7040

TGFB1

It has recently been suggested by several investigators that the epithelial-mesenchymal transition-inducing capacity of TGFbetas contributes to invasive transition of tumors at later stages of carcinogenesis. In the present study, we examined the possibility of TGFbeta1-stimulated epithelial-mesenchymal transition in SiHa cell line, detailed molecular events in the process, and its possible contribution to the invasive transition of tumors. TGFbeta1-induced epithelial-mesenchymal transition of SiHa cells was based on morphological and biochemical criteria; actin stress fiber formation, focal translocalization of integrin alphav, talin, and vinculin, fibronectin-based matrix assembly at the cell periphery, and translocalization and down-regulation of E-cadherin. TGFbeta1 also stimulated surface expression of integrin alphavbeta3 and FAK activation. Focal translocalization of integrin alphav preceded actin reorganization and fibronectin matrix assembly, and functional blocking of the integrin suppressed actin stress fiber formation. Furthermore, induction of actin reorganization and fibronectin matrix assembly by TGFbeta1 were shown to be mutually independent events. These changes were irreversible because 5 minutes pulse exposure to TGFbeta1 was sufficient to stimulate progress of actin reorganization and fibronectin matrix assembly. In further studies with raft culture, TGFbeta1 was found to stimulate invasion of SiHa cells into a type I collagen gel matrix. In conclusion, TGFbeta1 stimulated epithelial-mesenchymal transition of SiHa cells, indicating a positive role in the invasive transition of tumors.

Transforming growth factor-b1 (TGF-beta1), a multi-function polypeptide, is a double-edged sword in cancer. For some tumor cells, TGF-beta1 is a potent growth inhibitor and apoptosis inducer. More commonly, TGF-beta1 loses its growth-inhibitory and apoptosis-inducing effects, but stimulates the metastatic capacity of tumor cells. It is currently little known about TGF-beta1-promoted cell migration in hepatocellular carcinoma (HCC) cells, let alone its mechanism. In this study, we found that TGF-beta1 lost its tumor-suppressive effects, but significantly stimulated cell migration in SMMC-7721 human HCC cells. By FACS and Western blot analysis, we observed that TGF-beta1 enhanced the expression of alpha5beta1 integrin obviously, and subsequently stimulated cell adhesion onto fibronectin (Fn). Furthermore, we observed that TGF-beta1 could also promote SMMC-7721 cells adhesion onto laminin (Ln). Our data also provided evidences that TGF-beta1 induced epithelial-to-mesenchymal transformation (EMT) in SMMC-7721 cells. First, SMMC-7721 cells clearly switched to the spindle shape morphology after TGF-beta1 treatment. Furthermore, TGF-beta1 induced the down-regulation of E-cadherin and the nuclear translocation of beta-catenin. These results indicated that TGF-beta1-promoted cell adhesion and TGF-beta1-induced epithelial-to-mesenchymal transformation might be both responsible for TGF-beta1-enhanced cell migration.

In response to transforming growth factor beta (TGF-beta), Smad4 forms complexes with activated Smad2 and Smad3, which accumulate in the nucleus, where they both positively and negatively regulate TGF-beta target genes. mutation or deletion of Smad4 is found in about 50% of pancreatic tumors and in about 15% of colorectal tumors. As Smad4 is a central component of the TGF-beta/Smad pathway, we have determined whether Smad4 is absolutely required for all TGF-beta responses, to evaluate the effect of its loss during human tumor development. We have generated cell lines from the immortalized human keratinocyte cell line HaCaT or the pancreatic tumor cell line Colo-357, which stably express a tetracyline-inducible small interfering RNA targeted against Smad4. In response to tetracycline, Smad4 expression is effectively silenced. Large-scale microarray analysis identifies two populations of TGF-beta target genes that are distinguished by their dependency on Smad4. Some genes absolutely require Smad4 for their regulation, while others do not. Functional analysis also indicates a differential Smad4 requirement for TGF-beta-induced functions; TGF-beta-induced cell cycle arrest and migration, but not epithelial-mesenchymal transition, are abolished after silencing of Smad4. Altogether our results suggest that loss of Smad4 might promote TGF-beta-mediated tumorigenesis by abolishing tumor-suppressive functions of TGF-beta while maintaining some tumor-promoting TGF-beta responses.

During embryogenesis, epicardial cells undergo epithelial-mesenchymal transformation (EMT), invade the myocardium, and differentiate into components of the coronary vasculature, including smooth muscle cells. We tested the hypothesis that transforming growth factor-beta (TGFbeta) stimulates EMT and smooth muscle differentiation of epicardial cells. In epicardial explants, TGFbeta1 and TGFbeta2 induce loss of epithelial morphology, cytokeratin, and membrane-associated Zonula Occludens-1 and increase the smooth muscle markers calponin and caldesmon. Inhibition of activin receptor-like kinase (ALK) 5 blocks these effects, whereas constitutively active (ca) ALK5 increases cell invasion by 42%. Overexpression of Smad 3 did not mimic the effects of caALK5. Inhibition of p160 rho kinase or p38 MAP kinase prevented the loss of epithelial morphology in response to TGFbeta, whereas only inhibition of p160 rho kinase blocked TGFbeta-stimulated caldesmon expression. These data demonstrate that TGFbeta stimulates loss of epithelial character and smooth muscle differentiation in epicardial cells by means of a mechanism that requires ALK5 and p160 rho kinase.CI - 2005 Wiley-Liss, Inc.

BACKGROUND & AIMS: How hepatocellular carcinoma (HCC) cells acquire the ability to invade surrounding tissue is unknown, but epithelial mesenchymal transition (EMT) likely plays a role. We investigate how transforming growth factor (TGF)-beta1 and extracellular matrix protein Laminin-5 (Ln-5) induce EMT and cancer invasion. METHODS: Snail, Slug, E-cadherin, beta-catenin and Ln-5 were investigated on HCC tissues and on HCC cell lines. RESULTS: We show that in HCC but not in peritumoral tissue of the same HCC patients, Ln-5, Snail, and Slug are up-regulated, E-cadherin is down-regulated and beta-catenin is translocated into the nuclei. In vitro, HCC "invasive" cells, partially EMT-transformed, show low levels of E-cadherin. In presence of Ln-5, Snail, and Slug are up-regulated, E-cadherin is down-regulated, beta-catenin is translocated into the nuclei, and cells undergo a dramatic morphological change, becoming scattered and undergoing a complete EMT. This effect is reversed by anti-alpha3 but not by anti-alpha6 integrin blocking antibody. HCC "noninvasive" cells are not EMT-transformed, and have constitutively high levels of E-cadherin. In presence of Ln-5, cells undergo partial EMT, Snail, and Slug are up-regulated, E-cadherin is down-regulated but cells do not scatter. However, the presence of both Ln-5 and TGF-beta1 completes the EMT process, beta-catenin is translocated into the nuclei, cells scatter and become invasive, recalling the "invasive" cells. In this case, too, the effect is reversed by anti-alpha3 integrin blocking antibody. CONCLUSIONS: Our study shows that Ln-5 and TGF-beta1 cooperatively induce EMT in HCC, suggesting the microenvironment as a potential target for new biological therapies.

There is increasing evidence that epithelial to mesenchymal transition (EMT) is involved in cancer progression. Because local invasion and metastasis occurs early in the pathogenesis of esophageal adenocarcinoma, we hypothesized that EMT may be important in this disease. Using immunohistochemistry in a well-characterized set of adenocarcinoma tissues, we showed down-regulation of epithelial markers (E-cadherin and cytokeratin 18) and up-regulation of mesenchymal markers (vimentin and alpha-smooth muscle actin) with concomitant transforming growth factor-beta1 (TGF-beta1) expression at the invasive margin compared with the central tumor. A panel of esophageal cell lines was examined for the ability of TGF-beta1 to induce EMT in vitro. TE7 cells were selected as a model because TGF-beta1 (0-5 ng/mL) treatment induced morphologic and molecular expression changes suggestive of EMT. In TE7 cells, these TGF-beta1-induced changes were reversed by 100 ng/mL of bone morphogenetic protein 7 (BMP7), another member of the TGF-beta1 superfamily. EMT was mediated via canonical TGF-beta1 signaling with concomitant up-regulation of SMAD-interacting protein 1. Alterations in functional variables (aggregation, wounding, motility, and invasion) following TGF-beta1 treatment were consistent with a more invasive phenotype. These functional changes were reversed by BMP7 and SMAD4 RNA interference in vitro. These data suggest that TGF-beta1-mediated EMT may be relevant in esophageal carcinogenesis.

Idiopathic pulmonary fibrosis (IPF) comprises an aggregate of mesenchymal cells. However, the cellular origin of these mesenchymal phenotypes remains unclear. Transforming growth factor beta1 (TGF-beta1) has been known as the main cytokine involved in the pathogenesis of IPF. We examined whether the potent fibrogenic cytokine TGF-beta1 could induce the epithelial-to-mesenchymal transition (EMT) in the human alveolar epithelial cell line, A549, and determined whether snail expression is associated with the phenotypic changes observed in the A549 cells. EMT was investigated with cells morphology changes under phase-contrast microscopy, western blotting, and indirect immunofluorescence stains. E-cadherin and transcription factor, snail, were also evaluated by measuring mRNA levels using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The data showed that TGF-beta1 induced A549 cells with epithelial cell characteristics to undergo EMT in a concentration-dependent manner. Following TGF-beta1 treatment, A549 cells induced EMT characterized by cells morphological changes, loss of epithelial markers Ecaherin and cytokeratin, increased stress fiber reorganization by F-actin, and cytokeratin replacement by vimentin. Although IL-1beta failed to induce A549 cells to undergo EMT, the combination of TGF-beta1 and IL-1beta showed synergy effects in cells morphology changes and the expression of mesenchymal markers. The snail expression study using RT-PCR analysis provided that loss of E-cadherin expression was associated with snail expression. Stimulation of A54 cells with TGF-beta1 plus IL-1beta revealed a higher level of snail expression. Our data showed that EMT of A549 cells might be closely associated with snail expression.

The aberrant expression of transforming growth factor (TGF)-beta1 in the tumor microenvironment and fibrotic lesions plays a critical role in tumor progression and tissue fibrosis by inducing epithelial-mesenchymal transition (EMT). EMT promotes tumor cell motility and invasiveness. How EMT affects motility and invasion is not well understood. Here we report that HDAC6 is a novel modulator of TGF-beta1-induced EMT. HDAC6 is a microtubule-associated deacetylase that predominantly deacetylates nonhistone proteins, including alpha-tubulin, and regulates cell motility. We showed that TGF-beta1-induced EMT is accompanied by HDAC6-dependent deacetylation of alpha-tubulin. Importantly, inhibition of HDAC6 by small interfering RNA or the small molecule inhibitor tubacin attenuated the TGF-beta1-induced EMT markers, such as the aberrant expression of epithelial and mesenchymal peptides, as well as the formation of stress fibers. Reduced expression of HDAC6 also impaired the activation of SMAD3 in response to TGF-beta1. Conversely, inhibition of SMAD3 activation substantially impaired HDAC6-dependent deacetylation of alpha-tubulin as well as the expression of EMT markers. These findings reveal a novel function of HDAC6 in EMT by intercepting the TGF-beta-SMAD3 signaling cascade. Our results identify HDAC6 as a critical regulator of EMT and a potential therapeutic target against pathological EMT, a key event for tumor progression and fibrogenesis.

The subepithelial fibrosis component of airway remodeling in asthma is mediated through induction of transforming growth factor-beta1 (TGF-beta1) expression with consequent activation of myofibroblasts to produce extracellular matrix proteins. The number of myofibroblasts is increased in the asthmatic airway and is significantly correlated with the thickness of lamina reticularis. However, much is still unknown regarding the origin of bronchial myofibroblasts. Emerging evidence suggests that myofibroblasts can derive from epithelial cells by an epithelial-to-mesenchymal transition (EMT). In this study we investigated whether TGF-beta1 could induce bronchial epithelial EMT in the human bronchial epithelial cell. Cultured human bronchial epithelial cells, 16HBE-14o, were stimulated with 10 ng/ml TGF-beta1. Morphologic changes were observed and stress fiber by actin reorganization was detected by indirect immunostaining. The expression of alpha-SMA (alpha-smooth muscle actin) and the epithelial cell marker E-cadherin were detected in those 16HBE-14o cells after TGF-beta1 stimulation for 72 h, using immunostaining and RT-PCR. The contents of collagen I were determined by radioimmunoassay, and the levels of endogenous TGF-beta1 were measured with ELISA. Human bronchial epithelial cells stimulated with TGF-beta1 were converted from a "cobblestone" epithelial structure into an elongated fibroblast-like shape. Incubation of human bronchial epithelial cells with TGF-beta1 induced de novo expression of alpha-SMA, increased formation of stress fiber by F-actin reorganization, and loss of epithelial marker E-cadherin. Moreover, a significant increase in the levels of collagen I and endogenous TGF-beta1 released from bronchial epithelial cells stimulated with TGF-beta1 were observed. These results suggested that human bronchial epithelial cells, under stimulation of TGF-beta1, underwent transdifferentiation into myofibroblasts.

PURPOSE: Recent studies have showed that epithelial-mesenchymal transition (EMT) is a key process of glomerular and tubulointerstitial pathology in many chronic kidney diseases. However, there are no data of EMT in humane autosomal dominant polycystic kidney disease (ADPKD). PATIENTS AND METHODS: ADPKD kidneys (N = 5) with end stage renal disease (ESRD) and control kidneys (N = 4) were analyzed immnunohistochemically. We evaluated alpha-SMA, E-cadherin, vimentin, TGF-beta1 and Smad 2/3 expression in ADPKD and compared them with those in control kidney. These immunohistochemical findings were quantitatively analyzed by computer-assisted image analyzer and positive tubules (%). RESULTS: There were severe interstitial fibrosis and proliferation of alpha-SMA+ myofibroblasts in ADPKD. Cystic tubular epithelial cells in ADPKD lost epithelial marker (E-cadherin) and expressed mesenchymal markers (alpha-SMA, vimentin). There were significant increases of alpha-SMA (34.3 +/- 11.7% vs 0.9 +/- 1.5%), vimentin (19.9 +/- 3.9% vs 3.3 +/- 1.4%), TGF-beta1 (5.42 +/- 2.83% vs 0%) and Smad 2/3 (3.4 +/- 1.7% vs 0.7 +/- 0.6%) in ADPKD kidneys compared with control kidneys evidenced by computer-assisted image analyzer. When we analyze the positive tubules (%), the results were the same as computer-assisted image analyzer. CONCLUSION: Our results showed that the end stage of ADPKD is associated with TGF-beta, Smad 2/3 and markers of EMT. It suggests that TGF-beta mediated EMT has a role in progression of ADPKD.

Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology characterized by the development of subpleural foci of myofibroblasts that contribute to the exuberant fibrosis noted in the pulmonary parenchyma. Pleural mesothelial cells (PMC) are metabolically dynamic cells that cover the lung and chest wall as a monolayer and are in intimate proximity to the underlying lung parenchyma. The precise role of PMC in the pathogenesis of pulmonary parenchymal fibrosis remains to be identified. Transforming growth factor (TGF)-beta1, a cytokine known for its capacity to induce proliferative and transformative changes in lung cells, is found in significantly higher quantities in the lungs of patients with IPF. High levels of TGF-beta1 in the subpleural milieu may play a key role in the transition of normal PMC to myofibroblasts. Here we demonstrate that PMC activated by TGF-beta1 undergo epithelial-mesenchymal transition (EMT) and respond with haptotactic migration to a gradient of TGF-beta1 and that the transition of PMC to myofibroblasts is dependent on smad-2 signaling. The EMT of PMC was marked by upregulation of alpha-smooth muscle actin (alpha-SMA), fibroblast specific protein-1 (FSP-1), and collagen type I expression. Cytokeratin-8 and E-cadherin expression decreased whereas vimentin remained unchanged over time in transforming PMC. Knockdown of smad-2 gene by silencing small interfering RNA significantly suppressed the transition of PMC to myofibroblasts and significantly inhibited the PMC haptotaxis. We conclude that PMC undergo EMT when exposed to TGF-beta1, involving smad-2 signaling, and PMC may be a possible source of myofibroblasts in IPF.

BACKGROUND: Chronic persistent asthma is characterized by ongoing airway inflammation and airway remodeling. The processes leading to airway remodeling are poorly understood, and there is increasing evidence that even aggressive anti-inflammatory therapy does not completely prevent this process. We sought to investigate whether TGFbeta1 stimulates bronchial epithelial cells to undergo transition to a mesenchymal phenotype, and whether this transition can be abrogated by corticosteroid treatment or enhanced by the pro-inflammatory cytokine IL-1beta. METHODS: BEAS-2B and primary normal human bronchial epithelial cells were stimulated with TGFbeta1 and expression of epithelial and mesenchymal markers assessed by quantitative real-time PCR, immunoblotting, immunofluorescence microscopy and zymography. In some cases the epithelial cells were also incubated with corticosteroids or IL-1beta. Results were analyzed using non-parametric statistical tests. RESULTS: Treatment of BEAS-2B or primary human bronchial epithelial cells with TGFbeta1 significantly reduced the expression level of the epithelial adherence junction protein E-cadherin. TGFbeta1 then markedly induced mesenchymal marker proteins such as collagen I, tenascin C, fibronectin and alpha-smooth muscle actin mRNA in a dose dependant manner. The process of mesenchymal transition was accompanied by a morphological change towards a more spindle shaped fibroblast cell type with a more motile and invasive phenotype. Corticosteroid pre-treatment did not significantly alter the TGFbeta1 induced transition but IL-1beta enhanced the transition. CONCLUSION: Our results indicate, that TGFbeta1 can induce mesenchymal transition in the bronchial epithelial cell line and primary cells. Since asthma has been strongly associated with increased expression of TGFbeta1 in the airway, epithelial to mesenchymal transition may contribute to the contractile and fibrotic remodeling process that accompanies chronic asthma.

Recently, epithelial-mesenchymal transition (EMT) has been reported to contribute to tissue fibrosis through enhanced transforming growth factor (TGF)-beta1 signaling. tumor necrosis factor (TNF)-alpha has also been implicated in tissue fibrosis. Therefore, the authors investigated whether TNF-alpha affected TGF-beta1-induced EMT. Cultured alveolar epithelial cells (A549 cells) were stimulated with TGF-beta1 (5 ng/mL), with/without TNF-alpha (10 ng/mL). TGF-beta1 induced EMT of A549 cells, with loss of E-cadherin and acquisition of vimentin. Combination of TNF-alpha with TGF-beta1 enhanced EMT, causing morphological changes, while quantitative polymerase chain reaction (PCR) showed suppression of E-cadherin mRNA and expression of vimentin mRNA. In addition, the gel contraction method revealed that cells that had undergone EMT acquired cell contractility, which is a feature of mesenchymal cells. Stimulation with TGF-beta1 induced cell contraction, as did TNF-alpha. Moreover, costimulation with TGF-beta1 and TNF-alpha enhanced the cell contraction. Although IFN-gamma suppressed spontaneous cell contraction, it did not suppress cell contraction, which was induced by TGF-beta1. In conclusion, TNF-alpha enhances not only EMT but also cell contraction induced by TGF-beta1. EMT might contribute to tissue fibrosis through induction of cell contraction.

Id1 (inhibitor of differentiation 1) is a member of the bHLH protein family. Consistent with its role in promoting proliferation and inhibiting differentiation, Id1 expression is low or negligible in normal prostate epithelial cells but is high in prostate cancer. Ectopic expression of Id1 in normal prostate epithelial cells could therefore provide a model for understanding early events involved in initiation of prostate cancer. Over-expression of Id1 immortalized but did not transform ventral prostate epithelial cells (Id1-RPE). Immortalization was associated with decreased Cdkn2a, Cdkn1a, androgen receptor and increased Tert expression. Gene expression profiling over successive doublings was used to identify transcriptomic changes involved during immortalization (Tieg, Jun, alpha actin, Klf10, Id2) and in maintaining the immortalized phenotype (Igfbp3, Igfbp5, Mmp2, Tgfb3). Network analysis indicated that Id1 promotes cancer/tumor morphology, cell cycle and epithelial to mesenchymal transition by influencing AP1, tnf, tgfbeta, PdgfBB and estradiol pathways. During immortalization, the expression of majority of differentially expressed genes reduced over progressive doublings suggesting a decline in transcriptional regulatory mechanisms. The associated molecular/gene expression profile of Id1-RPE cells provides an opportunity to understand the molecular pathways associated with prostate epithelial cell survival and proliferation.

BACKGROUND: Transforming growth factor beta (TGF-beta) acts as a tumor promoter by inducing epithelial-mesenchymal transition (EMT), which leads to a motile phenotype, enabling invasion and metastasis of cancer cells. cancer-related inflammation, mediated by prostaglandins, has been proposed as a critical mechanism in conversion of benign cells to malignant. OBJECTIVE: Induction of cyclooxygenase 2 (COX-2), producer of prostaglandins, is thought to be a prerequisite for TGF-beta-induced EMT in benign cells. We used HaCaT derivatives, representative of skin cancer progression, to investigate TGF-beta1 mediated EMT response, and the role of COX-2 in it. METHODS: Effect of TGF-beta1 was investigated by analyzing cell proliferation, morphology and protein expression. Chemotaxis and scratch-wound assays were used to study migration. RESULTS: TGF-beta1 caused proliferation arrest of benign and malignant HaCaT cells, and changed the epithelial morphology of benign and low-grade malignant cells, but not metastatic cells, to mesenchymal spindle-shape. epithelial junction proteins ZO-1 and E-cadherin were downregulated in all cell lines in response to TGF-beta1, but mesenchymal markers were not induced, suggesting a partial EMT response. COX-2 and migration were induced only in benign HaCaT derivatives. Malignant derivatives did not induce COX-2 in response to TGF-beta 1 treatment, thus emphasizing the role of inflammation in EMT response of benign cells. CONCLUSIONS: TGF-beta1 operates via distinct mechanisms in inducing EMT and metastasis, and supporting this we show that TGF-beta1 induces COX-2 and promotes the migration of benign cells, but does not further augment the migration of malignant cells, indicating their resistance to TGF-beta1 in the context of motility.CI - 2010 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland 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.

Transforming growth factor (TGF)-beta1 is a key mediator of lung remodeling and fibrosis. epithelial cells are both a source of and can respond to TGF-beta1 with epithelial-to-mesenchymal transition (EMT). We recently determined that TGF-beta1-induced EMT in lung epithelial cells requires the presence of c-Jun N-terminal kinase (JNK) 1. Because TGF-beta1 signals via Smad complexes, the goal of the present study was to determine the impact of JNK1 on phosphorylation of Smad3 and Smad3-dependent transcriptional responses in lung epithelial cells. Evaluation of JNK1-deficient lung epithelial cells demonstrated that TGF-beta1-induced terminal phosphorylation of Smad3 was similar, whereas phosphorylation of mitogen-activated protein kinase sites in the linker regions of Smad3 was diminished, in JNK1-deficient cells compared with wild-type cells. In comparison to wild-type Smad3, expression of a mutant Smad3 in which linker mitogen-activated protein kinase sites were ablated caused a marked attenuation in JNK1 or TGF-beta1-induced Smad-binding element transcriptional activity, and expression of plasminogen activator inhibitor-1, fibronectin-1, high-mobility group A2, CArG box-binding factor-A, and fibroblast-specific protein-1, genes critical in the process of EMT. JNK1 enhanced the interaction between Smad3 and Smad4, which depended on linker phosphorylation of Smad3. Conversely, Smad3 with phosphomimetic mutations in the linker domain further enhanced EMT-related genes and proteins, even in the absence of JNK1. Finally, we demonstrated a TGF-beta1-induced interaction between Smad3 and JNK1. Collectively, these results demonstrate that Smad3 phosphorylation in the linker region and Smad transcriptional activity are directly or indirectly controlled by JNK1, and provide a putative mechanism whereby JNK1 promotes TGF-beta1-induced EMT.

