, LCN2 promoted SLUG expression via activating ERK signaling pathway. CONCLUSION: LCN2 plays an important role in promoting cell migration and invasion of prostate cancer by inducing EMT through the ERK/SLUG axis. Therefore, targeted inhibition of LCN2 may represent a therapeutic strategy to prevent the metastasis of prostate cancer.CI - (c) 2015 Wiley Periodicals, Inc.

epithelial-mesenchymal transition (EMT) has been recognized as a key element of cell migration, invasion, and drug resistance in several types of cancer. In this study, our aim was to clarify microRNAs (miRNAs)-related mechanisms underlying EMT followed by acquired resistance to chemotherapy in glioblastoma (GBM). We used multiple methods to achieve our goal including microarray analysis, qRT-PCR, western blotting analysis, loss/gain-of-function analysis, luciferase assays, drug sensitivity assays, wound-healing assay and invasion assay. We found that miR-203 expression was significantly lower in imatinib-resistant GBM cells (U251AR, U87AR) that underwent EMT than in their parental cells (U251, U87). Ectopic expression of miR-203 with miRNA mimics effectively reversed EMT in U251AR and U87AR cells, and sensitized them to chemotherapy, whereas inhibition of miR-203 in the sensitive lines with antisense oligonucleotides induced EMT and conferred chemoresistance. SNAI2 was identified as a direct target gene of miR-203. The knockdown of SNAI2 by short hairpin RNA (shRNA) inhibited EMT and drug resistance. In GBM patients, miR-203 expression was inversely related to SNAI2 expression, and those tumors with low expression of miR-203 experienced poorer clinical outcomes. Our findings indicate that re-expression of miR-203 or targeting SNAI2 might serve as potential therapeutic approaches to overcome chemotherapy resistance in GBM.

Homeoproteins, a family of transcription factors that have conserved homeobox domains, play critical roles in embryonic development in a wide range of species. Accumulating studies have revealed that homeoproteins are aberrantly expressed in multiple tumors and function as either tumor promoters or suppressors. In this study, we show that two homeoproteins, HOXB13 and ALX4, are associated with epithelial to mesenchymal transition (EMT) and invasion of ovarian cancer cells. HOXB13 and ALX4 formed a complex in cells, and exogenous expression of either protein promoted EMT and invasion. Conversely, depletion of either protein suppressed invasion and induced reversion of EMT. SLUG is a C2H2-type zinc-finger transcription factor that promotes EMT in various cell lines. Knockdown of HOXB13 or ALX4 suppressed SLUG expression, and exogenous expression of either protein promoted SLUG expression. Finally, we showed that SLUG expression was essential for the HOXB13- or ALX4-mediated EMT and invasion. Our results show that HOXB13/SLUG and ALX4/SLUG axes are novel pathways that promote EMT and invasion of ovarian cancer cells.

CD133-positive pancreatic cancer is correlated with unfavorable survival despite current development of therapy. Slug acts as a master regulator of epithelial-mesenchymal transition (EMT) which is the essential process in cancer progression. The aim of this study was to investigate the role of Slug in gemcitabine treatment for CD133-positive pancreatic cancer cells. We used a previously established pancreatic cancer cell line expressing high level of CD133 (Capan-1M9), which also expresses high level of Slug. We generated Slug knock-down subclone (shSlug M9) from this cell line, and compared expression of EMT-related genes, migration, invasion and gemcitabine resistance between two cell lines. Slug knock-down in CD133-positive pancreatic cancer cell line led to the reduction of migration and invasion ability. Furthermore, Slug knock-down sensitized CD133-positive pancreatic cancer cell line to gemcitabine. These results suggest that Slug plays an important role in not only invasion ability through EMT but also gemcitabine resistance of CD133-positive pancreatic cancer cells.