The histopathology of idiopathic pulmonary fibrosis (IPF) includes the presence of myofibroblasts within so-called fibroblastic foci, and studies suggest that lung myofibroblasts may be derived from epithelial cells through epithelial--mesenchymal transition (EMT). Transforming growth factor (TGF)-beta1 is expressed and/or activated in fibrogenesis, and induces EMT in lung epithelial cells in a dose-dependent manner. A higher occurrence of Epstein-Barr virus (EBV) has been reported in the lung tissue of patients with IPF. EBV expresses latent membrane protein (LMP) 1 during the latent phase of infection, and may play a role in the pathogenesis of pulmonary fibrosis inasmuch as LMP-1 may act as a constitutively active TNF-alpha receptor. Our data show a remarkable increase in mesenchymal cell markers, along with a concurrent reduction in the expression of epithelial cell markers in lung epithelial cells cotreated with LMP-1, and very low doses of TGF-beta1. This effect was mirrored in lung epithelial cells infected with EBV expressing LMP1 and cotreated with TGF-beta1. LMP1 pro-EMT signaling was identified, and occurs primarily through the nuclear factor-kappaB pathway and secondarily through the extracellular signal--regulated kinase (ERK) pathway. Activation of the ERK pathway was shown to be critical for aspects of TGF-beta1-induced EMT. LMP1 accentuates the TGF-beta1 activation of ERK. Together, these data demonstrate that the presence of EBV-LMP1 in lung epithelial cells synergizes with TGF-beta1 to induce EMT. Our in vitro data may help to explain the observation that patients with IPF demonstrating positive staining for LMP1 in lung epithelial cells have a more rapid demise than patients in whom LMP1 is not detected.

epithelial-mesenchymal transition (EMT) is suggested to be crucial for the development of an invasive and metastatic carcinoma cell phenotype. Therefore, the definition of this phenotype is of great clinical interest. We recently evidenced vimentin positive cells in oral squamous cell carcinoma (OSCC) invasive front expressing laminin gamma2 chain mRNA implicating an EMT origin of these cells. To further elucidate the nature of these cells, we have investigated the relation between EMT criteria and laminin-332 expression in a cell culture model of transforming growth factor beta-1 (TGFbeta1)/epithelial growth factor (EGF) long time co-stimulation. We demonstrate that in contrast to TGFbeta1 or EGF alone, co-stimulation induces phenotype transition in OSCC cells which fulfils the criteria of EMT in terms of vimentin up-regulation and E-cadherin down-regulation on protein level as well as cell scattering. Furthermore, cells displayed a strongly enhanced invasiveness and adhesion to type I-IV collagens. Phenotype transition is accompanied by an enhanced expression of laminin-332, especially of its gamma2 chain. We further analyse the expression of extracellular matrix related genes by RT-PCR profiling. With respect to strongly enhanced proteins, data confirm the EMT phenotype of co-stimulated OSCC cells and expression of laminin-332. Furthermore, alpha catenin, collagen type 16, the integrin alpha7 and beta1 chains, and MMP11 are suggested as candidates with potential role in EMT in OSCC. In summary we are able to show that EMT in OSCC is mediated by multiple growth factors and is accompanied by laminin gamma2 chain up-regulation evidencing the existence of an intermediate Vim(+) /Ln332(+) EMT phenotype as seen in situ.CI - (c) 2010 John Wiley & Sons A/S.

The epithelial-mesenchymal transition (EMT) is associated with progression and metastasis of epithelial ovarian cancer (EOC). Snail and Slug (two members of the Snail family of transcription factors) down-regulate the expression of the adhesion molecule E-cadherin and thus function as positive regulators of EMT. Their expression is associated with a more invasive phenotype of EOC. However, how their expression in EOC cells is regulated needs to be further defined. Here, we show that transforming growth factor beta (TGFbeta) and epidermal growth factor (EGF) synergistically induce the expression of Slug and Snail at both mRNA and protein levels in an EOC cell line OVCA429 cells. Using specific chemical inhibitors, we demonstrate that Slug and Snail expression induced by TGFbeta is mediated by TGFbeta/ALK5 pathway, and EGF-induced expression of Slug and Snail is MEK1/2-dependent. Interestingly, TGFbeta-induced Slug expression is also MEK1/2-dependent. Further, we demonstrate that combined TGFbeta and EGF stimulation is more potent than either alone in repressing the expression of E-cadherin. Functionally, combined stimulation of TGFbeta and EGF enhances the mobility of OVCA429 cells and induces the production of MMP2 by OVCA429 cells more potently than either alone. Taken together, our data demonstrate that TGFbeta and EGF signaling pathways synergistically induce EMT and render EOC cells a more invasive phenotype.CI - Copyright (c) 2010 Elsevier Inc. All rights reserved.

Idiopathic pulmonary fibrosis is characterized by myofibroblast accumulation, extracellular matrix (ECM) remodeling, and excessive collagen deposition. ECM-producing myofibroblasts may originate from epithelial cells through epithelial to mesenchymal transition (EMT). TGF-beta1 is an inducer of EMT in pulmonary epithelial cells in vitro and in vivo, though the mechanisms are unclear. We hypothesized that TGF-beta1 induced EMT through Smad-dependent and -independent processes. To test this hypothesis, we studied the roles and mechanisms of TGF-beta1-induced Smad and p38 mitogen-activated protein kinase (MAPK) signaling in EMT-related changes in pulmonary epithelial cells. Exposure of pulmonary epithelial 1HAEo(-) cells to TGF-beta1 resulted in morphological and molecular changes of EMT over a 96-h period; loss of cell-cell contact, cell elongation, down-regulation of E-cadherin, up-regulation of fibronectin, and up-regulation of collagen I. Both Smad2/3 and p38 MAPK signaling pathways were activated by TGF-beta1. However, neither Smad2/3 nor p38 MAPK were required for the down-regulation of E-cadherin, yet p38 MAPK was associated with fibronectin up-regulation. Both Smad2/3 and p38 MAPK had a role in regulation of TGF-beta1-induced collagen expression. Furthermore, these data demonstrate that Smads and p38 MAPK differentially regulate EMT-related changes in pulmonary epithelial cells.CI - Copyright (c) 2010 Wiley-Liss, Inc.

Transforming growth factor-beta1 (TGF-beta1) potently induces the epithelial-mesenchymal transition (EMT) during tumoral progression. Although Sky-interacting protein (SKIP) regulates TGF-beta1-induced Smad activation, its role in the induction of cell malignance remains uncertain. We found that TGF-beta1 increases SKIP expression in PDV cells. In cells stably transfected with SKIP antisense, AS-S, Smad3 activation decreased, along with an inhibition of TGF-beta1-induced EMT, and the cells were sensitized to the TGF-beta1-dependent inhibition of proliferation. Also, AS-S cells showed a weaker migration and invasion response. Moreover, TGF-beta1-induced urokinase-type plasminogen activator expression was inhibited, concomitantly with a TGF-beta1-independent increment of the plasminogen-activator inhibitor-1 expression. Thus, these results suggest that SKIP is required for EMT and invasiveness induced by TGF-beta1 in transformed cells.CI - Copyright (c) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The activity of a variety of extracellular signaling factors is tightly regulated by proteins containing A Disintegrin And a Metalloprotease domain (ADAM) metalloproteases through limited proteolysis. Thus, the identification of ADAM substrates may unveil novel components and mechanisms of cell signaling pathways. We report the identification of the transmembrane protein vasorin (VASN), a transforming growth factor-beta (TGFbeta) trap, as a substrate of ADAM17. The metalloprotease efficiently generates a soluble fragment encompassing the extracellular domain of VASN. Despite the importance of TGFbeta in normal development and tumor progression, the regulation of VASN is completely unknown. Here, we show that only the soluble form of VASN inhibits TGFbeta and that the secretion of VASN is tightly controlled by ADAM17. Hence, inhibition of ADAM17 leads to the upregulation of TGFbeta signaling. Adding a new level of complexity to the function of ADAM17, we finally show that, through the cleavage of VASN, the metalloprotease controls TGFbeta-mediated epithelial-to-mesenchymal transition.

OBJECTIVES: * To quantify fibrotic lesions in renal tissues obtained from patients with large calculi and to evaluate association with renal function. * Presence of epithelial-mesenchymal transition (EMT) in stone-containing renal tissues was investigated. PATIENTS, SUBJECTS AND METHODS: * In all, 50 patients with nephrolithiasis with large calculi and matched healthy controls (37) were recruited. * Plasma creatinine (Cr) and corrected Cr clearance (CCr) were determined in all subjects. * Of the 50 patients, 38 had renal tissue available for histological analysis. Fibrosis was assessed by Masson's trichrome staining. Co-expression of epithelial cytokeratins and mesenchymal markers [alpha-smooth muscle actin (alphaSMA) and vimentin] in renal tubular cells was detected by dual immunofluorescence staining. * expression of fibronectin, transforming growth factor beta(1) (TGF-beta(1)) and CD68 were investigated. RESULTS: * Overall, the kidney function of the patients was significantly reduced, indicated by increased plasma Cr and decreased corrected CCr compared with healthy controls. * Inflammation grading in renal tissues of the patients was correlated with the percentage of the fibrotic area. Renal fibrosis was inversely correlated with renal function. * Cytokeratins co-expressed with alphaSMA and vimentin were found in nephrolithiatic renal tubular cells, and these cells strongly expressed fibronectin and TGF-beta(1). * Infiltration of CD68-positive cells was a common finding in the inflamed renal sections. CONCLUSIONS: * Kidneys of large stone-forming patients had robust signs of inflammation and fibrosis, and there was a close correlation of renal fibrosis with renal dysfunction. * This is the first study to show evidence for renal tubular cells showing signs of EMT in large stone-containing kidneys. Plausibly, TGF-beta(1) triggers EMT, which at least in part contributes to large stone-induced renal fibrosis.CI - (c) 2011 THE AUTHORS. BJU INTERNATIONAL (c) 2011 BJU INTERNATIONAL.

Contrary to the long believed hypothesis, it is now evident that breast cancer cells can disseminate from the early phases of the oncogenesis; and that such early disseminated cells sometimes survive at the sites of dissemination and may outgrow after a long latency of years and decades. For cancer cells to leave their origin, they must at least transiently loosen their adhesion with adjacent epithelial cells and stroma, and become motile while avoiding anoikis. Such processes resemble epithelial-mesenchymal transdifferentiation (EMT), which normally takes place in situations such as embryogenesis and wound healing. Interestingly, the occurrence of an EMT-like process in breast cancer cells has been implicated in the generation of cancer stem-like cells, in which TGFbeta1 signalling often plays core roles. Here, I discuss the current knowledge regarding cancerous EMT and its signalling pathways with the aim to consider the possible mechanisms of early dissemination, and also the generation of cancer stem-like cells in mammary tumour.

development of metastasis is a major cause of death for squamous cell carcinoma of the head and neck (SCCHN) patients. epithelial to mesenchymal transition (EMT) is now regarded as a correlate of tumor metastasis. Given that transforming growth factor-beta1 (TGF-beta1) is an important inducer of EMT, we examined the effects of TGF-beta1 on the human SCCHN cell line Tu686. We found that TGF-beta1 mediated cell morphological changes. Phase-contrast microscopy revealed a loss of the adherent phenotype with cellular elongation, decrease in cell-to-cell contact, and the induction of a fibroblast-like state. Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that TGF-beta1 could induce down-regulation of the epithelial marker E-cadherin and up-regulation of the mesenchymal marker vimentin in Tu686 cells in a concentration- and time-dependent manner. Wound- healing and transwell invasion assay indicated that TGF-beta1 promoted Tu686 cell migration and invasion dramatically. In addition, these changes were mediated via canonical TGF-beta/Smad signaling with concomitant up-regulation of phosphorylated Smad2. Smad2 RNAi abrogated both expression and functional effects of TGF-beta1 on Tu686 cells. In conclusion, the present study demonstrates that TGF-beta1 could induce EMT in the SCCHN cell line via the TGF-beta/Smad signaling pathway. More importantly, a cell model for EMT was established, which is valuable for future studies on the metastasis of SCCHN.

Endometrial cancer is among the three most common cancers in females in industrialized countries. In the majority of cases, the tumor is confined to the uterus at the time of diagnosis and presents a good prognosis. However, after primary surgery, 15% to 20% of these tumors recur and have limited response to systemic therapy. We carried out gene expression profiling of high-risk recurrence endometrial cancers to identify new therapeutic approaches targeting the molecular pathways involved in the acquisition of an aggressive tumor phenotype. A microarray gene-expression analysis on a total of 51 human endometrial carcinomas revealed 77 genes specifically altered in high-risk recurrence tumors (P < 0.001). The bioinformatics analysis of gene-gene interactions and molecular relationships among these genes pointed to a prominent role for TGF-beta1 signaling in the acquisition of an aggressive phenotype. We further showed that TGF-beta1 has a principal role at the initiation of endometrial carcinoma invasion through the promotion of the epithelial to mesenchymal transition that leads to the acquisition of an invasive phenotype in HEC-1A and RL95-2 cells. Impairment of this initial step with SB-431542, a specific TGF-beta1 inhibitor, precluded further persistent endometrial carcinoma invasion. In conclusion, we showed that the characterization of the molecular changes associated with the acquisition of an aggressive phenotype represents a realistic strategy for the rational identification and characterization of new potential therapeutic targets in an effort to improve the clinical management and the outcome of high-risk endometrial cancer patients.CI - (c)2011 AACRFAU - Muinelo-Romay, Laura

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.

Lung cancer is a highly malignant carcinoma, and most deaths of lung cancer are caused by metastasis. The alterations associated with epithelial-to-mesenchymal transition (EMT) may be related to the cancer cell metastasis. Nevertheless, the mechanism of lung cancer metastasis remains unclear. We conducted a study in vitro to investigate whether transforming growth factor-beta1 (TGF-beta1) could induce changes of, such as cell morphology, expression of relative protein markers, and cellular motile and invasive activities. In this research, the changes of cell morphology were first investigated under a phase contrast microscope, then western blotting was employed to detect the expression of E-cadherin, vimentin, and fibronectin, and finally cell motility and invasion were evaluated by cell wound-healing as well as invasion assays. The data indicated that human lung adenocarcinoma cell lines, A-549 and PC-9 cells of epithelial cell characteristics, were induced to undergo EMT by TGF-beta1. Following TGF-beta1 treatment, cells showed dramatic morphological changes assessed by phase contrast microscopy, accompanied by decreased epithelial marker E-cadherin and increased mesenchymal markers vimentin and fibronectin. More importantly, cell motility and invasion were also enhanced in the EMT process. These results indicated that TGF-beta1 may promote lung adenocarcinoma invasion and metastasis via the mechanism of EMT.

Metastasis of tumor cells is associated with epithelial-to-mesenchymal transition (EMT), which is a process whereby epithelial cells lose their polarity and acquire new features of mesenchyme. EMT has been reported to be induced by transforming growth factor-beta1 (TGF-beta1), but its mechanism remains elusive. In this study, we performed a study to investigate whether PI3K/Akt and MAPK/Erk1/2 signaling pathways involved in EMT in the human lung cancer A549 cells. The results showed that after treated with TGF-beta1 for 48 h, A549 cells displayed more fibroblast-like shape, lost epithelial marker E-cadherin and increased mesenchymal markers Vimentin and Fibronectin. Moreover, TGF-beta1-induced EMT after 48 h was accompanied by increased of cell migration and change of Akt and Erk1/2 phosphorylation. In addition, EMT was reversed by PI3K inhibitor LY294002 and MEK1/2 inhibitor U0126, which suggested that A549 cells under stimulation of TGF-beta1 undergo a switch into mesenchymal cells and PI3K/Akt and MAPK/Erk1/2 signaling pathways serve to regulate TGF-beta1-induced EMT of A549 cells.

Neuropilins, initially characterized as neuronal receptors, act as co-receptors for cancer related growth factors and were recently involved in several signaling pathways leading to cytoskeletal organization, angiogenesis and cancer progression. Then, we sought to investigate the ability of neuropilin-2 to orchestrate epithelial-mesenchymal transition in colorectal cancer cells. Using specific siRNA to target neuropilin-2 expression, or gene transfer, we first observed that neuropilin-2 expression endows HT29 and Colo320 for xenograft formation. Moreover, neuropilin-2 conferred a fibroblastic-like shape to cancer cells, suggesting an involvement of neuropilin-2 in epithelial-mesenchymal transition. Indeed, the presence of neuropilin-2 in colorectal carcinoma cell lines was correlated with loss of epithelial markers such as cytokeratin-20 and E-cadherin and with acquisition of mesenchymal molecules such as vimentin. Furthermore, we showed by surface plasmon resonance experiments that neuropilin-2 is a receptor for transforming-growth factor-beta1. The expression of neuropilin-2 on colon cancer cell lines was indeed shown to promote transforming-growth factor-beta1 signaling, leading to a constitutive phosphorylation of the Smad2/3 complex. Treatment with specific TGFbeta-type1 receptor kinase inhibitors restored E-cadherin levels and inhibited in part neuropilin-2-induced vimentin expression, suggesting that neuropilin-2 cooperates with TGFbeta-type1 receptor to promote epithelial-mesenchymal transition in colorectal cancer cells. Our results suggest a direct role of NRP2 in epithelial-mesenchymal transition and highlight a cross-talk between neuropilin-2 and TGF-beta1 signaling to promote cancer progression. These results suggest that neuropilin-2 fulfills all the criteria of a therapeutic target to disrupt multiple oncogenic functions in solid tumors.

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.

Zinc finger E-box-binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in TGF-beta-mediated senescence, epithelial-to-mesenchymal transition (EMT), and cancer stem cell functions. ZEBs are negatively regulated by members of the miR-200 microRNA family, but precisely how tumor cells expressing ZEBs emerge during invasive growth remains unknown. Here, we report that NOTCH3-mediated signaling prevents expansion of a unique subset of ZEB-expressing cells. ZEB expression was associated with the lack of cellular capability of undergoing NOTCH3-mediated squamous differentiation in human esophageal cells. Genetic inhibition of the Notch-mediated transcriptional activity by dominant-negative Mastermind-like 1 (DNMAML1) prevented squamous differentiation and induction of Notch target genes including NOTCH3. Moreover, DNMAML1-enriched EMT-competent cells exhibited robust upregulation of ZEBs, downregulation of the miR-200 family, and enhanced anchorage-independent growth and tumor formation in nude mice. RNA interference experiments suggested the involvement of ZEBs in anchorage-independent colony formation, invasion, and TGF-beta-mediated EMT. Invasive growth and impaired squamous differentiation were recapitulated upon Notch inhibition by DNMAML1 in organotypic three-dimensional culture, a form of human tissue engineering. Together, our findings indicate that NOTCH3 is a key factor limiting the expansion of ZEB-expressing cells, providing novel mechanistic insights into the role of Notch signaling in the cell fate regulation and disease progression of esophageal squamous cancers.CI - (c)2011 AACR.

Tissue transglutaminase (TG2), an enzyme involved in cell proliferation, differentiation and apoptosis is overexpressed in ovarian carcinomas, where it modulates epithelial-to-mesenchymal transition (EMT) and promotes metastasis. Its regulation in ovarian cancer (OC) remains unexplored. Here, we show that transforming growth factor (TGF)-beta, a cytokine involved in tumor dissemination is abundantly secreted in the OC microenvironment and induces TG2 expression and enzymatic activity. This is mediated at transcriptional level by SMADs and by TGF-beta-activated kinase 1-mediated activation of the nuclear factor-kappaB complex. TGF-beta-stimulated OC cells aggregate as spheroids, which enable peritoneal dissemination. We show that TGF-beta-induced TG2 regulates EMT, formation of spheroids and OC metastasis. TG2 knock-down in OC cells decreases the number of cells harboring a cancer stem cell phenotype (CD44+/CD117+). Furthermore, CD44+/CD117+ cells isolated from human ovarian tumors express high levels of TG2. In summary, TGF-beta-induced TG2 enhances ovarian tumor metastasis by inducing EMT and a cancer stem cell phenotype.

epithelial-to-mesenchymal transition (EMT) plays a crucial role in the progression of renal interstitial fibrosis, which finally leads to renal failure. Erbin, a member of LAP family, is recently reported to inhibit Smads and ERK pathway which are two important types of intracellular signaling involved in TGF-beta1-induced EMT. However, the role of Erbin in the regulation of EMT and the underlying mechanisms remain to be fully understood. To that end, we aimed to evaluate the expression of Erbin in renal interstitial fibrosis and the potential role of Erbin in tubular EMT stimulated by TGF-beta1. In this study we demonstrated that the expression of Erbin was upregulated in the tubular epithelia of 5/6-nephrectomized rats. We also showed here that TGF-beta1 upregulated Erbin expression in NRK52E cells during their EMT phenotype acquisition. Importantly, elevated expression of Erbin inhibited ERK signaling and partial reversed EMT stimulated by TGF-beta1. In the mean time, reducing Erbin expression enhanced ERK phosphorylation, promoted the E-cadherin suppression, and induced alpha-SMA expression and fibronection secretion in response to TGF-beta1, which could be rescued if cells were treated with the inhibitor of MEK1/2 U0126. However, in the absence of TGF-beta1, Erbin failed to affect ERK activation and EMT process. These results suggest that Erbin is a negative feedback molecule induced by TGF-beta1 and inhibits TGF-beta1-induced EMT via ERK signaling pathway.

AIMS/HYPOTHESIS: A key pathology in diabetic nephropathy is tubulointerstitial fibrosis. The condition is characterised by increased deposition of the extracellular matrix, fibrotic scar formation and declining renal function, with the prosclerotic cytokine TGF-beta1 mediating many of these catastrophic changes. Here we investigated whether TGF-beta1-induced epithelial-to-mesenchymal transition (EMT) plays a role in alterations in cell adhesion, cell coupling and cell communication in the human renal proximal tubule. METHODS: Whole-cell and cell compartment abundance of E-cadherin, N-cadherin, snail, vimentin, beta-catenin and connexin-43 was determined in human kidney cell line (HK)2 and human proximal tubule cells with or without TGF-beta1, using western blotting and immunocytochemistry, followed by quantification by densitometry. The contribution of connexin-43 in proximal tubule cell communication was quantified using small interfering RNA knockdown, while dye-transfer was used to assess gap junctional intercellular communication (GJIC). Functional tethering was assessed by single-cell force spectroscopy with or without TGF-beta1, or by immunoneutralisation of cadherin ligation. RESULTS: High glucose (25 mmol/l) increased the secretion of TGF-beta1 from HK2 cells. Analysis confirmed early TGF-beta1-induced morphological and phenotypical changes of EMT, with altered levels of adhesion and adherens junction proteins. These changes correlated with impaired cell adhesion and decreased tethering between coupled cells. Impaired E-cadherin-mediated adhesion reduced connexin-43 production and GJIC, these effects being mimicked by neutralisation of E-cadherin ligation. Upregulation of N-cadherin failed to restore adhesion or connexin-43-mediated GJIC. CONCLUSIONS/INTERPRETATION: We provide compelling evidence that TGF-beta1-induced EMT instigates a loss of E-cadherin, cell adhesion and ultimately of connexin-mediated cell communication in the proximal tubule under diabetic conditions; these changes occur ahead of overt signs of renal damage.

Fibrotic remodelling of lung parenchymal and airway compartments is the major contributor to life-threatening organ dysfunction in chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) and Chronic Obstructive Pulmonary disease (COPD). Since transforming growth factor-beta1 (TGF-beta1) is believed to play a key role in disease pathogenesis and markers of oxidative stress are also commonly detected in bronchoalveolar lavage (BAL) from such patients we sought to investigate whether both factors might be interrelated. Here we investigated the hypothesis that oxidative stress to the lung epithelium promotes fibrotic repair by driving epithelial-to-mesenchymal transition (EMT) via the augmentation of TGF-beta1. We show that in response to 400muM hydrogen peroxide (H(2)O(2)) A549 cells, used a model for alveolar epithelium, and human primary bronchial epithelial cells (PBECs) undergo EMT displaying morphology changes, decreased expression of epithelial markers (E-cadherin and ZO-1), increased expression of mesenchymal markers (vimentin and alpha-smooth muscle actin) as well as increased secretion of extracelluar matrix components. The same oxidative stress also promotes expression of TGF-beta1. Inhibition of TGF-beta1 signalling as well as treatment with antioxidants such as phenyl tert-butylnitrone (PBN) and superoxide dismutase 3 (SOD3) prevent the oxidative stress driven EMT-like changes described above. Interventions also inhibited EMT-like changes. This study identifies a link between oxidative stress, TGF-beta1 and EMT in lung epithelium and highlights the potential for antioxidant therapies to limit EMT and its potential contribution to chronic lung disease.CI - Copyright (c) 2011 Elsevier Inc. All rights reserved.

The epithelial-to-mesenchymal transition (EMT) plays crucial roles in embryonic development, wound healing, tissue repair, and cancer progression. Results of this study show how transforming growth factor beta1 (TGF-beta1) down-regulates expression of N-acetylglucosaminyltransferase III (GnT-III) during EMT-like changes. Treatment with TGF-beta1 resulted in a decrease in E-cadherin expression and GnT-III expression, as well as its product, the bisected N-glycans, which was confirmed by erythro-agglutinating phytohemagglutinin lectin blot and HPLC analysis in human MCF-10A and mouse GE11 cells. In contrast with GnT-III, the expression of N-acetylglucosaminyltransferase V was slightly enhanced by TGF-beta1 treatment. Changes in the N-glycan patterns on alpha3beta1 integrin, one of the target proteins for GnT-III, were also confirmed by lectin blot analysis. To understand the roles of GnT-III expression in EMT-like changes, the MCF-10A cell was stably transfected with GnT-III. It is of particular interest that overexpression of GnT-III influenced EMT-like changes induced by TGF-beta1, which was confirmed by cell morphological changes of phase contrast, immunochemical staining patterns of E-cadherin, and actin. In addition, GnT-III modified E-cadherin, which served to prolong E-cadherin turnover on the cell surface examined by biotinylation and pulse-chase experiments. GnT-III expression consistently inhibited beta-catenin translocation from cell-cell contact into the cytoplasm and nucleus. Furthermore, the transwell assay showed that GnT-III expression suppressed TGF-beta1-induced cell motility. Taken together, these observations are the first to clearly demonstrate that GnT-III affects cell properties, which in turn influence EMT-like changes, and to explain a molecular mechanism for the inhibitory effects of GnT-III on cancer metastasis.

Transforming growth factor-beta (TGF-beta)-induced epithelial-mesenchymal transition (EMT) has been shown to be related to the pathogenesis of various diseases including lung cancer. Recently, microRNAs (miRNA) have been recognized as a new class of genes involved in human tumorigenesis. MiR-23a/24/27a is a miRNA cluster located in chromosome 19p13.12, which can function as an oncogene in several human cancers. In this study, we analyzed miR-23a/24/27a expression in 10 non-small cell cancer (NSCLC) cell lines by real-time PCR analysis. Correlation between expression of these miRNAs and TGF-beta/Smad signaling was evaluated. We found that miR-23a could be regulated by TGF-beta1 in a Smad-dependent manner in A549 lung adenocarcinoma cells showing the EMT phenomenon. Knockdown of miR-23a partially restored E-cadherin expression under conditions of TGF-beta1 stimulation. In contrast, overexpression of miR-23a could suppress E-cadherin expression and stimulate EMT. Furthermore, A549 cells with overexpressed miR-23a were more resistant to gefitinib compared to the parental cells. These findings suggest that miR-23a regulates TGF-beta-induced EMT by targeting E-cadherin in lung cancer cells and may be useful as a new therapeutic target in NSCLC.

Although the epithelial-mesenchymal transition (EMT) is a normal process that occurs during development, it is thought to be associated with cancer progression and metastasis. Emerging evidence links mesenchymal stem cells (MSCs) in the tumor microenvironment with the occurrence of EMT in cancer progression. In this study, the human breast cancer cell line MCF7 was co-cultured with human adipose-derived MSCs (hAD-MSCs) in a transwell system. Co-cultured cells were analyzed for changes in cellular morphology, EMT markers, protein expression and tumor characteristics. We found that co-cultured MCF7 cells underwent EMT and established a stable mesenchymal phenotype after prolonged co-culturing. Here, we demonstrate that paracrine transforming growth factor-beta1 (TGF-beta1) secreted by hAD-MSCs regulated the establishment of EMT in MCF7 cells by targeting the ZEB/miR-200 regulatory loop. The downregulation of paracrine TGF-beta1 levels can inhibit and reverse the EMT progress by downregulating ZEB1/2 and upregulating miR-200b and miR-200c. The maintenance of a stable mesenchymal state by MCF7 cells required the establishment of autocrine TGF-beta signaling to drive and sustain ZEB expression, which had been initiated by the prolonged co-culturing with hAD-MSCs. These results suggest that MSCs may promote breast cancer metastasis by stimulating and facilitating the EMT process.

SARA has been shown to be a regulator of epithelial cell phenotype, with reduced expression during TGF-beta1-mediated epithelial-to-mesenchymal transition. Examination of the pathways that might play a role in regulating SARA expression identified phosphatidylinositol 3-kinase (PI3K) pathway inhibition as sufficient to reduce SARA expression. The mechanism of PI3K inhibition-mediated SARA down-regulation differs from that induced by TGF-beta1 in that, unlike TGF-beta1, PI3K-dependent depletion of SARA was apparent within 6 h and did not occur at the mRNA or promoter level but was blocked by inhibition of proteasome-mediated degradation. This effect was independent of Akt activity because neither reducing nor enhancing Akt activity modulated the expression of SARA. Therefore, this is likely a direct effect of p85alpha action, and co-immunoprecipitation of SARA and p85alpha confirmed that these proteins interact. Both SARA and PI3K have been shown to be associated with endosomes, and either LY294002 or p85alpha knockdown enlarged SARA-containing endocytic vesicles. Inhibition of clathrin-mediated endocytosis blocked SARA down-regulation, and a localization-deficient mutant SARA was protected against down-regulation. As inhibiting PI3K can activate the endosomal fusion-regulatory small GTPase Rab5, we expressed GTPase-deficient Rab5 and observed endosomal enlargement and reduced SARA protein expression, similar to that seen with PI3K inhibition. Importantly, either interference with PI3K via LY294002 or p85alpha knockdown, or constitutive activity of the Rab5 pathway, enhanced the expression of smooth muscle alpha-actin. Together, these data suggest that although TGF-beta1 can induce epithelial-to-mesenchymal transition through reduction in SARA expression, SARA is also basally regulated by its interaction with PI3K.

Chronic renal failure (CRF) mainly results from kidney fibrosis. epithelial-to-mesenchymal transition (EMT) occurs in stressed tubular epithelial cells and contributes to renal fibrosis. Transforming growth factor-beta1 (TGF-beta1) has been shown to initiate and complete the whole EMT process. Peroxisome proliferators-activated receptor-gamma (PPAR-gamma) exerts anti-inflammatory, anti-fibrotic and vaculo-protective effects on different renal diseases. Telmisartan is a member of angiotensin II (Ang II) receptor blocker (ARB) family. Recent studies show that Telmisartan has a partial agonistic effect on PPAR-gamma. Therefore, we tested the hypothesis that Telmisartan reverses the progression of induced EMT by TGF-beta1 in cultured human renal proximal tubular epithelial (HK-2) cells. Cultured HK-2 cells were treated with TGF-beta1 (3 ng/ml), a combination of TGF-beta1 and Telmisartan (10-200 umol/L) and a combination of TGF-beta1, Telmisartan and GW9662, a PPAR-gamma antagonist for 48 hours. EMT was determined by quantitative real-time PCR analysis of E-cadherin (E-cad), Connective Tissue Growth Factor (CTGF) and PPAR-gamma transcript expression and immunocytochemical analysis of E-cad, alpha-Smooth Muscle Actin (alpha-SMA) and PPAR-gamma protein expression. TGF-beta1 induced phenotypic EMT in cultured HK-2 cell line via significantly reduced E-cad expression and significantly increased CTGF, alpha-SMA expression in association with the loss of epithelial morphology. Telmisartan reversed all EMT markers in a dose-dependent manner which was inhibited by PPAR antagonist GW9662. In the present study, it was suggested that Telmisartan attenuated TGF-beta1 induced EMT by agonistic activation of PPAR-gamma.

Human amniotic epithelial cells (hAECs) have been the object of intense research due to their potential therapeutic use. In this paper, we present molecular evidence of a bona fide epithelial to mesenchymal transition (EMT) undergone by hAECs. Amniotic membrane (AM)-derived hAECs showed the presence of typical epithelial markers such as E-cadherin and cytokeratins. hAECs in culture, however, underwent morphological changes acquiring a mesenchymal shape. epithelial cell markers were lost and typical mesenchymal markers, such as vimentin and alpha-SMA, appeared. Several genes associated with EMT, such as SNAI1, MMP9, PAI1, or ACTA2, increased their expression. The expression of the transcription activators KLF4 or MTA3 was consistent with the downregulation of CDH1. We have shown that hAECs undergo EMT due to the autocrine production of TGF-beta. Furthermore, the addition of the TGF-beta receptor I (ALK5) inhibitor SB-431542 or TGF-beta neutralizing antibody to hAECs prevented EMT and preserved the hAECs' epithelial phenotype. Altogether, these results suggest that cultured hAECs undergo EMT through the autocrine production of TGF-beta.

Metastasis is unequivocally the most lethal aspect of breast cancer and the most prominent feature associated with disease recurrence, the molecular mechanisms whereby epithelial-to-mesenchymal transition (EMT) mediates the initiation and resolution of breast cancer metastasis remains poorly understood. Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine that is intimately involved in regulating numerous physiological processes, including cellular differentiation, homeostasis and EMT. Recent findings have implicated high levels of TGF-beta1 were associated with poor outcome, whereas inhibition of TGF-beta signaling reduces metastasis in breast cancer, suggesting that the chemo-therapeutic targeting of TGF-beta1 or TGF-beta signaling may offer new inroads in ameliorating metastatic disease in breast cancer patients. In this study, we showed immunohistochemical evidence for EMT, which is associated with TGF-beta1 expression, at the invasion front of breast cancer in vivo. The data also indicated that human breast cancer cell lines, MCF-7 and MDA-MB-435S, of epithelial cell characteristics were induced to undergo EMT by TGF-beta1 and dependent on the Smad2 signaling pathway. Following TGF-beta1 treatment, cells showed dramatic morphological changes assessed by phase contrast microscopy, accompanied by decreased epithelial marker and increased mesenchymal markers. Importantly, cell invasion was also enhanced in the EMT process, while knockdown of the Smad2 gene by silencing siRNA partially inhibited these effects in MDA-MB435S (P<0.05). These data suggested that EMT of breast cancer induced by TGF-beta1 is dependent on Smad2 signaling and promotes breast cancer cell metastasis.

Mitochondria dysfunction has been reported in various kidney diseases but how it leads to kidney fibrosis and how this is regulated is unknown. Here we found that mitochondrial uncoupling protein 2 (UCP2) was induced in kidney tubular epithelial cells after unilateral ureteral obstruction in mice and that mice with ablated UCP2 resisted obstruction-induced kidney fibrosis. We tested this association further in cultured NRK-52E cells and found that TGF-beta1 remarkably induced UCP2 expression. Knockdown of UCP2 largely abolished the effect of TGF-beta1, whereas overexpression of UCP2 promoted tubular cell phenotype changes. Analysis using a UCP2 mRNA-3'-untranslated region luciferase construct showed that UCP2 mRNA is a direct target of miR-30e. MiR-30e was downregulated in tubular cells from fibrotic kidneys and TGF-beta1-treated NRK-52E cells. A miR-30e mimic significantly inhibited TGF-beta1-induced tubular-cell epithelial-mesenchymal transition, whereas a miR-30e inhibitor imitated TGF-beta1 effects. Finally, genipin, an aglycone UCP2 inhibitor, significantly ameliorated kidney fibrosis in mice. Thus, the miR-30e/UCP2 axis has an important role in mediating TGF-beta1-induced epithelial-mesenchymal transition and kidney fibrosis. Targeting this pathway may shed new light for the future of fibrotic kidney disease therapy.

The stem cell marker nestin is an intermediate filament protein that plays an important role in cell integrity, migration, and differentiation. Nestin expression occurs in approximately one third of pancreatic ductal adenocarcinoma (PDAC), and its expression strongly correlates with tumor staging and metastasis. Little is known about the mechanisms by which nestin influences PDAC progression. Here, nestin overexpression in PDAC cells increased cell motility and drove phenotypic changes associated with the epithelial-mesenchymal transition (EMT) in vitro; conversely, knockdown of endogenous nestin expression reduced the migration rate and reverted cells to a more epithelial phenotype. Mouse xenograft studies showed that knockdown of nestin significantly reduced tumor incidence and volume. Nestin protein expression was associated with Smad4 status in PDAC cells; hence, nestin expression might be regulated by the TGF-beta1/Smad4 pathway in PDAC. We examined nestin expression after TGF-beta1 treatment in human pancreatic cancer PANC-1 and PANC-1 shSmad4 cells. The TGF-beta1/Smad4 pathway induced nestin protein expression in PDAC cells in a Smad4-dependent manner. Moreover, increased nestin expression caused a positive feedback regulator of the TGF-beta1 signaling system. In addition, hypoxia was shown to induce nestin expression in PDAC cells, and the hypoxia-induced expression of nestin is mediated by the TGF-beta1/Smad4 pathway. Finally, the antimicrotubule inhibitors, cytochalasin D and withaferin A, exhibited anti-nestin activity; these inhibitors might be potential antimetastatic drugs. Our findings uncovered a novel role of nestin in regulating TGF-beta1-induced EMT. Anti-nestin therapeutics may serve as a potential treatment for PDAC metastasis.CI - (c)2013 AACRFAU - Su, Huei-Ting

epithelial-mesenchymal transition (EMT) is an important cellular event in organogenesis, cancer and renal tubulointerstitial fibrosis. Transforming growth factor-beta1 (TGF-beta1) is the key inducer of EMT and the p38 mitogen-activated protein kinases (p38 MAPK), an major intracellular signal transduction pathway is involved in TGF-beta1-induced EMT. Astrocyte elevated gene-1 (AEG-1) represents an chief genetic determinant regulating multiple events in tumorigenesis. Our present study is to explore the role of AEG-1 in TGF-beta1-induced p38 MAPK activation and EMT process in human renal tubular epithelial (HK-2) cells. The protein expressions of AEG-1, the markers of EMT and p38 phosphorylation were measured by Western blot. The protein expression of AEG-1 was increased in HK-2 cells treated with TGF-beta1. Knockdown of AEG-1 potently inhibited phosphorylation of p38 MAPK and reversed TGF-beta1-induced EMT. Over-expression of AEG-1 via AEG-1 transfection elicited p38 MAPK phosphorylation and promoted EMT. The effects of AEG-1 during EMT were blocked by a p38-specific inhibitor. Our findings suggest that AEG-1 plays an important role in TGF-beta1-induced EMT through activation of p38 MAPK in proximal tubular epithelial cells.CI - (c) 2013 International Federation for Cell Biology.

The interactions between kidney and thyroid functions have been known for many years, but how the thyroid affects the kidney function is largely unknown. Here we analyzed the role of T3 on the tubular epithelial-to-mesenchymal transition (EMT), which is recognized to play pivotal roles in the process of renal fibrosis. T3 was found to significantly inhibit the TGFbeta1 induced EMT in human proximal tubular epithelial cell line HK-2. Meanwhile, T3 induced the expression of miR34a. Molecularly, the T3 receptor could directly bind the T3R recognition motif at the -1505 to -1526bp and -604 to -609bp regions in the miR34a promoter and transcriptionally activate the expression of miR34a upon T3 treatment. Inhibition of the miR34a by miR34a knockdown nearly blocked the effects of T3 on EMT. Taken together, our study here revealed that thyroid hormone T3 could inhibit TGFbeta1 induced renal tubular epithelial to mesenchymal transition by increasing miR34a expression.CI - Copyright (c) 2013 Elsevier Inc. All rights reserved.

The epithelial-mesenchymal transition (EMT) is a process associated with the metastasis of solid tumors as well as with the acquisition of resistance to standard anticancer modalities. A major initiator of EMT in carcinoma cells is TGF-beta, which has been shown to induce the expression of several transcription factors ultimately responsible for initiating and maintaining the EMT program. We have previously identified Brachyury, a T-box transcription factor, as an inducer of mesenchymal features in human carcinoma cells. In this study, a potential link between Brachyury and TGF-beta signaling has been investigated. The results show for the first time that Brachyury expression is enhanced during TGF-beta1-induced EMT in various human cancer cell lines, and that a positive feedback loop is established between Brachyury and TGF-beta1 in mesenchymal-like tumor cells. In this context, Brachyury overexpression is shown to promote upregulation of TGF-beta1 at the mRNA and protein levels, an effect mediated by activation of the TGF-beta1 promoter in the presence of high levels of Brachyury. Furthermore, inhibition of TGF-beta1 signaling by a small-molecule inhibitor of TGF-beta receptor type I decreases Brachyury expression, induces a mesenchymal-to-epithelial transition, and renders cancer cells more susceptible to chemotherapy. This study thus has implications for the future development of clinical trials using TGF-beta inhibitors in combination with other anticancer agents.

Both SKP2 (S-phase kinase-associated protein 2) and transforming growth factor-beta1 (TGF-beta1) play important roles in cancer metastasis through different mechanisms: TGF-beta1 via induction of epithelial-mesenchymal transition (EMT) and SKP2 via downregulating p27(kip1). Recent studies indicated that c-Myc and Akt1 were active players in metastasis. In this study we demonstrated a crosstalk between these pathways. Specifically, we found that TGF-beta1 treatment increased SKP2 expression accompanied with increased phosphorylation of Akt1 and c-Myc protein accumulation during EMT. We demonstrated that Akt1 was required for TGF-beta1-mediated SKP2 upregulation and that c-Myc transcription factor specifically bound to the promoter of SKP2 for its enhanced transcription. Analysis of 25 samples of normal human skin, nevi, and melanomas revealed a positive correlation between c-Myc and SKP2 accumulation. Furthermore, accumulation of SKP2 and c-Myc proteins was significantly higher in metastatic melanoma samples as compared with that in primary melanomas, which again was higher than that in normal skin or nevi. In summary, our results integrated TGF-beta1 signals to SKP2 via Akt1 and c-Myc during EMT, and provided, to our knowledge, a previously unreported mechanistic molecular event for TGF-beta1-induced metastasis in human melanoma.

Severe asthma is a chronic airway disease characterized by the Th2/Th17-polarized inflammation along with permanent airway remodeling. Despite past extensive studies, the exact role for Th2 and Th17 cytokines in asthmatic pathoetiology, particularly in the pathogenesis of bronchial epithelial-mesenchymal transition (EMT), is yet to be fully addressed. We herein conducted studies in 16-HBE cells and demonstrated that Th2-derived IL-4 and Th17-derived IL-17A provide a chronic inflammatory milieu that favors TGF-beta1 to induce bronchial EMT. A synergic action was noted between TGF-beta1, IL-4 and IL-17A in terms of induction of EMT. IL-4 and IL-17A synergized with TGF-beta1 to induce epithelial cells re-entering cell cycle, and to promote epithelial to mesenchymal morphological transistion, and by which they enhanced the capacity of TGF-beta1 to suppress E-cadherin expression, and to induce a-SMA expression in epithelial cells. Mechanistic studies revealed that this synergic action is coordinated by the regulation of ERK1/2 activity. Our results not only provide a novel insight into the understanding of the mechanisms underlying airway remodeling in asthmatic condition, but also have the potential for developing more effective therapeutic strategies against severe asthmatics in clinical settings.

epithelial-to-mesenchymal transition (EMT) is a process for fully differentiated epithelial cells to undergo a phenotypic change to fibroblasts via diverse intracellular signaling pathways. While the pivotal role of fibroblasts in renal fibrosis is widely accepted, their origin remains undefined. In addition, although a large number of studies have provided evidence of EMT in human kidney diseases, specific signaling pathways leading to EMT have not yet been discovered in humans. To evaluate the origin of interstitial fibroblasts and signaling pathways involved in the EMT process, we analyzed the differential expression of EMT-related molecules in paraffin-fixed sections from 19 human fibrotic kidneys and 4 control kidneys. In human fibrotic kidneys, tubular epithelial cells (TECs) with intact tubular basement membrane (TBM) showed loss or down-regulation of an epithelial marker (E-cadherin), de novo expression of mesenchymal markers (vimentin and fibronectin), and significant up-regulation of inducers and mediators controlling the EMT process (transforming growth factor-beta1 (TGF-beta1), p-Smad2/3, beta1-integrin, p38 mitogen-activated protein kinase (MAPK), WNT5B and beta-catenin) in the areas of interstitial inflammation and fibrosis, compared with their expression in control kidneys. In conclusion, the type II EMT process in humans is thought to be an adaptive response of TECs to chronic injury and is regulated by interconnections of TGF-beta/Smad, integrin/integrin-linked kinase (ILK) and wnt/beta-catenin signaling pathways.

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.

BACKGROUND: Allergic asthma is characterized by airway inflammation in response to antigen exposure, leading to airway remodeling and lung dysfunction. epithelial-mesenchymal transition (EMT) may play a role in airway remodeling through the acquisition of a mesenchymal phenotype in airway epithelial cells. TGF-beta1 is known to promote EMT; however, other cytokines expressed in severe asthma with extensive remodeling, such as IL-22, may also contribute to this process. In this study, we evaluated the contribution of IL-22 to EMT in primary bronchial epithelial cells from healthy and asthmatic subjects. METHODS: Primary bronchial epithelial cells were isolated from healthy subjects, mild asthmatics and severe asthmatics (n=5 patients per group). The mRNA and protein expression of epithelial and mesenchymal cell markers and EMT-associated transcription factors was evaluated following stimulation with TGF-beta1, IL-22 and TGF-beta1+IL-22. RESULTS: Primary bronchial epithelial cells stimulated with TGF-beta1 underwent EMT, demonstrated by decreased expression of epithelial markers (E-cadherin and MUC5AC) and increased expression of mesenchymal markers (N-cadherin and vimentin) and EMT-associated transcription factors. IL-22 alone had no effect on epithelial or mesenchymal gene expression. However, IL-22+TGF-beta1 promoted the expression of some EMT transcription factors (Snail1 and Zeb1) and led to a more profound cadherin shift, but only in cells obtained from severe asthmatics. CONCLUSION: The impact of IL-22 on airway epithelial cells depends on the cytokine milieu and the clinical phenotype of the patient. Further studies are required to determine the molecular mechanism of IL-22 and TGF-beta1 cooperativity in driving EMT in primary human bronchial epithelial cells.