BACKGROUND: Circulating tumor cells (CTCs) play a crucial role in tumor dissemination and are an independent survival predictor in breast cancer (BC) patients. epithelial to mesenchymal transition (EMT) is involved in cancer invasion and metastasis. The aim of this study was to assess correlation between CTCs and expression of EMT transcription factors TWIST1 and SLUG in breast tumor tissue. METHODS: This study included 102 early BC patients treated by primary surgery. Peripheral blood mononuclear cells (PBMC) were depleted of hematopoietic cells using RossetteSep negative selection kit. RNA extracted from CD45-depleted PBMC was interrogated for expression of EMT (TWIST1, SNAIL1, SLUG, FOXC2 and ZEB1) and epithelial (KRT19) gene transcripts by qRT-PCR. expression of TWIST1 and SLUG in surgical specimens was evaluated by immunohistochemistry and quantified by multiplicative score. RESULTS: CTCs were detected in 24.5 % patients. CTCs exhibiting only epithelial markers were present in 8.8 % patients, whereas CTCs with only EMT markers were observed in 12.8 % of pts and CTCs co-expressing both markers were detected in 2.9 % pts. We observed lack of correlation between CTCs and expression of TWIST1 and SLUG in breast cancer cells or cancer associated stroma. Lack of correlation was observed for epithelial CTCs as well as for CTCs with EMT. CONCLUSIONS: In this translational study, we showed a lack of association between CTCs and expression of EMT-inducing transcription factors, TWIST1 and SLUG, in breast tumor tissue. Despite the fact that EMT is involved in cancer invasion and metastasis our results suggest, that expression of EMT proteins in unselected tumor tissue is not surrogate marker of CTCs with either mesenchymal or epithelial features.

BACKGROUND & AIMS: Gallbladder carcinoma (GBC) is the most common malignancy of the biliary tract and one of the most lethal forms of human cancer. However, there is limited information about the molecular pathogenesis of GBC. Here, we examined the functional role of the tumor suppressor N-myc downstream-regulated gene 2 (NDRG2) and the underlying molecular mechanisms of disease progression in GBC. METHODS: Clinical correlations between NDRG2 expression and clinicopathological factors were determined by immunohistochemical analysis of tumor tissues from 86 GBC patients. Biological functions of NDRG2 and NDRG2-mediated signaling pathways were determined in GBC cell lines with NDRG2 knockdown or overexpression. RESULTS: Loss of NDRG2 expression was an independent predictor of decreased survival and was significantly associated with a more advanced T stage, higher cellular grade, and lymphatic invasion in patients with GBC. GBC cells with loss of NDRG2 expression showed significantly enhanced proliferation, migration, and invasiveness in vitro, and tumor growth and metastasis in vivo. Loss of NDRG2 induced the expression of matrix metalloproteinase-19 (MMP-19), which regulated the expression of Slug at the transcriptional level. In addition, MMP-19-induced Slug, increased the expression of a receptor tyrosine kinase, Axl, which maintained Slug expression through a positive feedback loop, and stabilized epithelial-mesenchymal transition of GBC cells. CONCLUSIONS: The results of our study help to explain why the loss of NDRG2 expression is closely correlated with malignancy of GBC. These results strongly suggest that NDRG2 could be a favorable prognostic indicator and promising target for therapeutic agents against GBC.CI - Copyright (c) 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

UNLABELLED: The transcription factor sex determining region (Y SRY)-box 2 (SOX2) is known to play a crucial role in the maintenance of self renewal or pluripotency of undifferentiated embryonic and neuronal stem cells. An elevated expression of SOX2 has been correlated with poor prognosis of esophageal squamous cell carcinoma (ESCC). We sought to investigate the mechanism(s) by which SOX2 modulates the ESCC metastasis. The SOX2 coding DNA sequence was inserted into pCMV vector and stably transfected in ESCC cells (Eca-109). The effect of SOX2 over expression was evaluated on cell migration, invasion and epithelial to mesenchymal transition (EMT). We also measured the expression of Slug to explore if this transcription factor is involved in SOX2-mediated regulation of cell migration/invasion and EMT. In addition, we determined the role of STAT3/HIF-1alpha to further probe the mechanism of SOX2-mediated metastasis via Slug. Our results demonstrated that SOX2 over expressing Eca-109 cells showed an enhanced cell migration/invasion. Moreover, these cells exhibited the EMT characteristics, that is, a significantly suppressed expression of the epithelial cells marker with a concomitant enhancement of those of the mesenchymal markers. An increased expression of Slug in SOX2 over expressing cells suggested the involvement of this transcription factor in SOX2-regulated metastasis. Whereas the expressions of STAT3/HIF-1alpha were found to be up-regulated in SOX2 expressing cells, blockade of these transcription factors resulted in the inhibition of Slug expression at both protein and mRNA levels. CONCLUSION: These results suggest that SOX2 promoted the metastasis of ESCC, at least in part, by modulating Slug expression through the activation of STAT3/HIF-1alpha signaling.