Synovial sarcoma (SS) is a malignant tumor of soft tissue and is noted for late local recurrence and metastasis. Aberrant epithelial-mesenchymal transition (EMT) has been implicated in the pathogenesis of diverse human malignancies. Immunohistochemical techniques were used to assess EMT-related proteins (E-cadherin, N-cadherin, beta-catenin, Snail, and Slug) and the TGF-beta1 pathway (TGF-beta1 and Smad2/3) proteins expression in different histological subtypes and epithelial mesenchymal compositions of SS. The expression of cell-surface (E-cadherin) and cytoskeletal proteins (beta-catenin) were higher significantly in biphasic SSs (BSSs) (70.4%, 51.9%) than MFSSs (both for 10%). Among monophasic fibrous SSs (MFSSs) samples, E-cadherin protein expression was negatively correlated with expression Snail, Slug, TGF-beta1, and Smad2/3. The expression levels of Snail and Smad2/3 were correlated with the pTNM stage (I-II vs. III-IV; P=0.047, P=0.021) and TGF-beta1 exhibited a tendency toward a positive correlation with pTNM stage (I-II vs. III-IV; P=0.052), but did not correlate with the histological grade (p>0.05). Interestingly, our data showed that expression of E-cadherin protein correlated with greater survival in SS patients. Overexpression of Snail, and TGF-beta1 is associated with suppressed expression of E-cadherin in MFSSs, which supports the hypothesis that the MFSS subtype may have developed via neoplastic EMT.

Ovarian cancer is a leading cause of cancer deaths among women. Effective targets to treat advanced epithelial ovarian cancer (EOC) and biomarkers to predict treatment response are still lacking because of the complexity of pathways involved in ovarian cancer progression. Here we show that miR-181a promotes TGF-beta-mediated epithelial-to-mesenchymal transition via repression of its functional target, Smad7. miR-181a and phosphorylated Smad2 are enriched in recurrent compared with matched-primary ovarian tumours and their expression is associated with shorter time to recurrence and poor outcome in patients with EOC. Furthermore, ectopic expression of miR-181a results in increased cellular survival, migration, invasion, drug resistance and in vivo tumour burden and dissemination. In contrast, miR-181a inhibition via decoy vector suppression and Smad7 re-expression results in significant reversion of these phenotypes. Combined, our findings highlight an unappreciated role for miR-181a, Smad7, and the TGF-beta signalling pathway in high-grade serous ovarian cancer.

A growing body of evidence supports that the epithelial-to-mesenchymal transition (EMT), which occurs during cancer development and progression, has a crucial role in metastasis by enhancing the motility of tumor cells. Transforming growth factor-beta (TGF-beta) is known to induce EMT in a number of cancer cell types; however, the mechanism underlying this transition process is not fully understood. In this study we have demonstrated that TGF-beta upregulates the expression of tumor suppressor protein Par-4 (prostate apoptosis response-4) concomitant with the induction of EMT. Mechanistic investigations revealed that exogenous treatment with each TGF-beta isoform upregulates Par-4 mRNA and protein levels in parallel levels of phosphorylated Smad2 and IkappaB-alpha increase. Disruption of TGF-beta signaling by using ALK5 inhibitor, neutralizing TGF-beta antibody or phosphoinositide 3-kinase inhibitor reduces endogenous Par-4 levels, suggesting that both Smad and NF-kappaB pathways are involved in TGF-beta-mediated Par-4 upregulation. NF-kappaB-binding sites in Par-4 promoter have previously been reported; however, using chromatin immunoprecipitation assay we showed that Par-4 promoter region also contains Smad4-binding site. Furthermore, TGF-beta promotes nuclear localization of Par-4. Prolonged TGF-beta3 treatment disrupts epithelial cell morphology, promotes cell motility and induces upregulation of Snail, vimentin, zinc-finger E-box binding homeobox 1 and N-Cadherin and downregulation of Claudin-1 and E-Cadherin. Forced expression of Par-4, results in the upregulation of vimentin and Snail expression together with increase in cell migration. In contrast, small interfering RNA-mediated silencing of Par-4 expression results in decrease of vimentin and Snail expression and prevents TGF-beta-induced EMT. We have also uncovered a role of X-linked inhibitor of apoptosis protein in the regulation of endogenous Par-4 levels through inhibition of caspase-mediated cleavage. In conclusion, our findings suggest that Par-4 is a novel and essential downstream target of TGF-beta signaling and acts as an important factor during TGF-beta-induced EMT.

BACKGROUND: Transforming growth factor beta 1 (TGFbeta1) can induce epithelial-mesenchymal transition (EMT) in various human cancers, but the complex mechanisms underlying this have not been fully elucidated. Inhibitor of DNA binding 1 (Id-1) has been identified as a novel marker of ovarian cancer progression. This study aims to investigate the role of Id-1 in TGFbeta1-induced EMT in human ovarian cancer cells. METHODS: Ovarian cancer cells expressing or not expressing Id-1 were incubated with TGFbeta1. Changes in the EMT markers E-cadherin, vimentin, N-cadherin, Id-1, and miR-29b were detected using western blotting and qPCR analyses. Wound healing, transwell migration, and invasion assays were performed in cells where Id-1 was either knocked down or overexpressed. The effects of transfecting miR-29b mimics and inhibitors on Id-1 mRNA and protein expression were assessed. The interaction between miR-29b and Id-1 was confirmed using a luciferase reporter assay. RESULTS: Id-1 expression was increased and miR-29b expression was repressed in TGFbeta1-responsive ovarian cancer cells. Id-1 overexpression increases and Id-1 knockdown decreases cell migration and invasion capacities. Id-1 silencing leads to a partial blocking of TGFbeta1-induced EMT. miR-29b negatively regulates Id-1 expression. Direct binding of miR-29b to the 3'UTR region of Id-1 was confirmed using a luciferase reporter assay. CONCLUSION: Id-1, a protein repressed by miR-29b, facilitates TGFbeta1-induced EMT in human ovarian cancer cells and represents a promising therapeutic target for treating ovarian cancer.CI - (c) 2014 S. Karger AG, Basel.

A consistent positive association between cigarette smoking and the human esophageal cancer has been confirmed all over the world. However, details in the association need to be more focused on and be identified. Recently, aberrantly expressed microRNAs (miRNAs) have been shown to be promising biomarkers for understanding the tumorigenesis of a wide array of human cancers, including the esophageal cancer, and the deregulation on the epithelial to mesenchymal transition (EMT) by miRNAs is involved in the tumorigenesis. In present study, we were going to identify the role of nicotine-induced miR-21 in the EMT of esophageal cells. We found that there was an overexpression of miR-21 in esophageal specimens, having an association with cigarette smoking, and the upregulation of miR-21 was also induced by nicotine in esophageal carcinoma cell line, EC9706. Moreover, the upregulated miR-21 by nicotine promoted EMT transforming growth factor beta (TGF-beta) dependently. Thus, the present study reveals a novel oncogenic role of nicotine in human esophageal cancer.

Transforming growth factor beta (TGF-beta) isoforms are secreted as inactive complexes formed through noncovalent interactions between the bioactive TGF-beta entity and its N-terminal latency-associated peptide prodomain. Extracellular activation of the latent TGF-beta complex is a crucial step in the regulation of TGF-beta function for tissue homeostasis. We show that the fibrinogen-like (FBG) domain of the matrix glycoprotein tenascin-X (TNX) interacts physically with the small latent TGF-beta complex in vitro and in vivo, thus regulating the bioavailability of mature TGF-beta to cells by activating the latent cytokine into an active molecule. Activation by the FBG domain most likely occurs through a conformational change in the latent complex and involves a novel cell adhesion-dependent mechanism. We identify alpha11beta1 integrin as a cell surface receptor for TNX and show that this integrin is crucial to elicit FBG-mediated activation of latent TGF-beta and subsequent epithelial-to-mesenchymal transition in mammary epithelial cells.

Although specific mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) identify tumors that are responsive to EGFR tyrosine kinase inhibitors (TKI), these genetic alterations are present in only a minority of patients. Patients with tumors expressing wild-type EGFR lack reliable predictive markers of their clinical response to EGFR TKIs. Although epithelial-mesenchymal transition (EMT) has been inversely correlated with the response of cancers to EGFR-targeted therapy, the precise molecular mechanisms underlying this association have not been defined and no specific EMT-associated biomarker of clinical benefit has been identified. Here, we show that during transforming growth factor beta (TGFbeta)-mediated EMT, inhibition of the microRNAs 200 (miR200) family results in upregulated expression of the mitogen-inducible gene 6 (MIG6), a negative regulator of EGFR. The MIG6-mediated reduction of EGFR occurs concomitantly with a TGFbeta-induced EMT-associated kinase switch of tumor cells to an AKT-activated EGFR-independent state. In a panel of 25 cancer cell lines of different tissue origins, we find that the ratio of the expression levels of MIG6 and miR200c is highly correlated with EMT and resistance to erlotinib. Analyses of primary tumor xenografts of patient-derived lung and pancreatic cancers carrying wild-type EGFR showed that the tumor MIG6(mRNA)/miR200 ratio was inversely correlated with response to erlotinib in vivo. Our data demonstrate that the TGFbeta-miR200-MIG6 network orchestrates the EMT-associated kinase switch that induces resistance to EGFR inhibitors, and identify a low ratio of MIG6 to miR200 as a promising predictive biomarker of the response of tumors to EGFR TKIs.CI - (c)2014 American Association for cancer Research.

epithelial mesenchymal transition (EMT) has been shown to play a role in cellular differentiation during deve-lopment and tumor invasion. However, the precise molecular mechanisms of EMT are not fully elucidated. Previous studies suggested that the mechanism underlying the possible involvement of ezrin in EMT process might be different from that of moesin, another ERM protein. In our study, we examined the role of ezrin in actin filament reorganization and cell meta-stasis during TGF-beta1-induced alveolar EMT. Suppressing ezrin expression limited morphological changes and actin filament remodeling, decreased cell migration and invasion during EMT. Immunofluorescence experiments indicated that EMT characteristics in lung cancer cells are associated to differential ezrin subcellular localization. We also found that podocalyxin interacted with ezrin after TGF-beta1 induction. Therefore, ezrin is an important regulator of the EMT process, and its function might possibly be mediated by the ezrin-podocalyxin interaction during TGF-beta1-induced alveolar EMT. Our finding provides important new insights into the mechanisms of action of the ERM proteins in the TGF-beta1-induced alveolar EMT.

The function of the epithelial-to-mesenchymal transition (EMT) during hepatocellular carcinoma (HCC) progression is well established. However, the regulatory mechanisms modulating this phenomenon remain unclear. Homeobox B9 (HOXB9) has been proposed as an oncogene in many cancer developments, but its function and underlying mechanisms in HCC metastasis remain unknown. HOXB9 modulates EMT through the transforming growth factor-beta1 (TGF-beta1) pathway, which is a recognized regulator of EMT in HCC cells. The knockdown of HOXB9 decreased the migration and invasion of HCC cells. Conversely, the HOXB9 overexpression led to an increase in the above-mentioned phenotypes in HCC cells. Further analysis of HOXB9-regulated cellular functions showed the ability of this transcription factor to induce EMT. Moreover, we demonstrated that the TGF-beta1 pathway is important in HOXB9-induced EMT in HCC cells. These findings define a novel cellular mechanism regulated by HOXB9, which controls EMT phenotype in HCC. This study is the first to illustrate the pivotal function of HOXB9 in regulating the metastatic behavior of HCC cells.

More and more studies have reported that epithelial-mesenchymal transition (EMT) involved in the process of cancer development and progression occurs. The EMT also plays an important role in the movement and transfer of the tumors. Transforming growth factor-beta (TGF-beta) could induce the EMT in some cancer cell types. However, the mechanism underlying this transition process has also not been entirely clarified. In this study, the results indicated that TGF-beta1-mediated EMT in the tumor was associated with the estrogen receptor (ER). The decreased expression of vimentin and snail resulted in the decrease of the ER expression by small interfering RNA-mediated silencing and preventing the TGF-beta-induced EMT. In conclusion, our results indicated that TGF-beta1 is an estrogen receptor signaling and essential novel downstream targets and could act as an important factor in the TGF-beta-induced EMT.

Recent studies implicate a role for cell mechanics in cancer progression. The epithelial-to-mesenchymal transition (EMT) regulates the detachment of cancer cells from the epithelium and facilitates their invasion into stromal tissue. Although classic EMT hallmarks include loss of cell-cell adhesions, morphology changes, and increased invasion capacity, little is known about the associated mechanical changes. Previously, force application on integrins has been shown to initiate cytoskeletal rearrangements that result in increased cell stiffness and a stiffening response. Here we demonstrate that transforming growth factor beta (TGF-beta)-induced EMT results in decreased stiffness and loss of the normal stiffening response to force applied on integrins. We find that suppression of the RhoA guanine nucleotide exchange factors (GEFs) LARG and GEF-H1 through TGF-beta/ALK5-enhanced proteasomal degradation mediates these changes in cell mechanics and affects EMT-associated invasion. Taken together, our results reveal a functional connection between attenuated stiffness and stiffening response and the increased invasion capacity acquired after TGF-beta-induced EMT.CI - (c) 2014 Osborne et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution-Noncommercial-Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

The epithelial-to-mesenchymal transition (EMT), a process involving the breakdown of cell-cell junctions and loss of epithelial polarity, is closely related to cancer metastasis and invasion. The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane protein expressed in the majority of normal epithelial tissues and overexpressed in the majority of human epithelial cancers including breast cancer. EpCAM plays an important role in cancer progression. We showed that EpCAM participated in TGF-beta1-induced EMT. TGF-beta1 treatment of MCF-7 breast cancer cells was shown to induce EpCAM expression, which promoted the EMT and cell migration. EpCAM overexpression further enhanced TGF-beta1-induced EMT and EpCAM knockdown inhibited TGF-beta1-induced EMT. We further demonstrated that TGF-beta1 treatment induced the phosphorylation of JNK that was in turn responsible for the increased expression of Jun and Fos. This result suggests an important role of the JNK to AP-1 signaling to EpCAM downstream of TGF-beta1 for the induction of EMT in the breast cancer cells. Collectively, our study highlights a novel function for EpCAM in TGF-beta1-induced EMT process and suggests that targeting of EpCAM may be an attractive strategy to treat breast cancer. This study implicates the potential value of EpCAM as a molecular marker for breast cancer.CI - (c) 2014 Wiley Periodicals, Inc.

epithelial-mesenchymal transition (EMT) promotes tumor invasion and metastasis, but the coordination and integration mechanisms of these processes are still not fully understood. In this study, we used a cross-species expression profiling strategy of Hela cells to determine an important genetic program transfers. In particular, we have discovered a new transfer function, which is not previously known about transcription factor forkhead box Q1 (FOXQ1). The shRNA anti-FOXQ1 gene was synthesized and transfected into the Hela and EpRas cells. RT-PCR assay was performed to detect the mRNA levels in cells. Cell adhesion and separation assay were used to examine the cell-cell adhesion and separation among cells. Wound healing assay was utilized to examine cell migration and invasion ability. Chromatin immunoprecipitation assay was used to investigate the interaction between E-cadherin and N-cadherin and FOXQ1 promoter region. The results indicated that ectopic expression of FOXQ1 increased cell migration and invasion in vitro, enhanced mammary epithelial cells in vivo lung metastasis, and triggered significant EMT. In contrast, the opposite effects in vitro and in vivo of FOXQ1 knockdown phenotypes were caused by these mechanisms. Notably, FOXQ1 repressed core EMT regulation of the expression of TGF-beta1. FOXQ1 protein directly interacts with E-cadherin and N-cadherin promoter region. And surveys show that FOXQ1 expression regulation by TGF-beta1 and blockade induced EMT both morphological and molecular levels. Our findings emphasize the feasibility of cross-species expression profiles, as a strategy to identify metastasis-related genes. The induction of EMT by FOXQ1 defines a new transfer function in promoting cancer behind possible mechanisms.

epithelial-to-mesenchymal transition (EMT) is essential for embryonic morphogenesis and wound healing and critical for tumour cell invasion and dissemination. The AP-1 transcription factor Fra-1 has been implicated in tumorigenesis and in tumour-associated EMT in human breast cancer. We observed a significant inverse correlation between Fra-1 mRNA expression and distant-metastasis-free survival in a large cohort of breast cancer patients derived from multiple array data sets. This unique correlation among Fos genes prompted us to assess the evolutionary conservation between Fra-1 functions in EMT of human and mouse cells. Ectopic expression of Fra-1 in fully polarized, non-tumourigenic, mouse mammary epithelial EpH4 cells induced a mesenchymal phenotype, characterized by a loss of epithelial and gain of mesenchymal markers. Proliferation, motility and invasiveness were also increased in the resulting EpFra1 cells, and the cells were tumourigenic and efficiently colonized the lung upon transplantation. Molecular analyses revealed increased expression of Tgfbeta1 and the EMT-inducing transcription factors Zeb1, Zeb2 and Slug. Mechanistically, Fra-1 binds to the tgfb1 and zeb2 promoters and to an evolutionarily conserved region in the first intron of zeb1. Furthermore, increased activity of a zeb2 promoter reporter was detected in EpFra1 cells and shown to depend on AP-1-binding sites. Inhibiting TGFbeta signalling in EpFra1 cells moderately increased the expression of epithelial markers, whereas silencing of zeb1 or zeb2 restored the epithelial phenotype and decreased migration in vitro and tumorigenesis in vivo. Thus Fra-1 induces changes in the expression of genes encoding EMT-related transcription factors leading to the acquisition of mesenchymal, invasive and tumorigenic capacities by epithelial cells. This study defines a novel function of Fra-1/AP-1 in modulating tgfb1, zeb1 and zeb2 expression through direct binding to genomic regulatory regions, which establishes a basis for future in vivo genetic manipulations and preclinical studies using mouse models.

The epithelial-mesenchymal transition (EMT) is an essential process for embryogenesis. It also plays a critical role in the initiation of tumor metastasis. Src homology 2 (SH2)-domain containing protein-tyrosine phosphatase-2 (SHP2) is a ubiquitously expressed protein-tyrosine phosphatase and is mutated in many tumors. However, its functional role in tumor metastasis remains largely unknown. We found that TGFbeta1-induced EMT in lung epithelial A549 cells was partially blocked when SHP2 was decreased by transfected siRNA. The constitutively active form (E76V) promoted EMT while the phosphatase-dead mutation (C459S) and the SHP2 inhibitor PHPS1 blocked EMT, which further demonstrated that the phosphatase activity of SHP2 was required for promoting TGFbeta1-induced EMT. Using the protein-tyrosine phosphatase domain of SHP2 as bait, we identified a novel SHP2-interacting protein Hook1. Hook1 was down-regulated during EMT in A549 cells. Overexpression of Hook1 inhibited EMT while knockdown of Hook1 promoted EMT. Moreover, both the protein-tyrosine phosphatase domain and N-terminal SH2 domain of SHP2 directly interacted with Hook1. Down-regulation of Hook1 increased SHP2 activity. These results suggested that Hook1 was an endogenous negative regulator of SHP2 phosphatase activity. Our data showed that the protein-tyrosine phosphatase SHP2 was involved in the process of EMT and Hook1 repressed EMT by regulating the activation of SHP2. SHP2-Hook1 complex may play important roles in tumor metastases by regulating EMT in cancer cells.CI - (c) 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

beta-Galactoside alpha2,6-sialyltranferase 1 (ST6GAL1) catalyzes the addition of terminal alpha2,6-sialylation to N-glycans. Increased expression of ST6GAL1 has been reported in diverse carcinomas and highly correlates with tumor progression. Here, we report that St6gal1 transcription and alpha2,6-sialylated N-glycans are up-regulated during TGF-beta-induced epithelial-mesenchymal transition (EMT) in GE11 cells, requiring the Sp1 element within the St6gal1 promoter. Knockdown of St6gal1 strongly suppressed TGF-beta-induced EMT with a concomitant increase in E-cadherin expression, a major determinant of epithelial cell adherens junctions. Conversely, overexpression of ST6GAL1 increased the turnover of cell surface E-cadherin and promoted TGF-beta-induced EMT. Overexpressing beta-galactoside alpha2,3-sialyltranferase 4 had little influence on EMT, indicating specificity for alpha2,6-sialylation. The basal mesenchymal phenotype of MDA-MB-231 human breast cancer cells was partially reversed by ST6GAL1 silencing. Moreover, ST6GAL1 knockdown inhibited the phosphorylation of Akt, but not Smad2, suggesting that ST6GAL1 contributes to EMT through a non-Smad signaling pathway. Taken together, our data indicate that ST6GAL1 promotes TGF-beta-dependent EMT as well as maintenance of the mesenchymal state by growth signaling, providing a plausible mechanism whereby up-regulated ST6GAL1 may promote malignant progression.CI - (c) 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The molecular regulation of the lung metastasis of pancreatic carcinoma (PCC) is not completely understood. Here, we show that the levels of phosphorylated SMAD3, ZEB1, ZEB2, Snail1, and Snail2 were significantly higher in PCC with lung metastasis than in PCC without lung metastasis. Overexpression of TGFbeta1 enhanced the invasiveness of PCC cells, while inhibition of TGFbeta1 decreased the invasiveness of PCC cells, which appeared to be conducted by activated TGFbeta receptor signaling-induced upregulation of ZEB1, ZEB2, Snail1, and Snail2, suggesting a process of epithelial-mesenchymal transition (EMT). Taken together, our study provides evidence that TGFbeta receptor signaling-induced EMT may be responsible for the increased PCC invasiveness to enhance its lung metastasis.

BACKGROUND: epithelial-to-mesenchymal transition (EMT) induced by TGF-beta1 is one of well-recognized factors contributing to renal fibrosis. However, the underlying molecular mechanisms of EMT are not fully understood. Brachyury, an evolutionarily conserved transcription factor, was recently identified as an important factor promoting EMT in human carcinoma cell lines. There is no evidence that Brachyury is involved in renal tubular EMT. RESULTS: Our results demonstrated that Brachyury was prominently induced in TGF-beta1-treated human proximal tubular epithelial (HK-2) cells and that this induction was accompanied by changes characteristic of EMT. Blockage of Brachyury expression by short interfering RNA (siRNA) in HK-2 cells effectively reversed the TGF-beta1-induced EMT phenotype. Brachyury induction repressed E-cadherin transcription; the E-cadherin promoter contains a Brachyury binding site, and decreased expression of E-cadherin occurred in Brachyury-overexpressing cells when they were transfected with reporter constructs using the promoter. This effect was partially mediated by Slug and Snail, as knockdown of Snail and Slug by siRNA effectively reversed Brachyury-mediated EMT and partially restored E-cadherin expression. The expression of Brachyury also presented in a rat model of obstructive nephropathy and in tubulointerstitial fibrosis tissues of IgA nephropathy, suggesting that it may have a role in EMT and renal fibrosis in vivo. CONCLUSION: Our results demonstrate for the first time that Brachyury plays an important role in regulating TGF-beta1-mediated renal EMT and could be an attractive target for progression of renal disease therapies.

Invasion and metastasis are the major causes of death in patients with esophageal squamous cell carcinoma (ESCC). epithelial-mesenchymal transition (EMT) is a critical step in tumor progression and transforming growth factor-beta1 (TGF-beta1) signaling has been shown to play an important role in EMT. In this study, we investigated how TGF-beta1 signaling pathways contributed to EMT in three ESCC cell lines as well as 100 patients of nomadic ethnic Kazakhs residing in northwest Xinjiang Province of China. In vitro analyses included Western blotting to detect the expression of TGF-beta1/Smad and EMT-associated proteins in Eca109, EC9706 and KYSE150 cell lines following stimulation with recombinant TGF-beta1 and SB431542, a potent inhibitor of ALK5 that also inhibits TGF-beta type II receptor. TGF-beta-activated Smad2/3 signaling in EMT was significantly upregulated as indicated by mesenchymal markers of N-cadherin and Vimentin, and in the meantime, epithelial marker, E-cadherin, was markedly downregulated. In contrast, SB431542 addition downregulated the expression of N-cadherin and Vimentin, but upregulated the expression of E-cadherin. Moreover, the TGF-beta1-induced EMT promoted invasion capability of Eca109 cells. tumor cells undergoing EMT acquire fibroblastoid-like phenotype. Expressed levels of TGF-beta1/Smad signaling molecules and EMT-associated proteins were examined using immunohistochemical analyses in 100 ESCC tissues of Kazakh patients and 58 matched noncancerous adjacent tissues. The results showed that ESCC tissues exhibited upregulated expression of TGF-beta1/Smad. We also analyzed the relationship between the above proteins and the patients' clinicopathological characteristics. The TGF-beta1/Smad signaling pathway in human Eca109 ESCC cells may carry similar features as in Kazakh ESCC patients, suggesting that TGF-beta1/Smad signaling pathway may be involved in the regulation of EMT in ethnic Kazakh patients with ESCC from Xinjiang, China.