epithelial to mesenchymal transition (EMT) is a critical step in the growth and dissemination of malignant diseases, including breast cancer. It is known that microRNAs (miRNAs) play important roles in the regulation of tumor properties in cancers. However, whether miR-497 contributes to EMT in breast cancer cells remains unknown. Our study demonstrated that the expression of miR-497 was significantly decreased in human breast cancer cell lines and breast cancer specimens. In breast cancer cells, EMT was inhibited and promoted by the over-expression as well as depletion of miR-497, respectively. Dual-Luciferase ReporterAassay confirmed that Slug was a direct target of miR-497. The upregulation of miR-497 in breast cancer cells suppressed cell proliferation and induced apoptosis both in vitro and in vivo. Correlation analysis indicated that miR-497 was highly negatively correlated with Slug expression in breast cancer specimens. The knockdown of Slug expression in breast cancer cells significantly suppressed cell proliferation and promoted apoptosis. Our results suggested that the expression of miR-497 is significantly correlated with EMT in breast cancer cells by regulating Slug at the transcriptional as well as translational levels. Therefore, targeting miR-497 may provide a novel strategy for the treatment of breast 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.

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 epithelial-mesenchymal transition (EMT) is thought to be essential for cancer metastasis. While chromatin remodeling is involved in EMT, which processes contribute to this remodeling remain poorly investigated. Recently, we showed that silencing or removal of the histone variant H2A.X induced mesenchymal-like characteristics, including activation of the EMT transcription factors, Slug and Zeb1 in human colon cancer cells. Here, we provide the evidence that H2A.X loss in human non-tumorigenic breast cell line MCF10A results in a robust EMT activation, as substantiated by a genome-wide expression analysis. Cells deficient for H2A.X exhibit enhanced migration and invasion, along with an activation of a set of mesenchymal genes and a concomitant repression of epithelial genes. In the breast model, the EMT-related transcription factor Twist1 cooperates with Slug to regulate EMT upon H2A.X Loss. Of interest, H2A.X expression level tightly correlates with Twist1, and to a lesser extent with Slug in the panel of human breast cancer cell lines of the NCI-60 datasets. These new findings indicate that H2A.X is involved in the EMT processes in cells of different origins but pairing with transcription factors for EMT may be tissue specific.

Gastric cancer (GC) remains a threat to public health with high incidence and mortality worldwide. Increasing evidence demonstrates that long non-coding RNAs (lncRNAs) play critical regulatory roles in cancer biology, including GC. Previous profiling study showed that lncRNA linc00261 was aberrantly expressed in GC. However, the role of linc00261 in GC progression and the precise molecular mechanism remain unknown. In this study, we report that linc00261 was significantly down-regulated in GC tissues and the expression level of linc00261 negatively correlated with advanced tumour status and clinical stage as well as poor prognostic outcome. In vitro functional assays indicate that ectopic expression of linc00261 suppressed cell invasion by inhibiting the epithelial-mesenchymal transition (EMT). By RNA pull-down and mass spectrum experiments, we identified Slug as an RNA-binding protein that binds to linc00261. We confirmed that linc00261 down-regulated Slug by decreasing the stability of Slug proteins and that the tumour-suppressive function of linc00261 can be neutralized by Slug. linc00261 may promote the degradation of Slug via enhancing the interaction between GSK3beta and Slug. Moreover, linc00216 overexpression repressed lung metastasis in vivo. Together, our findings suggest that linc00261 acts a tumour suppressor in GC by decreasing the stability of Slug proteins and suppressing EMT. By clarifying the mechanisms underlying GC progression, these findings may facilitate the development of novel therapeutic strategies for GC.CI - (c) 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

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.

Hypoxia is a major driving force of cancer invasion and metastasis. Here we show that death domain-associated protein (Daxx) acts to negatively regulate hypoxia-induced cell dissemination and invasion by inhibiting the HIF-1alpha/HDAC1/Slug pathway. Daxx directly binds to the DNA-binding domain of Slug, impeding histone deacetylase 1 (HDAC1) recruitment and antagonizing Slug E-box binding. This, in turn, stimulates E-cadherin and occludin expression and suppresses Slug-mediated epithelial-mesenchymal transition (EMT) and cell invasiveness. Under hypoxic conditions, stabilized hypoxia-inducible factor (HIF)-1alpha downregulates Daxx expression and promotes cancer invasion, whereas re-expression of Daxx represses hypoxia-induced cancer invasion. Daxx also suppresses Slug-mediated lung cancer metastasis in an orthotopic lung metastasis mouse model. Using clinical tumour samples, we confirmed that the HIF-1alpha/Daxx/Slug pathway is an outcome predictor. Our results support that Daxx can act as a repressor in controlling HIF-1alpha/HDAC1/Slug-mediated cancer cell invasion and is a potential therapeutic target for inhibition of cancer metastasis.

epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Despite the anticancer capabilities of emodin observed in many cancers, including EOC, the underlying molecular mechanism remains to be elucidated. A crucial link has been discovered between the acquisition of metastatic traits and the epithelial-mesenchymal transition (EMT). The present study aimed to determine whether emodin could inhibit the EMT of EOC cells and explore the underlying mechanism. The CCK-8 assay and transwell assay showed that emodin effectively repressed the abilities of proliferation, invasion, and migration in A2780 and SK-OV-3 cells. The Western blot showed that emodin upregulated epithelial markers (E-cadherin and Claudin) while it downregulated mesenchymal markers (N-cadherin and Vimentin) and transcription factor (Slug) in a dose-dependent fashion. After transfection of siRNA-Slug, both Slug and N-cadherin were downregulated in EOC cells while E-cadherin was upregulated, which was intensified by emodin. Besides, emodin decreased the expression of ILK, p-GSK-3beta, beta-catenin, and Slug. Transfection of siRNA-ILK also achieved the same effects, which was further strengthened by following emodin treatment. Nevertheless, SB216763, an inhibitor of GSK-3beta, could reverse the effects of emodin except for ILK expression. These findings suggest that emodin inhibited the EMT of EOC cells via ILK/GSK-3beta/Slug signaling pathway.

epithelial-mesenchymal transition (EMT) has been recognized as a key element of cell migration and invasion in lung cancer; however, the underlying mechanisms are not fully elucidated. Recently, emerging evidence suggest that miRNAs have crucial roles in control of EMT and EMT-associated traits such as migration, invasion and chemoresistance. Here, we found that miR-218 expression levels were significantly downregulated in lung cancer tissues compared with adjacent non-cancerous tissues, and the levels of miR-218 were significantly associated with histological grades and lymph node metastasis. Overexpression of miR-218 inhibited cell migration and invasion as well as the EMT process. Of particular importance, miR-218 was involved in the metastatic process of lung cancer cells in vivo by suppressing local invasion and distant colonization. We identified Slug and ZEB2 as direct functional targets of miR-218. Inverse correlations were observed between miR-218 levels and Slug/ZEB2 levels in cancer tissue samples. In addition, overexpression of miR-218 in H1299 increased chemosensitivity of cells to cisplatin treatment through suppression of Slug and ZEB2. These findings highlight an important role of miR-218 in the regulation of EMT-related traits and metastasis of lung cancer in part by modulation of Slug/ZEB2 signaling, and provide a potential therapeutic strategy by targeting miR-218 in NSCLC.

PURPOSE: Long non-coding RNA (LncRNA) urothelial carcinoma-associated 1 (UCA1) is reported to be dysregulated in hepatocellular carcinoma (HCC) progression. However, the functions of UCA1 in HCC still need further study. The aim is to detect the role of UCA1 involving in HCC cells proliferation and invasion, and epithelial-mesenchymal transition (EMT). METHODS: The quantitative real-time PCR was used to detect the UCA1 and miR-203 expression levels in 60 cases' HCC tissues and adjacent normal tissues. Western blotting analysis was performed to detect the EMT markers E-cadherin, Vimentin and transcription factor Snail1, Snail2 expression. Luciferase reporter assay, RNA immunoprecipitation (RIP) and pull-down assays were used to evaluate whether miR-203 was a target of UCA1. RESULTS: Our results showed that UCA1 was markedly upregulated in HCC tissues and higher UCA1 expression in HCC was positively associated with tumor size, vascular invasion and American Joint Committee on cancer (AJCC) stage (P < 0.05). Furthermore, gain-of-function and loss-of-function analysis showed that UCA1 knockdown inhibited HCC cells proliferation and invasion in vitro and xenograft tumour growth in vivo. Moreover, UCA1 overexpression promoted cell epithelial-mesenchymal transition (EMT) in HCC via effectively sponging to miR-203 and thereby activating the expression of transcription factor Snail2. CONCLUSIONS: Our results identified that UCA1/miR-203/Snail2 pathway might involve in HCC progression. Inhibition of UCA1 acted as a promising therapeutic target for HCC patients.