Idiopathic pulmonary fibrosis (IPF) is one of the most common and severe interstitial lung diseases. epithelial-to-mesenchymal transition (EMT) is a process whereby epithelial cells undergo transition to a mesenchymal phenotype. This process has been shown to contribute to IPF. MicroRNAs (miRNAs) are small non-coding RNAs of 18-24 nucleotides in length which regulate gene expression. Several studies have implicated miRNAs in EMT; however, specific miRNAs that regulate EMT in IPF have not yet been identified. In this study, we identified 6 up-regulated and 3 down-regulated miRNAs in a human lung epithelial cell EMT model using miRNA microarray and real-time PCR. Overexpression of one of these up-regulated miRNAs, miR-424, increased the expression of alpha-smooth muscle actin, an indicator of myofibroblast differentiation, but had no effects on the epithelial or mesenchymal cell markers. miR-424 enhanced the activity of the TGF-beta signaling pathway, as demonstrated by a luciferase reporter assay. Further experiments showed that miR-424 decreased the protein expression of Smurf2, a negative regulator of TGF-beta signaling, indicating that miR-424 exerts a forward regulatory loop in the TGF-beta signaling pathway. Our results suggest that miR-424 regulates the myofibroblast differentiation during EMT by potentiating the TGF-beta signaling pathway, likely through Smurf2.CI - Copyright (c) 2014 Elsevier Inc. All rights reserved.

Long noncoding RNAs (lncRNAs) are emerging as key regulators in various biological processes. epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by tumor cells to depart from the primary tumor site, invade surrounding tissue, and establish distant metastases. Transforming growth factor beta (TGFbeta) signaling has been shown to be a major inducer of EMT and to facilitate breast cancer metastasis. However, the role of lncRNAs in this process remains largely unknown. Here we report a genome-wide lncRNA profile in mouse mammary epithelial NMuMG cells upon TGFbeta induction of EMT. Among 10,802 lncRNAs profiled, over 600 were up-regulated and down-regulated during the EMT, respectively. Furthermore, we identify that lncRNA-HIT (HOXA transcript induced by TGFbeta) mediates TGFbeta function, i.e. depletion of lncRNA-HIT inhibits TGFbeta-induced migration, invasion, and EMT in NMuMG. LncRNA-HIT is also significantly elevated in the highly metastatic 4T1 cells. Knockdown of lncRNA-HIT in 4T1 results in decrease of cell migration, invasion, tumor growth, and metastasis. E-cadherin was identified as a major target of lncRNA-HIT. Moreover, lncRNA-HIT is conserved in humans and elevated expression associates with more invasive human primary breast carcinoma. Collectively, these data suggest that a subset of lncRNAs such as lncRNA-HIT play a significant role in regulation of EMT and breast cancer invasion and metastasis, and could be potential therapeutic targets in breast cancers.CI - (c) 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Human leukocyte antigen class I antigens (HLA-I) is essential in immune response by presenting antigenic peptides to cytotoxic T lymphocytes. Downregulation of HLA-I is observed in primary and metastatic prostate cancers, which facilitates them escape from immune surveillance, thereby promotes prostate cancer progression. In addition, elevated level of growth factors like TGF-beta or EGF in microenvironment is related to the prostate cancer deterioration. Thus, we wondered whether TGF-beta or EGF was involved in the regulation of HLA-I during the development of prostate cancer cells. In this study, we demonstrated that TGF-beta and EGF both downregulated the expression of HLA-I, thereby attenuated the cytotoxic T cell mediated lysis of prostate cancer cells. Next, we revealed that TGF-beta and EGF induced downregulation of HLA-I is associated with classical epithelial-mesenchymal transition (EMT) morphological changes and expression profiles. We further illustrated that overexpression of Snail is crucial for HLA-I downregulation and its association with EMT. At last, we discussed that NF-kappaB/p65 is the plausible target for Snail to induce HLA-I downregulation. Taken together, this is the first evidence to reveal that both TGF-beta and EGF can induce HLA-I downregulation which is then proven to be associated with EMT in prostate cancer cells. These discoveries provide a deeper understanding of growth factors induced immune escape and introduce potential therapeutic targets for prostate cancers.CI - Copyright (c) 2015 Elsevier Ltd. All rights reserved.

It has been shown that acquisition of epithelial-mesenchymal transition (EMT) and induction of cancer stem cell (CSC)-like properties contribute to metastasis of cancers in many studies; however, the molecular mechanisms underlying EMT and CSC phenotypes in liver cancer cells remain to be elucidated. MiR-155 is an important microRNA associated with tumour progression. Here, we report that miR-155 regulates not only the epithelial-mesenchymal transition but also the stem-like transition in liver cancer cells. Utilizing quantitative RT-PCR, we found that the expression of miR-155 is positively related to the levels of CD90, CD133 and Oct4 in enriched spheres. Up-regulated miR-155 significantly increases the population of stem-like CSCs among liver cancer cells and the ability to form tumour spheres. Additionally, miR-155 overexpression in cells significantly increases cell motility and invasion, as well as the epithelial-mesenchymal transition process. Conversely, suppression of miR-155 in cells had an opposite effect, which was partially rescued by the down-regulation of TP53INP1. Collectively, miR-155 promotes liver cancer cell EMT and CSCs, in part, via silencing TP53INP1. In addition, we found that TGF-beta1 indirectly regulates TP53INP1 expression via miR-155 in liver cancer cells. Taken together, our findings suggest that miR-155 regulates TP53INP1 expression, to induce the epithelial-mesenchymal transition and acquisition of a stem cell phenotype.CI - Copyright (c) 2015 Elsevier Ireland Ltd. All rights reserved.

BACKGROUND/AIMS: Since renal fibrosis always predisposes end-stage renal disease, elucidation of the molecular mechanisms that underlie the progression of renal fibrosis may substantially improve the understanding and treatment for renal failure. Previous studies have highlighted an important counteraction between transforming growth factor beta 1 (TGFbeta1) and bone morphogenic protein 7 (BMP7) in the epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells during chronic renal injury. Macrophages are also believed to play a critical role in renal fibrosis. However, the relationship between macrophages and EMT is unknown. METHODS: Here, we used a mouse unilateral ureteral obstruction (UUO) model to address to these questions, and analyzed macrophage and its subpopulations purified by flow cytometry. RESULTS: We found that the recruited macrophages are polarized to a M2 subtype after renal injury. M2 macrophages released high levels TGFbeta1 to suppress BMP7 to enhance EMT-induced renal fibrosis. Depletion of M2 macrophages, but not of M1 macrophages, specifically inhibited EMT, and subsequently the renal fibrosis. Adoptive transplantation of M2 macrophages deteriorated renal fibrosis. CONCLUSION: Thus, our study highlights M2 macrophages as a critical target for treating renal fibrosis.CI - (c) 2015 S. Karger AG, Basel.

Doxorubicin (Dox) is a commonly used chemotherapeutic drug in human colon cancer. However, it becomes increasingly ineffective with tumor progression, the underlying mechanism of which remains to be elucidated. Emerging evidence has led to the identification of an association between chemoresistance and the acquisition of epithelial-mesenchymal transition (EMT) in cancer. However, it remains to be elucidated whether this process is involved in the development of resistance to Dox in colon cancer. In HCT116 human colon cancer cells treated with Dox (50 nmol/l), EMT was induced, and transforming growth factor (TGF)beta signaling and multi-drug resistant plasma membrane glycoprotein levels were significantly increased. By contrast, silencing of Smad4, using stable RNA interference, inhibited TGFbeta signaling, reversed the process of EMT and markedly increased the sensitivity of HCT116 cells to Dox. The results of the present study suggested that the combination of Dox with the downregulation of TGFbeta signaling may be a potential novel therapeutic strategy with which to overcome chemoresistance during colon cancer chemotherapy.

BACKGROUND: The epithelial-mesenchymal transition (EMT) has been associated with the acquisition of migration, invasiveness, and metastasis traits. During tumor progression, EMT can be induced by transforming growth factor-beta (TGF-beta) signal that epithelial cells receive from their microenvironment. However, the master regulatory controls on TGF-beta-EMT axis are not understood. METHODS: The protein expression in human specimens was measured by immunohistochemical staining. E74-like factor 5 (Elf5) was silenced by short interfering RNAs in LNCaP cells and stably overexpressed by HA-tagged Elf5 cDNAs in 22Rv1 cells. These cells were used to study migration and anchorage-independent growth. RESULTS: Our data reveal that Elf5 results in the failure of mesenchymal morphogenesis, upregulation of EMT markers, spheres formation, and migration in the presence of TGF-beta. Furthermore, Elf5 blocks TGF-beta signaling, through decreasing drosophila mothers against decapentaplegic protein (SMAD3) activation by binding to it, one of the major effector of TGF-beta-induced EMT. Moreover, Elf5 can serve as a prognostic marker of metastasis-free survival in patients with TGF-beta-positive prostate cancer. CONCLUSIONS: Elf5 expression is inversely correlated with EMT. Elf5 inhibits TGF-beta-driven EMT via repressing SMAD3 phosphorylation in prostate cancer cells. In addition, Elf5 can be used as a biomarker of metastasis-free survival in patients with TGF-beta-positive prostate cancer.CI - (c) 2015 Wiley Periodicals, Inc.

BACKGROUND: epithelial-mesenchymal transition (EMT), in which polarized epithelial cells have mesenchymal cell phenotypes, is thought to be a key process of invasion and metastasis of cancer. Transforming growth factor beta-1 (TGF-beta1) is known to be carcinogenic and Helicobacter pylori is a predominant carcinogen of gastric cancer. Our study aimed to determine whether TGF-beta1 or H. pylori infection enhances EMT process and cytotoxin-associated gene E (CagE) is associated with EMT. MATERIALS AND METHODS: Human gastric cancer cell AGS and MKN45 were treated with recombinant TGF-beta1 or H. pylori including cagE-negative (DeltacagE) mutant. Besides the assessment of EMT-related markers expression levels by means of RT-qPCR, Western blot, and immunofluorescence assay, the induction of in vitro EMT on gastric cancer cells (AGS and MKN cell lines) was confirmed by wound-healing assay and invasion assay. RESULTS: When gastric cancer cells were treated with TGF-beta1 or various strains of cagE-positive H. pylori, EMT-related marker altered significantly. However, the DeltacagE mutant did not. Wound-healing assay and invasion assay showed enhanced migration ability of the cells treated with cagE-positive H. pylori but not in DeltacagE mutant. CONCLUSIONS: EMT induction in gastric cancer cells by TGF-beta1 was confirmed. Only infection with cagE-positive H. pylori upregulated the TGF-beta1-mediated EMT pathway and consequently promotes EMT. Therefore, H. pylori might induce TGF-beta1-mediated EMT associated with the cagE.CI - (c) 2015 John Wiley & Sons Ltd.

Transforming growth factor beta (TGFbeta) is believed to play a dual role in prostate cancer. Molecular mechanism by which TGFbeta1 suppresses early prostate tumor growth and induces epithelial-to-mesenchymal transition (EMT) in advanced stages is not known. We determined if P21-activated kinase1 (Pak1), which mediates cytoskeletal remodeling is necessary for the TGFbeta1 induced prostate cancer EMT. Effects of TGFbeta1 on control prostate cancer PC3 and DU145 cells and those with IPA 3 and siRNA mediated Pak1 inhibition were tested for prostate tumor xenograft in vivo and EMT in vitro. TGFbeta1 inhibited PC3 tumor xenograft growth via activation of P38-MAPK and caspase-3, 9. Long-term stimulation with TGFbeta1 induced PC3 and DU145 cell scattering and increased expression of EMT markers such as Snail and N-cadherin through tumor necrosis factor receptor-associated factor-6 (TRAF6)-mediated activation of Rac1/Pak1 pathway. Selective inhibition of Pak1 using IPA 3 or knockdown using siRNA both significantly inhibited TGFbeta1-induced prostate cancer cell EMT and expression of mesenchymal markers. Our study demonstrated that TGFbeta1 induces apoptosis and EMT in prostate cancer cells via activation of P38-MAPK and Rac1/Pak1 respectively. Our results reveal the potential therapeutic benefits of targeting TGFbeta1-Pak1 pathway for advanced-stage prostate cancer.CI - Copyright (c) 2015 Elsevier B.V. All rights reserved.

epithelial-mesenchymal transition (EMT) was initially recognized during organogenesis and has recently been reported to be involved in promoting cancer invasion and metastasis. Cooperation of transforming growth factor-beta (TGF-beta) and other signaling pathways, such as Ras and Wnt, is essential to inducing EMT, but the molecular mechanisms remain to be fully determined. Here, we reported that insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1), a potential tumor suppressor, controls EMT in colorectal cancer progression. We revealed the inhibitory role of IGFBP-rP1 through analyses of clinical colorectal cancer samples and various EMT and metastasis models in vitro and in vivo. Moreover, we demonstrated that IGFBP-rP1 suppresses EMT and tumor metastasis by repressing TGF-beta-mediated EMT through the Smad signaling cascade. These data establish that IGFBP-rP1 functions as a suppressor of EMT and metastasis in colorectal cancer.

epithelial-mesenchymal transition (EMT) is a fundamental process in embryonic development and organ formation. Aberrant regulation of EMT often leads to tumor progression. Changes in cell surface sialylation have recently been implicated in mediating EMT. Herein we report the visualization of dynamic changes of sialylation and glycoproteomic analysis of newly synthesized sialylated proteins in EMT by metabolic labeling of sialylated glycans with azides, followed by click labeling with fluorophores or affinity tags. We discovered that sialylation was down-regulated during EMT but then reverted and up-regulated in the mesenchymal state after EMT, accompanied by mRNA expression level changes of genes involved in the sialic acid biosynthesis. Quantitative proteomic analysis identified a list of sialylated proteins whose biosynthesis was dynamically regulated during EMT. Sialylation of cell surface adherent receptor integrin beta4 was found to be down-regulated, which may regulate integrin functions during EMT. Furthermore, a global sialylation inhibitor was used to probe the functional role of sialylation during EMT. We found that inhibition of sialylation promoted EMT. Taken together, our findings suggest the important role of sialylation in regulating EMT and imply its possible function in related pathophysiological events, such as cancer metastasis.CI - (c) 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The aim of the present study was to examine the role of protease-activated receptor-1 (PAR1)-stimulated platelet activation in the epithelial-mesenchymal transition (EMT) and migration of colon cancer cells, and to identify the underlying mechanisms. TFLLR-NH2, a PAR1 agonist, was used to activate platelets and the platelet supernatants were used to treat the SW620 colon cancer cell line. expression of E-cadherin and vimentin on SW620 cells was detected by immunofluorescence and western blotting, and the level of the transforming growth factor beta1 (TGF-beta1) was measured using ELISA following the activation of platelets by TFLLR-NH2. miR-200b expression was detected using quantitative PCR in SW620 cells. In order to investigate the chemotactic ability of the SW620 cells, the expression of CXC chemokine receptor type 4 (CXCR4) was measured by flow cytometry. Transwell migration assays were performed following exposure of the cells to the supernatant of PAR1-activated platelets. SW620 cells cultured in the supernatant of TFLLR-NH2-activated platelets upregulated E-cadherin expression and downregulated the vimentin expression. In the in vitro platelet culture system, a TFLLR-NH2 dose-dependent increase of secreted TGF-beta1 was detected in the supernatant. The activation of PAR1 on the platelets led to the inhibition of miR-200b expression in the SW620 cells that were cultured in platelet-conditioned media. The number of SW620 cells that penetrated through the Transwell membrane increased with the dose of TFLLR-NH2 used to treat the platelets. The percentage of CXCR4-positive SW620 cells was significantly higher when they were exposed to the supernatant of platelets cultured for 24 h with PAR1 agonist than when cultured in non-conditioned media (40.89 +/- 6.74 vs. 3.47 +/- 1.40%, P < 0.01). Platelet activation with a PAR1 agonist triggered TGF-beta secretion, which induced EMT of SW620 human colon cancer cells via the downregulation of miR-200b expression, and activated platelets had a chemotactic effect on colon cancer cells mediated by the upregulation of CXCR4 on the cell surface.

BACKGROUND: Chronic airway inflammatory disorders, such as asthma, are characterized by airway inflammation and remodeling. Chronic inflammation and damage to the airway epithelium cause airway remodeling, which is associated with improper epithelial repair, and is characterized by elevated expression of transforming growth factor-beta (TGF-beta). epithelial-mesenchymal transition (EMT) is an important mechanism during embryonic development and tissue remodeling whereby epithelial cells gain the capacity to increase motility by down-regulation of epithelial markers and up-regulation of mesenchymal markers. TGF-beta is a central inducer of EMT, and TGF-beta-induced EMT is enhanced by pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta. We investigated whether the pro-inflammatory cytokine TWEAK (TNF-like weak inducer of apoptosis) enhanced TGF-beta1-induced EMT in the human bronchial epithelial cell line BEAS-2B. METHODS: Quantitative RT-PCR and western blotting were used to define alterations in epithelial and mesenchymal marker expression in BEAS-2B cells. The cells were assessed for 48 h after stimulation with TGF-beta1 alone or in combination with TWEAK. RESULTS: TGF-beta1 induced spindle-like morphology and loss of cell contact, and reduced the expression of epithelial marker E-cadherin and increased the expression of mesenchymal markers N-cadherin and vimentin. Our data, for the first time, show that TWEAK reduced the expression of E-cadherin, and that co-treatment with TGF-beta1 and TWEAK enhanced the TGF-beta1-induced features of EMT. Moreover, hyaluronan synthase 2 expression was up-regulated by a combination with TGF-beta1 and TWEAK, but not TNF-alpha. We also demonstrated that the Smad, p38 MAPK, and NF-kappaB signaling pathways, and the transcriptional repressor ZEB2 might mediate N-cadherin up-regulation by TGF-beta1 in combination with TWEAK. CONCLUSIONS: These findings suggest that the pro-inflammatory cytokine TWEAK and TGF-beta1 have synergistic effects in EMT and may contribute to chronic airway changes and remodeling.

epithelial-mesenchymal transition (EMT) is a key event in cancer metastasis, which confers cancer cells with increased motility and invasiveness, and EMT is characterized by loss of epithelial marker E-cadherin and gain of mesenchymal marker N-cadherin. Transforming growth factor-beta (TGF-beta) signaling is a crucial inducer of EMT in various types of cancer. Ski is an important negative regulator of TGF-beta signaling, which interacts with SMADs to repress TGF-beta signaling activity. Although there is accumulating evidence that Ski functions as a promoter or suppressor in human types of cancer, the molecular mechanisms by which Ski affects TGF-beta-induced EMT and invasion in non-small cell lung cancer (NSCLC) are not largely elucidated. In the present study, we investigated the mechanistic role of Ski in NSCLC metastasis. Ski was significantly reduced in metastatic NSCLC cells or tissues when compared with non-metastatic NSCLC cells or tissues. Moreover, following TGF-beta stimulation Ski-silenced A549 cells had more significant features of EMT and a higher invasive activity when compared with A549 cells overexpressing Ski. Mechanistically, Ski-silenced and overexpressed A549 cells showed an increase and a reduction in the SMAD3 phosphorylation level, respectively. This was supported by plasminogen activator inhibitor-1 (PAI-1) promoter activity obtained in Ski-silenced and overexpressed A549 cells. However, after treatment of SIS3 (inhibitor of SMAD3 phosphorylation) followed by TGF-beta1 stimulation, we did not observe any effect of Ski on TGF-beta-induced EMT, and invasion in Ski-silenced and overexpressed A549 cells. In conclusion, our findings suggest that Ski represses TGF-beta-induced EMT and invasion by inhibiting SMAD-dependent signaling in NSCLC.

tumor cells frequently disseminate through the lymphatic system during metastatic spread of breast cancer and many other types of cancer. Yet it is not clear how tumor cells make their way into the lymphatic system and how they choose between lymphatic and blood vessels for migration. Here we report that mammary tumor cells undergoing epithelial-mesenchymal transition (EMT) in response to transforming growth factor-beta (TGF-beta1) become activated for targeted migration through the lymphatic system, similar to dendritic cells (DCs) during inflammation. EMT cells preferentially migrated toward lymphatic vessels compared with blood vessels, both in vivo and in 3D cultures. A mechanism of this targeted migration was traced to the capacity of TGF-beta1 to promote CCR7/CCL21-mediated crosstalk between tumor cells and lymphatic endothelial cells. On one hand, TGF-beta1 promoted CCR7 expression in EMT cells through p38 MAP kinase-mediated activation of the JunB transcription factor. Blockade of CCR7, or treatment with a p38 MAP kinase inhibitor, reduced lymphatic dissemination of EMT cells in syngeneic mice. On the other hand, TGF-beta1 promoted CCL21 expression in lymphatic endothelial cells. CCL21 acted in a paracrine fashion to mediate chemotactic migration of EMT cells toward lymphatic endothelial cells. The results identify TGF-beta1-induced EMT as a mechanism, which activates tumor cells for targeted, DC-like migration through the lymphatic system. Furthermore, it suggests that p38 MAP kinase inhibition may be a useful strategy to inhibit EMT and lymphogenic spread of tumor cells.

Maternal Id-like molecule (Maid) is a dominant negative helix-loop-helix protein that has been implicated in regulating gene expression as well as cell-cycle progression. Overexpressed Maid was previously shown to inhibit certain cellular responses induced by transforming growth factor-beta (TGF-beta), such as TGF-beta-induced cytostasis and cell motility, but not epithelial-mesenchymal transition (EMT). The role of endogenous Maid in regulating TGF-beta signalling, however, has not been elucidated. We have found evidence that endogenous Maid negatively regulates TGF-beta-induced cell motility. Maid knockdown enhanced TGF-beta-induced cell motility as measured by chamber migration and wound healing assays but did not affect cell motility induced by bone morphogenetic protein (BMP)-4. Endogenous Maid does not appear to be involved in regulating TGF-beta-induced cytostasis, resistance to apoptosis or EMT. Notably, Maid expression was induced in the delayed phase (later than 24 h) after TGF-beta stimulation whereas the expression of two other negative feedback regulators, Smad7 and SnoN, was induced as early as 1 h after stimulation. These findings indicate that Maid is a unique negative feedback regulator of TGF-beta signalling in its mode of action as well as the timing of its induction.CI - (c) The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

epithelial-mesenchymal transition (EMT), a process closely related to tumor development, is regulated by a variety of signaling pathways and growth factors, such as transforming growth factor-beta1 (TGF-beta1) and epidermal growth factor (EGF). Hyaluronan (HA) has been shown to induce EMT through either TGF-beta1 or EGF signaling and to be a regulator of the crosstalk between these two pathways in fibroblasts. In this study, in order to clarify whether HA has the same effect in tumor cells, we utilized the lung cancer cell line, A549, and the breast cancer cell line, MCF-7, and found that the effects of stimulation with TGF-beta1 were more potent than those of EGF in regulating the expression of EMT-associated proteins and in enhancing cell migration and invasion. In addition, we observed that TGF-beta1 activated EGF receptor (EGFR) and its downstream AKT and extracellular signal-regulated kinase (ERK) pathways. Furthermore, we found that TGF-beta1 upregulated the expression of hyaluronan synthases (HAS1, HAS2 and HAS3) and promoted the expression of CD44, a cell surface receptor for HA, which interacts with EGFR, resulting in the activation of the downstream AKT and ERK pathways. Conversely, treatment with 4-methylumbelliferone (4-MU; an inhibitor of HAS) prior to stimulation with TGF-beta1, inhibited the expression of CD44 and EGFR, abolished the interaction between CD44 and EGFR. Furthermore, the use of shRNA targeting CD44 impaired the expression of EGFR, deactivated the AKT and ERK pathways, reversed EMT and decreased the migration and invasion ability of cells. In conclusion, our data demonstrate that TGF-beta1 induces EMT by the transactivation of EGF signaling through HA/CD44 in lung and breast cancer cells.