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.

Chemokine (CC motif) ligand 18 (CCL18) is involved in remodeling of the tumor microenvironment and plays critical roles in oncogenesis, invasiveness, and metastasis. We previously investigated the overexpression of CCL18 in primary oral squamous cell carcinoma (OSCC) tissues and its association with advanced clinical stage in OSCC patients. However, the underlying mechanisms of this CCL18-derived activity remains unidentified. This study showed exogenous CCL18 increased cell migration and invasion and induced cell epithelial-mesenchymal transition (EMT), and that E-cadherin, an epithelial marker, decreased and N-cadherin, a mesenchymal marker, increased, compared to negative control in OSCC cells. Furthermore, we detected that CCL18 induced the acquisition of cancer stem(-like) cell characteristics in oral cancer cells, but also found a significantly positive correlation between the expression of CCL18 and Bmi-1 (P < 0.001) in OSCC surgical specimens by immunohistochemistry analysis. The expression of octamer-binding transcription factor 4 and Bmi-1 were significantly upregulated, and proportions of aldehyde dehydrogenase(high+) cells and CD133(+) cells were markedly increased in CCL18-treated cells compared to untreated cells. Sphere formation ability was observably enhanced when cells were continually exposed to high levels of CCL18. Moreover, CCL18 upregulated Slug expression by stimulating the mammalian target of rapamycin (mTOR) signaling pathway in OSCC cell lines. Inhibition of the mTOR pathway by INK128, or Slug knockdown by RNA interference, reversed CCL18-induced EMT and the stemness response at both molecular and functional levels. In conclusion, our data suggested that CCL18 upregulated Slug expression to promote EMT and stem cell-like features by activating the mTOR pathway in oral cancer. These findings provide new potential targets for the early diagnosis and treatment of OSCC.CI - (c) 2017 The Authors. cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese cancer Association.

Metastasis is a crucial impediment to the successful treatment for gastric cancer. SPOCK1 has been demonstrated to facilitate cancer metastasis in certain types of cancers; however, the role of SPOCK1 in the invasion and metastasis of gastric cancer remains elusive. SPOCK1 and epithelial-mesenchymal transition (EMT)-related biomarkers were detected by immunohistochemistry and Western blot in gastric cancer specimens. Other methods including stably transfected against SPOCK1 into gastric cancer cells, Western blot, migration and invasion assays in vitro and metastasis assay in vivo were also performed. The elevated expression of SPOCK1 correlates with EMT-related markers in human gastric cancer tissue, clinical metastasis and a poor prognosis in patients with gastric cancer. In addition, knockdown of SPOCK1 expression significantly inhibits the invasion and metastasis of gastric cancer cells in vitro and in vivo, inversely, SPOCK1 overexpression results in the opposite effect. Interestingly, SPOCK1 expression has no effect on cell proliferation in vitro and in vivo. Regarding the mechanism(s) of SPOCK1-induced cells invasion and metastasis, we prove that Slug-induced EMT is involved in SPOCK1-facilitating gastric cancer cells invasion and metastasis. The elevated SPOCK1 expression is closely correlated with cancer metastasis and patient survival, and SPOCK1 promotes the invasion and metastasis of gastric cancer through Slug-mediated EMT, thereby possibly providing a novel therapeutic target for gastric cancer.CI - (c) 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

High metastatic ability and poor clinical outcome are the most known clinical features of the triple-negative breast tumors. Given that the tumor cells undergoing epithelial-mesenchymal transition (EMT) often gain malignant and invasive features, we have investigated the possibility of EMT reversal in triple-negative breast cancer cells by targeting the epigenetic-modifying enzymatic complexes named histone deacetylases (HDACs) and examined the possible mechanism underlying the HDACs-based inversion in model MDA-MB-231 cells. Cells were treated with a maximal tolerable 200 nM concentrations of classical HDACs inhibitor Trichostatin A (TSA) for 48 h and afterwards the invasiveness and immigration of the cells were evaluated in TransWell Invasion Scratch Wound Healing assays. Then, in treated and control cells, quantitative real time-PCR reacions were performed for assessing the gene expression of EMT biomarkers E-cadherin, Vimentin and transcriptional factor Slug. After TSA treatment, the invasion and migration properties MDA-MB-231 cells significantly decreased, gene expression of E-cadherin was significantly up-regulated, while the levels of Slug and Vimentin encoding mRNAs were suppressed. We conclude that inhibition of HDACs in triple-negative breast cancer cells may lead to inversion of EMT and the decrease of invasiveness by down-regulating the gene expression of Slug. Since EMT is known as a pre-metastatic process, triple-negative breast tumors, the EMT reversal effects of HDACs inhibition may reduce tumor cell metastasis.

Spleen tyrosine kinase (SYK) is a nonreceptor tyrosine kinase with dual properties of an oncoprotein and an oncosuppressor in distinctive cell types. In solid cancers, two isoforms SYK(L) and SYK(S) of SYK were recently identified due to its alternative mRNA splicing. However, the cellular activity and the biological significance of the long isoform of SYK, SYK(L), is still not well defined in human lung cancers. Here, we describe an interaction between SYK(L) and the ubiquitously expressed transcription regulator Yin Yang 1 (YY1) in the nucleus, which suppresses the epithelial-to-mesenchymal transition (EMT) by inactivating SNAI2 (coding transcription factor SLUG) transcription. ChIP indicated that endogenous SYK(L) interacts directly with a YY1 binding cis-regulatory element in the SNAI2 promoter. Importantly, knockdown of YY1 activates SYK(L)-dependent EMT suppression in human lung cancer H1155 cells. We also found that the protein level of SYK(L) is markedly upregulated in various types of human lung cancers, and its nuclear localization is strongly correlated with clinical benefits of lung adenocarcinomas. Collectively, our data reveal a SYK(L)-dependent transcriptional regulation of EMT through SLUG as a potential biomarker for lung cancer aggressiveness.CI - (c) 2018 Federation of European Biochemical Societies.

The mammalian pancreas develops by the expansion and morphogenesis of the epithelial cells of the foregut endoderm via the sequential activation of transcription factors that direct differentiation into the various pancreatic lineages. Implicit in this growth and differentiation are the temporal and spatial processes of cell migration and three-dimensional organization, which cooperate to form a properly functioning organ. In many organ systems, such as the kidney, heart, and neural crest derivatives, migration and tissue morphogenesis is accomplished by the transient conversion of stationary epithelial cells to migratory mesenchymal-like cells in a process known as epithelial-mesenchymal transition (EMT). We report the identification of the expression of the transcription factor Snail2/Slug, a known inducer of EMT and cell movement, in both the endocrine and exocrine cells of the developing mouse pancreas. Snail2 is expressed in Neurogenin3-positive endocrine progenitor cells, and expression is maintained during endocrine cell differentiation where it becomes increasingly restricted to the insulin-producing beta cells and somatostatin-producing delta cells. In the adult pancreas, the expression of Snail2 is maintained at low but detectable levels in all beta cells, indicating a latent role for Snail2 in the mature islet. These findings of Snail2 expression during endocrine pancreas development are relevant to the recent evidence demonstrating the involvement of EMT in the expansion of human islet tissue in vitro. EMT-like events appear to be involved in the development of the mammalian pancreas in vivo.