BACKGROUND: In our previous study, we found that periostin was upregulated in prostate cancer, and its expression could be modulated by TGF-beta. TGF-beta could upregulate periostin expression in some cells, and both TGF-beta and periostin could induce epithelial mesenchymal transition (EMT). We aimed to study the effect of periostin in the process of TGF-beta-induced EMT in prostate cancer cells. METHODS: We constructed a lentivirus vector containing the periostin gene and transduced it into PC3 and DU145 cells. After confirming periostin overexpression by PCR and Western blotting, we used an MTT assay to establish a growth curve to measure cell proliferation. Additionally, we performed transwell and wound healing assays to measure cell invasion and migration, respectively. Lastly, we measured the expression of EMT associated factors using Western blot analysis to test the effect of periostin on EMT in prostate cancer cells. RESULTS: PCR and Western blot analyses confirmed that periostin was upregulated after infection with the periostin lentiviral vector. Periostin overexpression promoted increased cell proliferation, invasion, and migration as measured by MTT, transwell, and wound healing assays, respectively. Western blot analysis illustrated that periostin overexpression increased the expression of EMT associated factors, and periostin overexpression activated Akt and GSK-3beta, which could be inhibited using a PI3K inhibitor. Additionally, TGF-beta increased the levels of STAT3, Twist1 and periostin, while both STAT3 shRNA and Twist1 shRNA inhibited periostin expression. However, STAT3 shRNA also decreased Twist1 expression. Although reduction of STAT3, Twist1 or periostin levels with shRNA inhibited TGF-beta-induced overexpression of EMT associated factors, periostin overexpression could reverse such inhibition by interfering with STAT3 and Twist1. Similarly, periostin overexpression also reversed inhibition of cell invasion induced by interference of STAT3 and Twist1. CONCLUSION: Our findings indicate that periostin is an important mediator of TGF-beta-induced EMT and suggest that periostin is a potential therapeutic target for suppressing the metastatic progression of prostate cancer.CI - (c) 2015 S. Karger AG, Basel.

Keloid is a skin fibrotic disease with the characteristics of recurrence and invasion, its pathogenesis still remains unrevealed. The epithelial-mesenchymal transition (EMT) is critical for wound healing, fibrosis, recurrence, and invasion of cancer. We sought to investigate the EMT in keloid and the mechanism through which the EMT regulates keloid formation. In keloid tissues, the expressions of EMT-associated markers and transforming growth factor (TGF)-beta1/Smad3 signaling were examined by immunohistochemistry. In the keloid epidermis and dermal tissue, the expressions of genes related to the regulation of skin homeostasis, fibroblast growth factor receptor 2 (FGFR2) and p63, were analyzed using quantitative real-time polymerase chain reaction. The results showed that accompanying the loss of the epithelial marker E-cadherin and the gain of the mesenchymal markers fibroblast-specific protein 1 (FSP1) and vimentin in epithelial cells from epidermis and skin appendages, and in endothelial cells from dermal microvessels, enhanced TGF-beta1 expression and Smad3 phosphorylation were noted in keloid tissues. Moreover, alternative splicing of the FGFR2 gene switched the predominantly expressed isoform from FGFR2-IIIb to -IIIc, concomitant with the decreased expression of DeltaNp63 and TAp63, which changes might partially account for abnormal epidermis and appendages in keloids. In addition, we found that TGF-beta1-induced hair follicle outer root sheath keratinocytes (ORSKs) and normal skin epithelial cells underwent EMT in vitro with ORSKs exhibiting more obvious EMT changes and more similar expression profiles for EMT-associated and skin homeostasis-related genes as in keloid tissues, suggesting that ORSKs might play crucial roles in the EMT in keloids. Our study provided insights into the molecular mechanisms mediating the EMT pathogenesis of keloids.CI - (c) 2015 by the Wound Healing Society.

Peritoneal fibrosis resulting from long-term peritoneal dialysis is a major cause of failure of peritoneal ultrafiltration function and main reason of dropout from peritoneal dialysis. epithelial-mesenchymal transition (EMT) of peritoneal mesochelial cells (HPMCs) is a major contributor of peritoneal fibrosis. Recently, the association between histone acetylation and kinds of fibrosis including liver, lung and kidney fibrosis is well established. Thus, in this study we tried to profile whether histone acetylation is also operates EMT process in HPMCs and what's the regulatory mechanism. We established an EMT model of HPMCs through high glucose treatment. And hyperacetylation of H3 histone was found using western blot in EMT model. After treated with C646, a histone acetyltransferase (HAT) inhibitor, high glucose-induced EMT in HPMCs was counteracted. To further understand the molecular mechanism of C646 rescues high glucose-induced EMT, CHIP-qPCRwas used to examine the modulation of histone H3 acetylation at promoters of series signaling target genes. We found that the H3 acetylation level at TGF-beta1 gene promoter was down-regulation by C646 treatment. Moreover, we also found that TGF-beta1/Smad3 signaling was blocked. Hence, our results suggest that histone H3 acetylation activated TGF-beta1/Smad3 signaling during EMT of HPMCs, and C646 can rescue the mesenchymal phenotype transition. These findings may provide a novel pathogenic mechanism and therapeutic target for peritoneal fibrosis.

The epithelial-mesenchymal transition (EMT) is a key process in tumor invasion and migration. Transforming growth factor-betas (TGF-betas) are multifunctional growth factors and powerful modulators of the EMT. Here, we investigated the relationship between TGF-beta expression and invasion by treated triple-negative breast cancer (TNBC) cells. Our results show that invasion capacity of TNBC cells was markedly higher than that of non-TNBC cells. In addition, EMT-related gene signatures, including vimentin (vim), fibronectin (FN), snail, and slug were highly expressed in TNBC cells. Interestingly, our results show that TGF-beta1 and beta2 mRNA expression levels were higher in TNBC cells than those in non-TNBC cells. Thus, we examined the effect of the TGF-beta receptor I/II inhibitor LY2109761 on EMT-related gene expression and cell motility. Our data show that vim, FN, and slug mRNA expression levels dose-dependently decreased in response to LY2109761. TNBC cell motility also decreased in response to LY2109761. Finally, we investigated the effect of LY2109761 on TGF-beta1 or TGF-beta2-induced E-cadherin (E-cad), vim, and FN mRNA and protein expression. The reduction in E-cad and induction of vim and FN expression by TGF-beta1 or TGF-beta2 were completely reversed by LY2109761 treatment in HCC1806 TNBC cells. Taken together, we demonstrated that elevated TGF-beta expression triggers invasion and migration by TNBCs through the EMT process. Inhibiting the TGF-beta signaling pathway is considered a promising therapeutic strategy for treating TNBC.CI - Copyright (c) 2015 Elsevier Ltd. All rights reserved.

CCR7 is a G protein-coupled chemokine receptor. In this study, we used immunohistochemistry with tissue microarrays to measure CCR7 expression in tumor specimens from 122 patients with gastric cancer. We show that CCR7 expression is associated with lymph node metastasis (P = 0.022) and overall survival (OS; P = 0.025), and is an independent factor associated with poorer overall survival (P = 0.032). The CCR7 mechanism was predicted based on bioinformatic analysis and verified in gastric cancer cell lines and primary tumor samples. The data show that CCR7 contributes to TGF-beta1-induced epithelial-mesenchymal transition (EMT) and that the effects of TGF-beta1 are inhibited by a CCR7 neutralizing antibody or a NF-kappaB inhibitor. Increased TGF-beta1 expression was accompanied by nuclear localization of NF-kappaB-p65 and higher levels of the mesenchymal marker vimentin in human gastric cancer samples. We conclude that the CCR7 axis mediates TGF-beta1-induced EMT via crosstalk with NF-kappaB signaling, facilitating lymph node metastasis and poorer overall survival in patients with gastric cancer. These findings suggest CCR7 is a novel prognostic indicator and a potential target for gastric cancer therapy.

Transforming growth factor-beta1 (TGF-beta1) is a potent induction factor for epithelial-mesenchymal transition (EMT). mesenchymal-epithelial transition (MET), as the inverse process of EMT, has recently been reported to promote the induction of induced pluripotent stem cells (iPSCs). We have developed pyrrole-imidazole (PI) polyamide, a novel gene regulator that targets human TGF-beta1, and investigated its effects on the EMT/MET process. PI polyamide targeted to TGF-beta1 significantly inhibited the mRNA expression of TGF-beta1 and SNAI1 as an EMT marker and increased mRNA and protein expression of E-cadherin in human epithelial cells. To enhance the induction of iPSCs by the MET process, PI polyamide targeted to TGF-beta1 was applied to human fibroblasts transfected with exogenous reprogramming factors by Sendai virus vector and grown in human iPSCs. The PI polyamide significantly increased the number of alkaline phosphatase-positive colonies. The expression of undifferentiated markers was also observed in these colonies. These results suggest that PI polyamide targeted to human TGF-beta is a novel compound that can control the EMT/MET process of human epithelial cells and enhance the induction of human fibroblasts to iPSCs.CI - Copyright (c) 2015 Elsevier Ltd. All rights reserved.

epithelial-mesenchymal transition (EMT) is associated with the metastasis and poor prognosis of cervical cancer. However, the underlying mechanisms are poorly defined. In the present study, we investigated whether Twist plays a direct role in human cervical cancer using immunohistochemical and western blot analyses. Immunohistochemical analysis revealed that Twist is highly expressed in cervical cancer, which correlates with poor tumor pathological differentiation or lymph node metastasis (P<0.05). Depletion of Twist by stable shRNA-mediated knockdown decreased the migratory ability of cancer cell lines in vitro. Suppression or overexpression of Twist also resulted in an altered expression of the molecular mediators of EMT. Furthermore, exogenous TGF-beta promoted EMT by upregulating the expression of Twist through the TGF-beta/Smad3 pathway, and this effect was eliminated by Twist depletion in cancer cells as demonstrated in the in vitro study. The use of in vivo models revealed a decreased tumor proliferation potential in Twist-depleted cancer cells. The results suggested a novel function for Twist in the promotion of EMT via TGF-beta/Smad3 signaling pathway. Thus, Twist constitutes a potential therapeutic target in human cervical cancer.

Mitochondrial dysfunction has been found to be associated with various pathological conditions, particularly cancer. However, the mechanisms underlying tumor malignancy induced by mitochondrial dysfunction are not fully understood. In the present study, the effects of mitochondrial dysfunction on epithelial-mesenchymal transition (EMT), were investigated using mitochondrial-depleted rho(0) cells derived from the Hep3B hepatocarcinoma cell line. The Hep3B/rho(0) cells displayed the EMT phenotype with more aggressive migration and higher invasiveness compared to their parental cells. The Hep3B/rho(0) cells also showed typical expression pattern of EMT markers such as vimentin and E-cadherin. These phenotypes in Hep3B/rho(0) cells were mediated by increased transforming growth factor-beta (TGF-beta) through the canonical Smad-dependent signaling pathway. Additionally, TGF-beta signaling was activated via induction of c-Jun/AP-1 expression and activity. Therefore, mitochondrial dysfunction induces EMT through TGF-beta/Smad/Snail signaling via c-Jun/AP-1 activation. These results indicate that mitochondrial dysfunction plays an important role in the EMT process and could be a novel therapeutic target for malignant cancer therapy.

BACKGROUND: Increasing evidence suggests that transforming growth factor-beta 1 (TGF-beta1) triggers epithelial to mesenchymal transition (EMT) and facilitates breast cancer stem cell differentiation. Gelsolin (GSN) is a ubiquitous actin filament-severing protein. However, the relationship between the expression level of GSN and the TGF-beta signaling for EMT progression in breast cancer cells is not clear. RESULTS: TGF-beta1 acted on MDA-MB231 breast cancer cells by decreasing cell proliferation, changing cell morphology to a fibroblast-like shape, increasing expressions for CD44 and GSN, and increasing EMT expression and cell migration/invasion. Study with GSN overexpression (GSN op) in both MDA-MB231 and MCF-7 cells demonstrated that increased GSN expression resulted in alterations of cell proliferation and cell cycle progression, modification of the actin filament assembly associated with altering cell surface elasticity and cell detachment in these breast cancer cells. In addition, increased cell migration was found in GSN op MDA-MB231 cells. Studies with GSN op and silencing by small interfering RNA verified that GSN could modulate the expression of vimentin. Sorted by flow cytometry, TGF-beta1 increased subpopulation of CD44+/CD22- cells increasing their expressions for GSN, Nanog, Sox2, Oct4, N-cadherin, and vimentin but decreasing the E-cadherin expression. Methylation specific PCR analysis revealed that TGF-beta1 decreased 50 % methylation but increased 3-fold unmethylation on the GSN promoter in CD44+/CD22- cells. Two DNA methyltransferases, DNMT1 and DNMT3B were also inhibited by TGF-beta1. CONCLUSIONS: TGF-beta1 induced epigenetic modification of GSN could alter the EMT process in breast cancer cells.

BACKGROUND: Flot2, a highly conserved protein of the SPFH domain containing proteins family, has recently been identified as oncogene to be involved in the tumorigenesis and metastasis of several cancers including gastric cancer. However, the underlying molecular mechanism of Flot2 in gastric cancer (GC) is largely unknown. METHODS: qRT-PCR and western blot was performed to detect miR-449a and Flot2 expression in GC cell lines and Normal human gastric epithelial cells. Then, luciferase reporter assay was used to elucidate whether Flot2 is a target gene of miR-449a. Finally, the roles and mechanism of miR-449a in regulation of tumor invasion were further investigated. RESULTS: In this study, miR-449a expression was downregulated and Flot2 was upregulated in all GC cell lines as compared with that in GES-1. luciferase reporter assay identified Flot2 as a novel direct target of miR-449a. miR-449a regulated GC cell invasion by suppressing Flot2 expression. expression analysis of a set of epithelial-mesenchymal transition (EMT) markers showed that miR-449a reduced the expression of mesenchymal markers (vimentin and N-cadherin) and induced the expression of epithelial marker (E-cadherin), which was consistent with silenced Flot2. Moreover, Flot2 is necessary for TGF-beta-induced EMT in GC cells. CONCLUSIONS: Our results demonstrated that miR-449a suppressed Flot2 expression results in decreased cell invasion through repressing TGF-beta-mediated-EMT, and provides a new theoretical basis to further investigate miR-449a-regulated Flot2 as a potential biomarker and a promising approach for GC treatment.

The level of microRNA-93 (miR-93) in tumors has been recently reported to be negatively correlated with survival of lung cancer patients. Considering that the most devastating aspect of lung cancer is metastasis, which can be promoted by transforming growth factor-beta (TGF-beta)-induced epithelial-to-mesenchymal transition (EMT), we sought to determine whether miR-93 is involved in this process. Here, we report that a previously unidentified target of miR-93, neural precursor cell expressed developmentally downregulated gene 4-like (NEDD4L), is able to mediate TGF-beta-mediated EMT in lung cancer cells. miR-93 binds directly to the 3'-UTR of the NEDD4L messenger RNA (mRNA), leading to a downregulation of NEDD4L expression at the protein level. We next demonstrated that the downregulation of NEDD4L enhanced, while overexpression of NEDD4L reduced TGF-beta signaling, reflected by increased phosphorylation of SMAD2 in the lung cancer cell line after TGF-beta treatment. Furthermore, overexpression of miR-93 in lung cancer cells promoted TGF-beta-induced EMT through downregulation of NEDD4L. The analysis of publicly available gene expression array datasets indicates that low NEDD4L expression correlates with poor outcomes among patients with lung cancer, further supporting the oncogenic role of miR-93 in lung tumorigenesis and metastasis.

Smad and STAT proteins are critical signal transducers and transcription factors in controlling cell growth and tumorigenesis. Here we report that the STAT3 signaling pathway attenuates transforming growth factor-beta (TGF-beta)-induced responses through a direct Smad3-STAT3 interplay. Activated STAT3 blunts TGF-beta-mediated signaling. Depletion of STAT3 promotes TGF-beta-mediated transcriptional and physiological responses, including cell cycle arrest, apoptosis and epithelial-to-mesenchymal transition. STAT3 directly interacts with Smad3 in vivo and in vitro, resulting in attenuation of the Smad3-Smad4 complex formation and suppression of DNA-binding ability of Smad3. The N-terminal region of DNA-binding domain of STAT3 is responsible for the STAT3-Smad3 interaction and also indispensable for STAT3-mediated inhibition of TGF-beta signaling. Thus, our finding illustrates a direct crosstalk between the STAT3 and Smad3 signaling pathways that may contribute to tumor development and inflammation.

MicroRNA-663 (miR-663) has been detected in a large variety of tumor types; however, it still holds both tumor suppressive and oncogenic roles in different tumor types. The miRNA-CHIP microarray assay revealed downregulation of miR-663 in papillary thyroid carcinoma (PTC); however, the effect of miR-663 on PTC cell invasion and migration remains unknown. Accordingly, this study aimed to investigate the potential involvement of miR-663 in PTC. In this study, miR-663 expression level was measured via quantitative real-time PCR in 91 pairs of human PTC and adjacent normal tissues and in two human PTC cell lines. The effect of miR-663 on PTC cell invasion and migration were studied by transwell and wound healing assays. In addition, the miR-663 target was searched and the underlying mechanism was clarified by reporter assay and rescue experiment. The current study confirmed that miR-663 expression was inhibited in PTC tissue samples and PTC cell lines. There were statistically significant differences in expression of miR-663 with regard to age and tumor size. Upregulation of miR-663 suppressed PTC cell invasion and migration. Further study showed that transforming growth factor beta 1 (TGFbeta1) was the direct target of miR-663 and mediated the effect of miR-663 on PTC development. By targeting TGFbeta1, miR-663 efficiently regulates the expression of epithelial-mesenchymal transition (EMT) markers and matrix metalloproteinases (MMPs). The data indicated that miR-663 may suppress tumor invasion and migration by targeting TGFbeta1 and regulate EMT progress of PTC cells.

mesenchymal stem cells from human adipose tissue (hASCs) are proposed as suitable tools for soft tissue engineering and reconstruction. Although it is known that hASCs have the ability to home to sites of inflammation and tumor niche, the role of inflammatory cytokines in the hASCs-affected tumor development is not understood. We found that interferon-gamma (IFN-gamma) and/or tumor necrosis factor-alpha (TNF-alpha) prime hASCs to produce soluble factors which enhance MCF-7 cell line malignancy in vitro. IFN-gamma and/or TNF-alpha-primed hASCs produced conditioned media (CM) which induced epithelial to mesenchymal transition (EMT) of MCF-7 cells by reducing E-Cadherin and increasing Vimentin expression. Induced EMT was accompanied by increased invasion, migration, and urokinase type-plasminogen activator (uPA) expression in MCF-7 cells. These effects were mediated by increased expression of transforming growth factor-beta1(TGF-beta1) in cytokines-primed hASCs, since inhibition of type I TGF-beta1 receptor on MCF-7 cells and neutralization of TGF-beta1 disabled the CM from primed hASCs to increase EMT, cell migration, and uPA expression in MCF-7 cells. Obtained data suggested that IFN-gamma and/or TNF-alpha primed hASCs might enhance the malignancy of MCF-7 cell line by inducing EMT, cell motility and uPA expression in these cells via TGF-beta1-Smad3 signalization, with potentially important implications in breast cancer progression.CI - (c) 2016 International Union of Biochemistry and Molecular Biology.

TGF-beta and cancer progression share a multifaceted relationship. Despite the knowledge of TGF-beta biology in the development of cancer, several factors that mediate the cancer-promoting role of TGF-beta continue to be identified. This study aimed to identify and characterise novel factors potentially related to TGF-beta-mediated tumour aggression in breast cells. We treated the human mammary epithelial cell line MCF10A with TGF-beta and identified TGF-beta-dependent upregulation of PRRX2, the gene encoding paired-related homeobox 2 transcription factor. Overexpression of PRRX2 enhanced migration, invasion and anchorage-independent growth of MCF10A cells and induced partial epithelial mesenchymal transition (EMT), as determined by partial fibroblastoid morphology of cells, upregulation of EMT markers and partially disrupted acinar structure in a three-dimensional culture. We further identified PLAT, the gene encoding tissue-type plasminogen activator (tPA), as the highest differentially expressed gene in PRRX2-overexpressing MCF10A cells, and demonstrated direct binding and transactivation of the PLAT promoter by PRRX2. Furthermore, PLAT knockdown inhibited PRRX2-mediated enhanced migration and invasion, suggesting that tPA may mediate PRRX2-induced migration and invasion. Finally, the significant correlation of PRRX2 expression with poor survival in 118 primary breast tumour samples (P = 0.027) and the increased PRRX2 expression in metaplastic breast carcinoma samples, which is pathogenetically related to EMT, validated the biological importance of PRRX2-enhanced migration and invasion and PRRX2-induced EMT. Thus, our data suggest that upregulation of PRRX2 may be a mechanism contributing to TGF-beta-induced invasion and EMT in breast cancer. (c) 2016 Wiley Periodicals, Inc.CI - (c) 2016 Wiley Periodicals, Inc.

BACKGROUND: IL-27 is a multifunctional cytokine that has both pro-inflammatory and anti-inflammatory functions. Although IL-27 has been shown to potently inhibit lung fibrosis, the detailed mechanism of IL-27 in this process is poorly understood. epithelial-mesenchymal transition (EMT) is one of the key mechanisms involved in pulmonary fibrosis. We assessed the effects of IL-27 on TGF-beta1-induced EMT in alveolar epithelial cells. METHODS: A549 cells (a human AEC cell line) were incubated with TGF-beta1, IL-27, or both TGF-beta1 and IL-27, and changes in E-cadherin, beta-catenin, vimentin and a-SMA levels were measured using real-time PCR, western blotting and fluorescence microscopy. The related proteins in the JAK/STAT and TGF-beta/Smad signalling pathways were examined by western blot. RESULTS: IL-27 increased the expression of epithelial phenotypic markers, including E-cadherin and beta-catenin, and inhibited mesenchymal phenotypic markers, including vimentin and a-SMA in A549 cells. Moreover, TGF-beta1-induced EMT was attenuated by IL-27. Furthermore, we found that TGF-beta1 activated the phosphorylation of JAK1, STAT1, STAT3, STAT5, Smad1, Smad3 and Smad5, and IL-27 partially inhibited these changes in this process. When cells were treated with the STAT3 specific inhibitor wp1006 and the Smad3 specific inhibitor SIS3, the inhibition of EMT by IL-27 was significantly strengthened. CONCLUSION: Our results suggest that IL-27 attenuates epithelial-mesenchymal transition in alveolar epithelial cells in the absence or presence of TGF-beta1 through the JAK/STAT and TGF-beta/Smad signalling pathways.

The signaling adaptor protein Crk has been shown to play an important role in various human cancers. However, its regulatory machinery is not clear. Here, we demonstrated that Crk induced EMT in A549 human lung adenocarcinoma cells through differential regulation of Rac1/Snail and RhoA/Slug, leading to decreased expression of E-cadherin and increased N-cadherin, fibronectin, and MMP2 expression. cancer cells with mesenchymal features produced TGF-beta and also increased the levels of TGF-beta receptor. TGF-beta increased the endogenous levels of Crk and also augmented Crk-dependent expression of Snail and Slug, and conversely TGF-beta receptor inhibitor suppressed the levels of Snail and Slug. Overexpression of Crk was observed at the invasive front of human lung cancer tissues and was significantly associated with poor prognosis. Thus, TGF-beta and Crk collaborate to form a positive feedback loop to facilitate EMT, which may lead to the malignancy of human cancers possibly being affected by their microenvironment.