Animal models of acute renal injury suggest that the epidermal growth factor receptor (EGFR) axis may have a beneficial role in the recovery from acute renal injury, but recent reports describe detrimental effects of EGFR activation in chronic renal injury. expression of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) increases following renal injury, but the effects of this sustained upregulation have not been well studied. Here, stable overexpression of soluble HB-EGF (sHB-EGF) in mouse inner medullary collecting duct (IMCD) cells led to marked phenotypic changes: sHB-EGF-expressing cells demonstrated a fibroblast-like morphology, did not form epithelial sheets, exhibited cytoplasmic projections, decreased expression of epithelial markers, and increased expression of fibroblast-specific protein-1. They also demonstrated anchorage-independent growth and formed tumors when injected subcutaneously into nude mice. Quantitative RT-PCR and a luciferase reporter assay suggested that sHB-EGF repressed transcription of E-cadherin, and a concomitant TGF-beta-independent upregulation of the E-cadherin repressor Snail-2 was observed. Stable downregulation of Snail-2 in sHB-EGF-overexpressing cells restored epithelial characteristics (E-cadherin and cytokeratin expression) but did not alter their anchorage-independent growth. In summary, sustained exposure to sHB-EGF induces epithelial-to-mesenchymal transition of IMCD cells, in part by upregulating the E-cadherin transcriptional repressor Snail-2.

BACKGROUND: epithelial-to-mesenchymal transition (EMT) is a series of molecular changes allowing epithelial cancer cells to acquire properties of mesenchymal cells: increased motility, invasion, and protection from apoptosis. Transcriptional regulators such as Slug mediate EMT, working in part to repress E-cadherin transcription. We report a novel, noninvasive in vivo rectal cancer model to explore the role of Slug in colorectal cancer (CRC) tumor development. METHODS: For the generation of DLD-1 cells overexpressing Slug (Slug DLD-1), a Slug or empty (Empty DLD-1) pCMV-3Tag-1 (kanamycin-resistant) vector was used for transfection. Cells were evaluated for Slug and E-cadherin expression, and cell migration and invasion. For the in vivo study, colon cancer cells (parental DLD-1, Slug DLD-1, empty DLD-1, and HCT-116) were submucosally injected into the posterior rectum of nude mice using endoscopic guidance. After 28 d, tumors were harvested and tissue was analyzed. RESULTS: Slug expression in our panel of colon cancer cell lines was inversely correlated with E-cadherin expression and enhanced migration/invasion. Slug DLD-1 cells demonstrated a 21-fold increased Slug and 19-fold decreased E-cadherin expression compared with empty DLD-1. Similarly, the Slug DLD-1 cells had significantly enhanced cellular migration and invasion. In the orthotopic rectal cancer model, Slug DLD-1 cells formed rectal tumors in 9/10 (90%) of the mice (mean volume = 458 mm(3)) compared with only 1/10 (10%) with empty DLD-1 cells. CONCLUSION: Slug mediates EMT with enhanced in vivo rectal tumor formation. Our noninvasive in vivo model enables researchers to explore the molecular consequences of altered genes in a clinically relevant rectal cancer in an effort to develop novel therapeutic approaches for patients with rectal cancer.CI - Published by Elsevier Inc.

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.

epithelial-mesenchymal transition (EMT) is implicated in various pathological processes within the prostate, including benign prostate hyperplasia (BPH) and prostate cancer progression. However, an ordered sequence of signaling events initiating carcinoma-associated EMT has not been established. In a model of transforming growth factor beta (TGFbeta)-induced prostatic EMT, SLUG is the dominant regulator of EMT initiation in vitro and in vivo, as demonstrated by the inhibition of EMT following Slug depletion. In contrast, SNAIL depletion was significantly less rate limiting. TGFbeta-stimulated KLF4 degradation is required for SLUG induction. expression of a degradation-resistant KLF4 mutant inhibited EMT, and furthermore, depletion of Klf4 was sufficient to initiate SLUG-dependent EMT. We show that KLF4 and another epithelial determinant, FOXA1, are direct transcriptional inhibitors of SLUG expression in mouse and human prostate cancer cells. Furthermore, self-reinforcing regulatory loops for SLUG-KLF4 and SLUG-FOXA1 lead to SLUG-dependent binding of polycomb repressive complexes to the Klf4 and Foxa1 promoters, silencing transcription and consolidating mesenchymal commitment. Analysis of tissue arrays demonstrated decreased KLF4 and increased SLUG expression in advanced-stage primary prostate cancer, substantiating the involvement of the EMT signaling events described in model systems.