BACKGROUND: In biliary atresia (BA), epithelial-mesenchymal hepatic progenitor cells (HPC) expressing the stem/progenitor cell marker PROMININ-1 (PROM1) undergo expansion and subsequent transdifferentiation into collagen-producing myofibroblasts within regions of evolving biliary fibrosis under the regulation of Transforming Growth Factor-beta (TGFbeta) signaling. We hypothesized that pro-inflammatory Toll-like Receptor-3 (TLR3) signal activation promotes the differentiation of PROM1+ HPC via TGFbeta pathway activation in vitro. METHODS: PROM1+ Mat1a(-/-) HPC were treated with a double-stranded RNA analog, polyionosinic-polycytidylic acid (Poly I:C), +/- small molecule inhibitors nafamostat, or SB431542. RESULTS: Poly I:C induced myofibroblastic-like morphologic changes, degradation of IkappaB-alpha consistent with TLR3-NFkappaB activation, a 15-fold increase in the expression of Vimentin, a 9-fold increase in Collagen-1a, a 4.6-fold increase in Snail at 24h (p<0.05), and an 8.2-fold increase in Prom1 at 72h (p<0.0001) by qPCR. Immunofluorescence demonstrated nuclear phosphorylated SMAD3, TLR3, and COLLAGEN-1alpha staining following Poly I:C treatment. Degradation of IkappaBalpha was inhibited by nafamostat. Co-treatment with either nafamostat or SB431542 blocked the morphologic change and abrogated the increased expression of Cd133, Collagen, Vimentin, and Snail1. CONCLUSIONS: TLR3 activation induces myofibroblastic differentiation of PROM1+ HPC in part via TGFbeta pathway activation to promote BA-associated biliary fibrosis.CI - Copyright (c) 2016 Elsevier Inc. All rights reserved.

Non-canonical transforming growth factor beta (TGFbeta) signaling through protein kinase B (Akt2) induces phosphorylation of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) at serine-43 (p-hnRNP E1). This post-translational modification (PTM) of hnRNP E1 promotes its dissociation from a 3' untranslated region (UTR) nucleic acid regulatory motif, driving epithelial to mesenchymal transition (EMT) and metastasis. We have identified an hnRNP E1 consensus-binding motif and genomically resolved a subset of genes in which it is contained. This study characterizes the binding kinetics of the consensus-binding motif and hnRNP E1, its various K-homology (KH) domains and p-hnRNP E1. Levels of p-hnRNP E1 are highly upregulated in metastatic cancer cells and low in normal epithelial tissue. We show a correlation between this PTM and levels of Akt2 and its activated form, phosphorylated serine-474 (p-Akt2). Using cellular progression models of metastasis, we observed a signature high level of Akt2, p-Akt2 and p-hnRNP E1 protein expression, coupled to a significantly reduced level of total hnRNP E1 in metastatic cells. Genes that are translationally silenced by hnRNP E1 and expressed by its dissociation are highly implicated in the progression of EMT and metastasis. This study provides insight into a non-canonical TGFbeta signaling cascade that is responsible for inducing EMT by aberrant expression of hnRNP E1 silenced targets. The relevance of this system in metastatic progression is clearly shown in cellular models by the high abundance of p-hnRNP E1 and low levels of hnRNP E1. New insights provided by the resolution of this molecular mechanism provide targets for therapeutic intervention and give further insight into the role of the TGFbeta microenvironment.CI - (c) The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

epithelial-mesenchymal transition (EMT) plays a critical role in the progression of epithelial ovarian cancer (EOC). However, the mechanisms that regulate EMT in EOC are not fully understood. Here, we report that activation of Notch1 induces EMT in EOC cells as evidenced by downregulation of E-cadherin and cytokeratins, upregulation of Slug and Snail, as well as morphological changes. Interestingly, activation of Notch1 increases TGFbeta/Smad signaling by upregulating the expression of TGFbeta and TGFbeta type 1 receptor. Time course experiments demonstrate that inhibition of Notch by DAPT (a gamma-secretase inhibitor) decreases TGFbeta-induced phosphorylation of receptor Smads at late, but not at early, timepoints. These results suggest that Notch activation plays a role in sustaining TGFbeta/Smad signaling in EOC cells. Furthermore, inhibition of Notch by DAPT decreases TGFbeta induction of Slug and repression of E-cadherin and knockdown of Notch1 decreases TGFbeta-induced repression of E-cadherin, indicating that Notch is required, at least in part, for TGFbeta-induced EMT in EOC cells. On the other hand, TGFbeta treatment increases the expression of Notch ligand Jagged1 and Notch target gene HES1 in EOC cells. Functionally, the combination of Notch1 activation and TGFbeta treatment is more potent in promoting motility and migration of EOC cells than either stimulation alone. Taken together, our results indicate that Notch and TGFbeta form a reciprocal positive regulatory loop and cooperatively regulate EMT and promote EOC cell motility and migration.CI - Copyright (c) 2016 Elsevier Inc. All rights reserved.

Transforming growth factor-beta (TGF-beta) signaling pathway is a key regulator of various cancer biologies, including cancer cell migration, invasion, angiogenesis, proliferation, as well as apoptosis, and it is one of indispensable signaling pathways during cancer metastasis. TGF-beta signaling pathway can regulate and be regulated by a series of molecular and signaling pathways where microRNAs (miRNAs) seem to play important roles. miRNAs are small non-coding RNAs that can regulate expressions of their target genes. Emerging evidence suggest that miRNAs participate in various biological and pathologic processes such as cancer cells apoptosis, proliferation, invasion, migration, and metastasis by influencing multiple signaling pathways. In this article, we focus on the interaction between miRNAs and TGF-beta in breast cancer (BC) metastasis through modulating invasion-metastasis-related factors, including epithelial-to-mesenchymal transition (EMT), cancer stem cells (CSCs), matrix metalloproteinase (MMP), tissue inhibitors of MMPs (TIMPs), cell adhesion molecules (CAMs), and tumor microenvironment (TME). Through a clear understanding of the complicated links between TGF-beta pathway and miRNAs, it may provide a novel and safer therapeutic target to prevent BC metastasis.

Hepatocellular carcinoma (HCC) is currently still a major cause of cancer-related deaths. Identifying early metastatic biomarkers and therapeutic targets for HCC is of great importance. Emerging evidence suggest that epithelial-mesenchymal transitions (EMTs) play important roles in tumor metastasis and recurrence. Understanding molecular mechanisms that regulate the EMT process is crucial for improving HCC. In this study, we find Ficolin-2 (FCN2) plays an essential role in metastasis and EMT of HCC. FCN2 expression is downregulated in HCC cells and tissues. Low level of FCN2 in HCCs is correlated with aggressive metastatic features, and would be a prognostic factor for overall disease-free survival of HCC patients. Ectopic expression of FCN2 markedly inhibits HCC cells migration, invasion as well as EMT in vitro and in vivo. Moreover, TGF-beta is found contribute to the function of FCN2 in suppressing metastasis and EMT of HCC. Collectively, our data suggest that FCN2 may have prognostic value in HCC metastasis. Additionally, the FCN2/ TGF-beta/EMT axis identified in this study provides novel insight into the mechanisms of HCC metastasis, which may facilitate the development of new therapeutics against HCC.CI - Copyright (c) 2016 Elsevier Ireland Ltd. All rights reserved.

Invasion into surrounding normal brain and resistance to genotoxic therapies are the main devastating aspects of glioblastoma (GBM). These biological features may be associated with the stem cell phenotype, which can be induced through a dedifferentiation process known as epithelial-mesenchymal transition (EMT). We show here that tumor cells around pseudopalisading necrotic areas in human GBM tissues highly express the most important EMT inducer, transforming growth factor (TGF-beta), concurrently with the EMT-related transcriptional factor, TWIST. In addition, the stem cell markers CD133 and alkaline phosphatase (ALPL) were also highly expressed around necrotic foci in GBM tissues. The high expression of TGF-beta around necrotic regions was significantly correlated with shorter progression-free survival and overall survival in patients with GBM. High expression of stem cell markers, ALPL, CD133, and CD44 was also correlated with poor outcomes. These results collectively support the hypothesis that tissue hypoxia induces the stem cell phenotype through TGF-beta-related EMT and contributes to the poor outcome of GBM patients.

The TGF-beta/Smad signaling pathway plays important roles in cancer cell proliferation, apoptosis, differentiation, angiogenesis and epithelial-mesenchymal transition (EMT), which is the key event in the early stages of cancer metastasis and enhances the capability of cell migration and invasion. Smad4 acts as the only Co-Smad of TGF/Smad signaling pathway and plays the key role in TGF-beta-mediated EMT. Nevertheless, the mRNA regulation mechanisms of Smad4 in human non-small cell lung cancer (NSCLC) remains largely unclear. Computational algorithms predicted that the 3'-UTR of Smad4 is a target of miR205. Here, we validated that miR205 could directly bind to 3'-UTR of Smad4 by luciferase assays. Moreover, we investigated the functional roles of miR205 and its molecular link to Smad4 in lung cancer cells. In this study, we confirmed that overexpression of miR205 suppressed the expression of Smad4, in turn, weakened the TGF-beta/Smad signaling pathway and inhibited TGF-beta/Smad4-induced EMT, invasion and migration ultimately. Furthermore, this study shows that miR205 can serve as a promising therapeutic target of highly aggressive NSCLC.

cancer metastasis is strongly correlated with epithelial-mesenchymal transition (EMT), in which transforming growth factor-beta (TGF-beta) signaling plays a central role. CD44 has emerged as a cancer stem cell (CSC) marker that strongly induces EMT together with TGF-beta1. This study aimed to investigate the link between high CD44 and TGF-beta1 levels during EMT in HCC cell lines. FACS analysis showed high expression of CD44 in TGF-beta1-positive SNU-368 cells and TGF-beta1-negative SNU-354 cells. SNU-368 CD44(+) cells showed EMT through up-regulation of the AKT/GSK-3beta/beta-catenin pathway. By comparison, SNU-354 CD44(+) cells showed only increased N-cadherin expression, which was not accompanied by a decrease in E-cadherin expression, and also down-regulated the AKT/GSK-3beta/beta-catenin pathway. However, TGF-beta1-stimulated SNU-354 cells (CD44/TGF-beta1(+)) exhibited lower E-cadherin and higher N-cadherin expression with increased AKT/GSK-3beta/beta-catenin pathway activity. CD44/TGF-beta1(+) SNU-354 cells also showed enhanced migration and formed larger spheres, while the TGF-beta1-induced stem cell properties returned to their original state with the TGF-beta1 inhibitor SB431542. SB431542-treated SNU-368 (CD44/TGF-beta1(-)) cells also showed diminished N-cadherin and AKT/GSK-3beta/beta-catenin pathway activity and further decreased cell motility in a wound healing assay. However, CD44 knockdown in SNU-354 cells did not induce EMT even after treatment with TGF-beta1. Finally, double inhibition of both CD44 and TGF-beta1 further decreased migration and sphere formation more strongly than a single inhibition in SNU-368 cells. In conclusion, the current study demonstrated the synergistic interactions between CD44 and TGF-beta1 in EMT induction and CSC properties through the AKT/GSK-3beta/beta-catenin pathway in HCC cells.CI - Copyright (c) 2016 Elsevier Inc. All rights reserved.

UNLABELLED: The airway epithelium is a semi-impermeable barrier whose disruption by growth factor reprogramming is associated with chronic airway diseases of humans. Transforming growth factor beta (TGFbeta)-induced epithelial mesenchymal transition (EMT) plays important roles in airway remodeling characteristic of idiopathic lung fibrosis, asthma and chronic obstructive pulmonary disease (COPD). Inflammation of the airways leads to airway injury and tumor necrosis factor alpha (TNFalpha) plays an important pro-inflammatory role. Little systematic information about the effects of EMT on TNFalpha signaling is available. Using an in vitro model of TGFbeta-induced EMT in primary human small airway epithelial cells (hSAECs), we applied quantitative proteomics and phosphoprotein profiling to understand the molecular mechanism of EMT and the impact of EMT on innate inflammatory responses. We quantified 7925 proteins and 1348 phosphorylation sites by stable isotope labeling with iTRAQ technology. We found that cellular response to TNFalpha is cell state dependent and the relative TNFalpha response in mesenchymal state is highly compressed. Combined bioinformatics analyses of proteome and phosphoproteome indicate that the EMT state is associated with reprogramming of kinome, signaling cascade of upstream transcription regulators, phosphor-networks, and NF-kappaB dependent cell signaling. BIOLOGICAL SIGNIFICANCE: epithelial mesenchymal transition and inflammation have important implications for clinical and physiologic manifestations of chronic airway diseases such as severe asthma, COPD, and lung fibrosis. Little systematic information on the interplay between EMT and innate inflammation is available. This study combined quantitative proteomics and phosphorproteomics approach to obtain systems-level insight into the upstream transcription regulators involved in the TGFbeta-induced EMT in primary human small airway epithelial cells and to elucidate how EMT impacts on the TNFalpha signaling pathways. The proteomics and phosphoproteomics analysis indicates that many signaling pathways involved in TGFbeta-induced EMT and EMT has profound reprogramming effects on innate inflammation response.CI - Copyright (c) 2016 Elsevier B.V. All rights reserved.

Osteosarcoma patients often exhibit pulmonary metastasis, which results in high patient mortality. Understanding the mechanisms of advanced metastasis in osteosarcoma cell is important for the targeted treatment and drug development. Our present study revealed that transforming growth factor-beta (TGF-beta) treatment can significantly promote the in vitro migration and invasion of human osteosarcoma MG-63 and HOS cells. The loss of epithelial characteristics E-cadherin (E-Cad) and up regulation of mesenchymal markers Vimentin (Vim) suggested TGF-beta induced epithelial-mesenchymal transition (EMT) of osteosarcoma cells. TGF-beta treatment obviously increased the expression of Snail, a key EMT-related transcription factor, in both MG-63 and HOS cells. Silencing of Snail markedly attenuated TGF-beta induced down regulation of E-cad and up regulation of Vim. TGF-beta treatment also significantly increased the expression and nuclear translocation of estrogen-related receptors alpha (ERRalpha), while had no obvious effect on the expression of ERalpha, ERbeta, or ERRgamma. Knock down of ERRalpha or its inhibitor XCT-790 significantly attenuated TFG-beta induced EMT and transcription of Snail in osteosarcoma cells. Collectively, our present study revealed that TGF-beta treatment can trigger the EMT of osteosarcoma cells via ERRalpha/Snail pathways. Our data suggested that ERRalpha/Snail pathways might be potential therapeutic targets of metastasis of osteosarcoma cells.

As one member of Forkhead box transcription factors, Forkhead box J2 (FOXJ2) has been found to be involved in epithelial-mesenchymal transition (EMT) process. However, the role and mechanism of FOXJ2 in non-small cell lung cancer (NSCLC) and EMT regulation have not been fully revealed. In this paper, it was revealed that the expression of FOXJ2 was lower in NSCLC samples compared with matched peritumoral lung tissue. We demonstrated that FOXJ2 expression was down-regulated by transforming growth factor-beta1 (TGF-beta1) treated, and overexpression of FOXJ2 inhibited TGF-beta1-induced EMT. Mechanistically, knocking out the expression of FOXJ2 promoted EMT by increasing the expression of Notch1 and NICD. This study implicates the potential value of FOXJ2 as a molecular marker for NSCLC.CI - (c) 2016 International Federation for Cell Biology.

During epithelial-mesenchymal transition (EMT), epithelial cells lose key phenotypic markers (e.g., E-cadherin and cytokeratin 18) and acquire mesenchymal markers (e.g., N-cadherin and vimentin). Although the loss of cytokeratin 18 is a hallmark of EMT, the regulatory role of cytokeratin 18 in EMT is not yet fully understood. Here, we report that cytokeratin 18 is involved in the regulation of transforming growth factor-beta1 (TGF-beta1)-induced EMT in breast epithelial cells. When MCF10A cells were treated with TGF-beta1 for 24 h, considerable morphological changes, indicative of the early stages of EMT (e.g., loss of cell-cell contact), were observed and cytokeratin 18 was downregulated. However, E-cadherin levels were not altered until a later time point. This suggests that cytokeratin 18 may play an active role during the earlier stages of EMT. Consistent with this notion, siRNA-mediated knockdown of cytokeratin 18 delayed TGF-beta1-mediated EMT, and the associated downregulation of E-cadherin reduced the phosphorylation/nuclear localization of smad 2/3 and decreased the expression levels of snail and slug (which inhibit E-cadherin expression in epithelial cells as an early response to TGF-beta1). Taken together, these results suggest that cytokeratin 18 critically contributes to initiating TGF-beta1-induced EMT via the smad 2/3-mediated regulation of snail and slug expression in breast epithelial cells.

According to recent research, Ras homolog gene family member C (RhoC) is confirmed to have a powerful regulatory effect on cell motility mediated by the cytoskeleton, and this process is closely associated with tumor invasion and metastasis. In addition, the epithelial-mesenchymal transition (EMT) process which causes cytoskeleton rearrangement, also plays a pivotal role in tumor invasion and metastasis.Consequently, in the present study, we aimed to ascertain whether RhoC has an effect on the EMT process induced by TGF-beta1 in lung adenocarcinoma cells and whether RhoC promotes tumor invasion by mediating the occurrence of EMT. Based on the findings, we demonstrated that RhoC was an essential mediator of the EMT process in lung adenocarcinoma cell line A549 which was evaluated by observing the morphological characteristics of the cells and by assessing the expression levels of two EMT marker proteins: E-cadherin and vimentin. During the process of EMT in the A549 cells induced by TGF-beta1 (5 ng/ml), upregulated RhoC protein and RhoC activity were detected, which was associated with the enhanced invasive capability of the cells in vitro. Conversely, downregulation of the expression of RhoC by shRNA markedly impeded EMT progression as well as the invasion of A549 cells. Our results may provide a novel target towards the prevention of metastasis in advanced lung adenocarcinoma.

Sec8 is one of the subunits of the exocyst, which is an evolutionarily conserved complex of eight proteins, comprising Sec3 (EXOC1), Sec5 (EXOC2), Sec6 (EXOC3), Sec8 (EXOC4), Sec10 (EXOC5), Sec15 (EXOC6), Exo70 (EXOC7), and Exo84 (EXOC8) subunits. Sec8 knockout mice embryos initiate gastrulation but are unable to progress beyond the primitive streak stage and die shortly. During embryonic development, the first epithelial-mesenchymal transition (EMT) event occurs at gastrulation. Sec8 may be involved in the early embryonic development through EMT. However, the function of Sec8 in EMT remains unclear. In the present study, it was found that Sec8 regulates N-cadherin expression by controlling Smad3 and Smad4 expression at the basal transcriptional level, thereby modulating cell migration and adhesion. Furthermore, Sec8 knockdown decreased CREB binding protein (CBP) expression at mRNA and protein levels. However, CBP knockdown did not affect Sec8 expression. These results indicated that Sec8 regulates N-cadherin expression by controlling Smad3 and Smad4 expression through CBP, thereby mediating the EMT.CI - Copyright A(c) 2016 Elsevier Inc. All rights reserved.

The epithelial-mesenchymal transition (EMT) is involved in many different types of cellular behavior, including liver fibrosis. In this report, we studied a novel function of RAR-related orphan receptor gamma (ROR-gamma) in hepatocyte EMT during liver fibrosis. To induce EMT in vitro, primary hepatocytes and FL83B cells were treated with TGF-beta1. expression of ROR-gamma was analyzed by Western blot in the fibrotic mouse livers and human livers with cirrhosis. To verify the role of ROR-gamma in hepatocyte EMT, we silenced ROR-gamma in FL83B cells using a lentiviral short hairpin RNA (shRNA) vector. The therapeutic effect of ROR-gamma silencing was investigated in a mouse model of TAA-induced fibrosis by hydrodynamic injection of plasmids. ROR-gamma expression was elevated in hepatocyte cells treated with TGF-beta1, and ROR-gamma protein levels were elevated in the fibrotic mouse livers and human livers with cirrhosis. Knockdown of ROR-gamma resulted in the attenuation of TGF-beta1-induced EMT in hepatocytes. Strikingly, ROR-gamma bound to ROR-specific DNA response elements (ROREs) in the promoter region of TGF-beta type I receptor (Tgfbr1) and Smad2, resulting in the downregulation of Tgfbr1 and Smad2 after silencing of ROR-gamma. Therapeutic delivery of shRNA against ROR-gamma attenuated hepatocyte EMT and ameliorated liver fibrosis in a mouse model of TAA-induced liver fibrosis. Overall, our results suggest that ROR-gamma regulates TGF-beta-induced EMT in hepatocytes during liver fibrosis. We suggest that ROR-gamma may become a potential therapeutic target in treating liver fibrosis. J. Cell. Biochem. 118: 2026-2036, 2017. (c) 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.CI - (c) 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.

Specificity protein1 (Sp1) is required for TGF-beta-induced epithelial-to-mesenchymal transition (EMT) which has been demonstrated to aggravate the progression of cancer including lung cancer. microRNA-29c (miR-29c) is identified to inhibit EMT, but the correlation between miR-29c and Sp1 in human lung cancer remain incompletely clarified. Here, we confirmed decreased expression of miR-29c and enhanced expression of Sp1 in lung cancer tissues (n = 20) and found that Sp1 could be targeted and inhibited by miR-29c. Besides, the expression of miR-29c was down-regulated in high-metastatic lung cancer cell lines and TGF-beta1-treated cells. The inhibition of miR-29c or overexpression of Sp1 in 95C and A549 cells dramatically enhanced the cell migration and invasion, and also induced the decrease in the expression of epithelial markers, e.g. thyroid transcription factor 1 (TTF-1) and E-cadherin, together with an increase in mesenchymal markers including vimentin, alpha-smooth muscle actin (alpha-SMA), which could be restored by overexpression of miR-29c mimics during the TGF-beta-induced EMT. Moreover, dual-luciferase reporter assay was performed and the results indicated that miR-29c/Sp1 could form an auto-regulatory loop with TGF-beta1, which impaired TGFB1 transcription. Furthermore, miR-29c overexpression could abrogate the tumor progression and inhibit the Sp1/TGF-beta expressions in vivo, indicating that miR-29c could be a tumor suppressor and repress the Sp1/TGF-beta axis-induced EMT in lung cancer.

epithelial-mesenchymal transition (EMT) is one of the decisive steps regulating cancer invasion and metastasis. However, the molecular mechanisms underlying this transition require further clarification. MDA-9/syntenin (SDCBP) expression is elevated in breast cancer patient samples as well as cultured breast cancer cells. Silencing expression of MDA-9 in mesenchymal metastatic breast cancer cells triggered a change in cell morphology in both 2D- and 3D-cultures to a more epithelial-like phenotype, along with changes in EMT markers, cytoskeletal rearrangement and decreased invasion. Conversely, over expressing MDA-9 in epithelial non-metastatic breast cancer cells instigated a change in morphology to a more mesenchymal phenotype with corresponding changes in EMT markers, cytoskeletal rearrangement and an increase in invasion. We also found that MDA-9 upregulated active levels of known modulators of EMT, the small GTPases RhoA and Cdc42, via TGFbeta1. Reintroducing TGFbeta1 in MDA-9 silenced cells restored active RhoA and cdc42 levels, modulated cytoskeletal rearrangement and increased invasion. We further determined that MDA-9 interacts with TGFbeta1 via its PDZ1 domain. Finally, in vivo studies demonstrated that silencing the expression of MDA-9 resulted in decreased lung metastasis and TGFbeta1 re-expression partially restored lung metastases. Our findings provide evidence for the relevance of MDA-9 in mediating EMT in breast cancer and support the potential of MDA-9 as a therapeutic target against metastatic disease.