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.

mutation in interferon regulatory factor 6 (IRF6) is known to cause syndromic and non-syndromic cleft lip/palate in human. In this study, we investigated the molecular mechanisms related to IRF6 during palatal fusion using palatal shelves organ culture. The results showed that ablation of Irf6 resulted in a delay in TGFbeta3-regulated palatal fusion. Ectopic expression of IRF6 was able to promote palatal fusion and rescue shTgfbeta3-induced fusion defect. These findings indicate that IRF6 is involved in TGFbeta3-mediated palatal fusion. Molecular analysis revealed that ectopic expression of IRF6 increased the expression of SNAI2, an epithelial mesenchymal transition (EMT) regulator, and diminished the expression of various epithelial markers, such as E-cadherin, Plakophilin and ZO-1. In addition, knockdown of Irf6 expression decreased SNAI2 expression, and restored the expression of ZO-1 and Plakophilin that were diminished by TGFbeta3. Blocking of Snai2 expression delayed palatal fusion and abolished the IRF6 rescuing effect associated with shTgfbeta3-induced fusion defect. These findings indicate that TGFbeta3 increases IRF6 expression and subsequently regulates SNAI2 expression, and IRF6 appears to regulate EMT during palatal fusion via SNAI2. Taken together, this study demonstrates that IRF6 is a mediator of TGFbeta3, which regulates EMT and fusion process during the embryonic palate development.

Vimentin has been shown to be involved in wound healing, but its functional contribution to this process is poorly understood. Here we describe a previously unrecognized function of vimentin in coordinating fibroblast proliferation and keratinocyte differentiation during wound healing. Loss of vimentin led to a severe deficiency in fibroblast growth, which in turn inhibited the activation of two major initiators of epithelial-mesenchymal transition (EMT), TGF-beta1 signaling and the Zinc finger transcriptional repressor protein Slug, in vimentin-deficient (VIM(-/-)) wounds. Correspondingly, VIM(-/-) wounds exhibited loss of EMT-like keratinocyte activation, limited keratinization, and slow reepithelialization. Furthermore, the fibroblast deficiency abolished collagen accumulation in the VIM(-/-) wounds. Vimentin reconstitution in VIM(-/-) fibroblasts restored both their proliferation and TGF-beta1 production. Similarly, restoring paracrine TGF-beta-Slug-EMT signaling reactivated the transdifferentiation of keratinocytes, reviving their migratory properties, a critical feature for efficient healing. Our results demonstrate that vimentin orchestrates the healing by controlling fibroblast proliferation, TGF-beta1-Slug signaling, collagen accumulation, and EMT processing, all of which in turn govern the required keratinocyte activation.

In this study, we investigated whether there is any association between the expression of FSCN1 and SNAI2 and the possible underlying mechanisms in head and neck squamous cell carcinoma (HNSC). In addition, we also investigated whether FSCN1 modulates epithelial-to-mesenchymal transition (EMT) in HNSC cells. Microarray data of dysregulated genes in HNSC were searched in GEO datasets. The association between FSCN1 expression and the 5-year/10-year overall survival (OS), as well as the correlation between the expression of FSCN1 and SOX2, MYBL2, SNAI2, STAT1, and SOX4, was analyzed based on data in TCGA HNSC cohort (TCGA-HNSC). The binding site of SNAI2 in FSCN1 promoter was verified using luciferase reporter assay. SCC9 and SCC15 cells were transfected with pCMV-SNAI2 or pCMV-FSCN1 expression vector or the empty control. Alteration of E-cadherin, Claudin 1, Vimentin, and N-cadherin was then quantified. Our results showed that FSCN1 is significantly upregulated in HNSC tissues compared with the normal control tissues. High FSCN1 expression is associated with worse 5-year and 10-year OS among the HNSC patients. Bioinformatic prediction showed a highly possible SNAI2 binding site in FSCN1 promoter and following luciferase reporter assay verified this site. SNAI2 overexpression significantly increased FSCN1 expression at both mRNA and protein level. FSCN1 overexpression reduced the expression of E-cadherin and Claudin 1, but increased the expression of Vimentin and N-cadherin in SCC9 and SCC-15 cells. Therefore, we infer that FSCN1 is a downstream effector of SNAI2 in promoting EMT in HNSC cells.CI - (c) 2017 International Federation for Cell Biology.