TGF-beta secreted by tumor stroma induces epithelial-to-mesenchymal transition (EMT) in cancer cells, a reversible phenotype linked to cancer progression and drug resistance. However, exposure to stromal signals may also lead to heritable changes in cancer cells, which are poorly understood. We show that epithelial cells failing to undergo proliferation arrest during TGF-beta-induced EMT sustain mitotic abnormalities due to failed cytokinesis, resulting in aneuploidy. This genomic instability is associated with the suppression of multiple nuclear envelope proteins implicated in mitotic regulation and is phenocopied by modulating the expression of LaminB1. While TGF-beta-induced mitotic defects in proliferating cells are reversible upon its withdrawal, the acquired genomic abnormalities persist, leading to increased tumorigenic phenotypes. In metastatic breast cancer patients, increased mesenchymal marker expression within single circulating tumor cells is correlated with genomic instability. These observations identify a mechanism whereby microenvironment-derived signals trigger heritable genetic changes within cancer cells, contributing to tumor evolution.CI - Copyright (c) 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

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.

The transforming growth factor beta (TGFbeta) pathway plays critical roles during cancer cell epithelial-mesenchymal transition (EMT) and metastasis. SMAD7 is both a transcriptional target and a negative regulator of TGFbeta signalling, thus mediating a negative feedback loop that may potentially restrain TGFbeta responses of cancer cells. Here, however, we show that TGFbeta treatment induces SMAD7 transcription but not its protein level in a panel of cancer cells. Mechanistic studies reveal that TGFbeta activates the expression of microRNA-182 (miR-182), which suppresses SMAD7 protein. miR-182 silencing leads to SMAD7 upregulation on TGFbeta treatment and prevents TGFbeta-induced EMT and invasion of cancer cells. Overexpression of miR-182 promotes breast tumour invasion and TGFbeta-induced osteoclastogenesis for bone metastasis. Furthermore, miR-182 expression inversely correlates with SMAD7 protein in human tumour samples. Therefore, our data reveal the miR-182-mediated disruption of TGFbeta self-restraint and provide a mechanism to explain the unleashed TGFbeta responses in metastatic cancer cells.

epithelial-mesenchymal transition (EMT) is a dynamic process through which epithelial cells transdifferentiate from an epithelial phenotype into a mesenchymal phenotype. Previous studies have demonstrated that both mechanical signaling and soluble growth factor signaling facilitate this process. One possible point of integration for mechanical and growth factor signaling is the extracellular matrix. Here we investigate the role of the extracellular matrix (ECM) protein fibronectin (FN) in this process. We demonstrate that inhibition of FN fibrillogenesis blocks activation of the Transforming Growth Factor-Beta (TGF-beta) signaling pathway via Smad2 signaling, decreases cell migration and ultimately leads to inhibition of EMT. Results show that soluble FN, FN fibrils, or increased contractile forces are insufficient to independently induce EMT. We further demonstrate that inhibition of latent TGF-beta1 binding to FN fibrils via either a monoclonal blocking antibody against the growth factor binding domain of FN or through use of a FN deletion mutant that lacks the growth factor binding domains of FN blocks EMT progression, indicating a novel role for FN in EMT in which the assembly of FN fibrils serves to localize TGF-beta1 signaling to drive EMT.CI - Copyright (c) 2017 Elsevier B.V. All rights reserved.

As part of its potential pro-tumorigenic actions, Transforming Growth Factor-(TGF)-beta induces epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) cells. Whether EMT induces changes in tumor cell plasticity has not been fully explored yet. Here, we analyze the effects of TGF-beta on the EMT and stem-related properties of HCC cells and the potential correlation among those processes. The translational aim of the study was to propose a TGF-beta/EMT/stem gene signature that would help in recognizing HCC patients as good candidates for anti-TGF-beta therapy. Results indicate that when TGF-beta induces EMT in HCC cells, a switch in the expression of stem genes is observed and their stemness potential and migratory/invasive capacity are enhanced. However, TGF-beta may induce a partial EMT in some epithelial HCC cells, increasing the expression of mesenchymal genes and CD44, but maintaining epithelial gene expression. epithelial cells show higher stemness potential than the mesenchymal ones, but respond to TGF-beta increasing their migratory and invasive capacity. In HCC patient samples, TGFB1 expression most frequently correlates with a partial EMT, increase in mesenchymal genes and CD44 expression, as well as maintenance or over-expression of epithelial-related genes.CI - Copyright (c) 2017 Elsevier B.V. All rights reserved.

Metastasis is one of the most decisive factors influencing CRC patient prognosis and current studies suggest that a molecular mechanism known as EMT broadly regulates cancer metastasis. NR2F2 is a key molecule in the development of CRC, but the roles and underlying mechanisms of NR2F2 in TGF-beta induced EMT in CRC remain largely unknown. In the current study, we were interested to examine the role of NR2F2 in the TGF-beta-induced EMT in CRC. Here, we found NR2F2 was upregulated in CRC cells and promotes TGF-beta-induced EMT in CRC. Using comparative miRNA profiling TGF-beta pre-treated CRC cells in which NR2F2 had been knocked down with that of control cells, we identified miR-21 as a commonly downregulated miRNA in HT29 cells treated with TGF-beta and NR2F2 siRNA, and its downregulation inhibiting migration and invasion of CRC cells. Moreover, we found NR2F2 could transcriptional activated miR-21 expression by binding to miR-21 promoter in HT29 by ChIP and luciferase assay. In the last, our data demonstrated that Smad7 was the direct target of miR-21 in CRC cells. Thus, NR2F2 could promote TGF-beta-induced EMT and inhibit Smad7 expression via transactivation of miR-21, and NR2F2 may be a new common therapeutic target for CRC.CI - Copyright (c) 2017 Elsevier Inc. All rights reserved.

Atrial natriuretic peptide (ANP) is increasingly expressed on airway and inhibits pulmonary arterial remodeling. However, the role of ANP in remodeling of respiratory system is still unclear. The role of ANP on airway remodeling and the possible mechanism was explored in this study. Both human bronchial epithelial 16HBE-14o cells and alveolar epithelial A549 cells were stimulated by TGF-beta1, ANP, cGMP inhibitor, PKG inhibitor, and cGMP analogue. The expressions of epithelial markers, mesenchymal markers, and Smad3 were assessed by quantitative real-time PCR and western blotting. Immunohistochemical staining was employed to assess Smad3 expression once it was silenced by siRNA in 16HBE-14o or A549 cells. Our results showed that the mRNA and protein expressions of E-Cadherin were decreased, whereas alpha-SMA expressions were increased after induction by TGF-beta1 in 16HBE-14o and A549 cells. The E-Cadherin expressions were increased and alpha-SMA expressions were decreased after ANP stimulation. Inhibition of cGMP or PKG decreased E-Cadherin expression but increased alpha-SMA expression, which could be reversed by cGMP analogue. Moreover, the phosphorylated Smad3 expression was consistent with alpha-SMA expression. After smad3 was silenced, Smad3 was mostly expressed in cytoplasm instead of nucleus as non-silenced cells during epithelial-mesenchymal transition (EMT). In conclusion, ANP inhibits TGF-beta1-induced EMT in 16HBE-14o and A549 cells through cGMP/PKG signaling, by which it targets TGF-beta1/Smad3 via attenuating phosphorylation of Smad3. These findings suggest the potential of ANP in the treatment on pulmonary diseases with airway remodeling.CI - Copyright (c) 2017 Elsevier Inc. All rights reserved.

Abnormal expression of high-mobility group box-1 (HMGB1) protein occurs in many tumors and is closely associated with tumor invasion and metastasis. However, a role for HMGB1 in epithelial-mesenchymal transition (EMT) in hypopharyngeal carcinoma has not been previously reported. We cultured cells of the hypopharyngeal carcinoma cell line FaDu in vitro and then treated them with 5 ng/ml TGF-beta1 for 48 h to induce EMT. Vimentin, Snail, and HMGB1 expression patterns were then detected using immunofluorescence staining; HMGB1 mRNA and protein expression were verified by RT-PCR and western blot analyses. HMGB1 was then silenced in FaDu cells using RNAi, followed by detection of Vimentin, Snail, and HMGB1 expressions by immunofluorescence staining. The mRNA expression levels of Vimentin, Snail, HMGB1, and E-cadherin were verified by RT-PCR, while protein expression of HMGB1 and receptor for advanced glycation end products (RAGE) were detected by western blot analysis. The biological behavior of FaDu cells was observed before and after HMGB1 silencing using wound healing and cell invasion assays. Following culture with 5 ng/ml TGF-beta1 for 48 h, the morphology of FaDu cells changed from a regular cobblestone-like appearance into a spindle-like shape. expression levels of Vimentin, Snail, and HMGB1 were upregulated at both mRNA and protein levels as determined by RT-PCR, immunofluorescence, and western blotting. After HMGB1 silencing, mRNA expression levels of the epithelial cell marker E-cadherin were upregulated. Meanwhile, expression levels of the mesenchymal markers Vimentin and Snail were decreased. Western blotting revealed that HMGB1 and RAGE were downregulated. RNAi-mediated inhibition of HMGB1 expression decreased the capacities of FaDu cells for invasion and metastasis as determined by wound healing and cell invasion assays. HMGB1 is essential for maintaining the interstitial cell phenotype in TGF-beta1-induced EMT of FaDu cells, and silencing HMGB1 greatly inhibits the invasive and metastatic ability of these cells.

AIM: This study investigated the effect of reactive oxygen species (ROS) on transforming growth factor (TGF)-beta-mediated epithelial-to-mesenchymal transition (EMT) in order to clarify the influence of ROS and TGFbeta on the induction of dysplasia and ultimately, tumorigenesis. MATERIALS AND METHODS: Confluent MCF-10A human mammary epithelial cells were treated with H2O2 for 1 h, then reseeded at low density in the presence of TGFbeta and cultured until confluence. RESULTS: Hydrogen peroxide (H2O2, 250 muM) enhanced TGFbeta-mediated EMT, as evidenced by increased expression of EMT-associated transcription factors, which was accompanied by increased nuclear localization of phosphorylated SMAD family member 2 (SMAD2) and up-regulation of the TGFbeta signaling pathway components mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK). Pharmacological inhibition of MEK/ERK signaling partly reversed the effects of H2O2 Conclusion: H2O2 enhances TGFbeta-mediated EMT via SMAD and MEK/ERK signaling.CI - Copyright(c) 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

The homeoprotein Bapx1 is an important regulator of gastroduodenal tract morphogenesis. Here, we investigated how Bapx1 influences gastric cancer (GC) prognosis and elucidated the underlying mechanisms. Bapx1 expression was greater in GC tissues compared to adjacent non-tumor tissues and expression was positively correlated with mortality, lymph node and distance metastasis. Silencing Bapx1 diminished cell invasion/migration and decreased mesenchymal phenotypes. Transforming growth factor-beta (TGF-beta) induced Bapx1 expression and epithelial-mesenchymal transition (EMT) in GC cells. However, down-regulated Bapx1 reversed TGF-beta induced invasion, migration, morphological changes, and EMT. In summary, Bapx1 indicates poor prognosis for GC by promoting tumor migration and invasion via TGF-beta-induced EMT.CI - Copyright (c) 2017 Elsevier Inc. All rights reserved.

PURPOSE: The aim of this study is to investigate the mechanisms of interactions between TGF-beta and Wnt/beta-catenin pathways that induce and regulate EMT and promote breast cancer cells to become resistant to treatment. METHODS: The effect of TGF-beta on Wnt/beta-catenin signaling pathway was examined by using a human Wnt/beta-catenin-regulated cDNA plate array and western blot analysis. The interaction of Twist at promoter of Wnt3 was examined by chromatin immunoprecipitation (ChIP) assay. Secreted Wnt3 level was determined by ELISA assay. RESULTS: HER2-overexpressing breast cancer cells treated with TGF-beta have a reduced response to trastuzumab and exhibited EMT-like phenotype. The TGF-beta-induced EMT in HER2-cells was concordant with upregulation of Wnt3 and beta-catenin pathways. The TGF-beta-induced induction of Wnt3 during EMT was found to be Smad3-dependent. ChIP analysis identified occupancy of Twist at promoter region of Wnt3. Knock-down of Twist by shRNA confirmed the significance of Twist in response to TGF-beta regulating Wnt3 during EMT. Subsequently, TGF-beta-induced matrix metalloproteinases, MMP1, MMP7, MMP9, MMP26, Vascular endothelial growth factors (VEGF), and activation of Wnt/beta-catenin signaling were repressed by the shRNA treatment. TGF-betaR1 ALK5 kinase inhibitor, A83-01 can effectively prevent the TGF-beta-induced Twist and Wnt3. Co-treating A83-01 and trastuzumab inhibited TGF-beta-induced cell invasion significantly in both trastuzumab responsive and resistant cells. CONCLUSIONS: Our data demonstrated an important interdependence between TGF-beta and Wnt/beta-catenin pathways inducing EMT in HER2-overexpressing breast cancer cells. Twist served as a linkage between the two pathways during TGF-beta-induced EMT. A83-01 could inhibit the TGF-beta-initiated pathway interactions and enhance HER2-cells response to trastuzumab treatment.

While TGF-beta1 is known to induce epithelial-mesenchymal transition (EMT), a major factor in the pathogenesis of proliferative vitreoretinopathy (PVR), in ARPE-19 cells. The molecular pathways involved in EMT formation have not yet to be fully characterized. In this study, we have found that TGF-beta1-mediated induction of EMT in ARPE-19 cells varied in a dose- and time-dependent manner. Specifically, TGF-beta1 inhibited GSK-3beta by accelerating phosphorylation at ser9. GSK-3beta inhibitor or knockdown of GSK-3beta resulted in enhanced TGF-beta1-mediated EMT, migration and collagen contraction in ARPE-19 cells, which were then abrogated by GSK-3beta overexpression and PI3K/AKT inhibitor. Importantly, GSK-3beta also mediated metabolic reprogramming in TGF-beta1-treated cells. Our results indicate that GSK-3beta plays a pivotal role in TGF-beta1-mediated EMT in ARPE-19 cells.CI - Copyright (c) 2017 Elsevier Inc. All rights reserved.

Signal transduction through the transforming growth factor-beta 1 (TGF-beta1) pathway affects epithelial to mesenchymal transition (EMT), partly by modulation of E-Cadherin expression. The concurrent impact of extracellular matrix driven regulation of integrin signaling on EMT has not been well characterized. We assessed the cumulative effect and molecular mechanisms of TGF-beta1 and integrin signal transduction on E-Cadherin in a renal cell cancer (RCC) model. Stimulation of RCC cells with TGF-beta1 demonstrated a three-fold increased expression of integrin alphav. A ligand of integrin alphav-beta3, (cyclopentapeptide containing Arginyl-Glycyl-Aspartic acid motif, RGD), was used to mimic integrin signaling. Treatment of cells with RGD and TGF-beta1 demonstrated significantly greater E-cadherin depletion than either ligand alone. This cooperative action on E-Cadherin expression is regulated by transcription factor Snai1 and is followed on a cellular level by increased cellular mobility as evidenced in a wound healing assay. Subsequent silencing of potential downstream mediators of the cumulative action of RGD and TGF-beta1 was carried out by small interfering RNA transfection and confirmed by Western blotting and/or RT-PCR. SiRNA mediated silencing of FAK and PINCH1 independently abrogated the cumulative effect of RGD and TGF-beta1 on E-Cadherin expression. We have identified a novel mechanism through which extracellular matrix event transduction by integrins further augments TGF-beta1 related effects on EMT. Molecular machinery involved in the integrin alphav-TGF-beta1 interplay may represent a therapeutic target in RCC.CI - Copyright (c) 2017 Elsevier Inc. All rights reserved.

Previous studies in our laboratory identified that 3-deazaneplanocin A (DZNep), a carbocyclic adenosine analog and histone methyl transferase inhibitor, suppresses TGFbeta-induced epithelial-to-mesenchymal (EMT) characteristics. In addition, DZNep epigenetically reprograms miRNAs to regulate endogenous TGFbeta1 levels via miR-663/4787-mediated RNA interference (Mol cancer Res. 2016 Sep 13. pii: molcanres.0083.2016) (1). Although DZNep also attenuates exogenous TGFbeta-induced EMT response, the mechanism of this inhibition was unclear. Here, DZNep induced miR-202-5p to target both TGFbeta receptors, TGFBR1 and TGFBR2, for RNA interference and thereby contributes to the suppression of exogenous TGFbeta-induced EMT in pancreatic cancer cells. Lentiviral overexpression of miR-202 significantly reduced the protein levels of both TGFbeta receptors and suppressed TGFbeta signaling and EMT phenotypic characteristics of cultured parenchymal pancreatic cancer cells. Consistently, transfection of anti-miRNAs against miR-202-5p resulted in increased TGFBR1 and TGFBR2 protein expressions and induced EMT characteristics in these cells. In stellate pancreatic cells, miR-202 overexpression slowed growth as well as reduced stromal extracellular membrane matrix protein expression. In orthotopic pancreatic cancer mouse models, both immunodeficient and immunocompetent, miR-202 reduced tumor burden and metastasis. Together, these findings demonstrate an alternative mechanism of DZNep in suppressing TGFbeta signaling at the receptor level and uncover the EMT-suppressing role of miR-202 in pancreatic cancer.Implications: These findings support the possibility of combining small molecule-based (e.g., DZNep analogs) or large molecule-based (e.g., miRNAs) epigenetic modifiers with conventional nucleoside analogs (e.g., gemcitabine, capecitabine) to improve the antimetastatic potential of current pancreatic cancer therapy. Mol cancer Res; 15(8); 1029-39. (c)2017 AACR.CI - (c)2017 American Association for cancer Research.

A high incidence of tumor recurrence and metastasis has been reported in breast cancer patients; nevertheless, the underlying molecular mechanisms are largely unknown. epithelial-mesenchymal transition (EMT), which is induced by transforming growth factor-beta (TGF-beta), has been implicated in tumorigenesis and breast cancer metastasis. EMT events are now directly associated with tumor metastasis, and this progress is dependent on the inflammatory microenvironment. Cytosolic phospholipase A2alpha (cPLA2alpha) has been shown to participate in a series of biological processes including inflammation and cancer development. However, the role and molecular mechanism of cPLA2alpha in breast cancer EMT and metastasis remain enigmatic. In this study, we found that cPLA2alpha was commonly overexpressed in most human breast cancer tissues and significantly correlated with a poor prognosis for human breast cancer. Functional studies demonstrated that cPLA2alpha overexpression was significantly associated with elevated migration and invasion in MDA-MB-231 and T47D cells. Conversely, reduced cPLA2alpha expression strongly attenuated metastasis and the EMT program of MDA-MB-231 cells. Further study found that knockdown of cPLA2alpha in MDA-MB-231 cells inhibited TGF-beta-induced EMT through the PI3K/Akt signaling pathway. Animal experiments revealed that cPLA2alpha downregulation in MDA-MB-231 cells markedly restrained tumorigenesis and metastasis in vivo. This study indicates the potential role of cPLA2alpha in breast cancer metastasis and indicates that this molecule is a promising therapeutic target for breast cancer.

Neuroblastoma is the second most common extracranial malignant solid tumor that occurs in childhood, and metastasis is one of the major causes of death in neuroblastoma patients. The epithelial-mesenchymal transition (EMT) is an important mechanism for both the initiation of tumor invasion and subsequent metastasis. Therefore, this study investigated the mechanism by which transforming growth factor (TGF)-beta1 induces EMT in human neuroblastoma cells. Using quantitative RT-qPCR and western blot analyses, we found that the mRNA and protein expression levels of E-cadherin were significantly decreased, whereas that of alpha-SMA was significantly increased after neuroblastoma cells were treated with different concentrations of TGF-beta1. A scratch test and Transwell migration assay revealed that cell migration significantly and directly correlated with the concentration of TGF-beta1 indicating that TGF-beta1 induced EMT in neuroblastoma cells and led to their migration. Inhibiting Smad2/3 expression did not affect the expression of the key molecules involved in EMT. Further investigation found that the expression of the glioblastoma transcription factor (Gli) significantly increased in TGF-beta1-stimulated neuroblastoma cells undergoing EMT, accordingly, interfering with Gli1/2 expression inhibited TGF-beta1-induced EMT in neuroblastoma cells. GANT61, which is a targeted inhibitor of Gli1 and Gli2, decreased cell viability and promoted cell apoptosis. Thus, TGF-beta1 induced EMT in neuroblastoma cells to increase their migration. Specifically, EMT induced by TGF-beta1 in neuroblastoma cells did not depend on the Smad signaling pathway, and the transcription factor Gli participated in TGF-beta1-induced EMT independent of Smad signaling.

Although it is well known that exaggerated proliferation, metastasis and the mesenchymal subtype is related with worst prognoses in glioblastoma (GBM) and that transforming growth factor-beta1 (TGF-beta1) is a potent factor in regulating the proliferation, migration and epithelial-mesenchymal transition (EMT) phenotype of GBM, the detailed mechanisms are still far from elucidated. MicroRNAs (miRNAs) are small non-coding RNAs which play critical roles in various diseases by regulating target gene expression. We report that miR-10b, a molecule downstream of TGF-beta1, is involved in TGF-beta1-regulated GBM cell proliferation, migration and EMT. We found that exposure of GBM cells to TGF-beta1 significantly upregulated miR-10b expression. Overexpression of miR-10b promotes GBM cell proliferation, migration and EMT, whereas depletion of miR-10b obtained reverse effects. Further studies uncovered that some tumor-associated genes including epithelial cadherin (E-cadherin), apoptotic protease activating factor 1 (Apaf-1) and phosphatase and tensin homolog (PTEN) are target genes of miR-10b. In human GBM xenografts, antagomiR directed against miR-10b markedly suppressed tumor growth, and the tumor volume shrunk from 1252.5+/-285 to 873.4+/-205 mm3 after antagomiR10b treatment for 3 weeks compared with the control group (P<0.01). Taken together, our data collectively demonstrate that the proliferation, migration and EMT features of GBM cells can be regulated by TGF-beta1 stimulation through controlling miR-10b. Thus, our findings provide a rationale for targeting TGF-beta1 or miR-10b for the treatment of GBM.

ASPP2 regulates cell polarity and cell-cell adhesion by binding to, and co-localizing with PAR3 at tight junctions. Here we show a novel role of ASPP2 in suppressing gastric cancer (GC) invasiveness. Immunoprecipitation and immunofluorescence analyses showed that ASPP2 promoted the recruitment of PAR3 to cell-cell junctions in GC cells. Diminished expression of ASPP2 and loss of junctional PAR3 localization were significantly associated with diffuse-type histology, deeper invasion depth, positive peritoneal dissemination and worse prognosis in primary GC. ASPP2 suppressed migration and invasion of GC cells in vitro and peritoneal dissemination of GC cells in vivo in a mouse model. ASPP2 suppressed epithelial-mesenchymal transition (EMT) induced by TGF-beta1-Smad2/3 signaling in GC cells through suppression of the degradation of Smad7, a negative regulator of TGF-beta1-Smad2/3 signaling, by interacting with the E3 ubiquitin ligase ITCH. In conclusion, ASPP2 suppresses invasion, peritoneal dissemination and TGF-beta1-induced EMT by inhibiting Smad7 degradation mediated by ITCH.CI - Copyright (c) 2017 Elsevier B.V. All rights reserved.

BACKGROUND/AIMS: The aggressive property of ovarian cancer (OC) in terms of epithelial-mesenchymal transition (EMT), proliferation and metastasis are of major concern. Different growth factors including TGFbeta are associated with regulating these molecular events but the underlying mechanisms remain unclear. The aim of this report is to decipher the regulation of EMT by co-activation of TGFbeta and Wnt signalling cascades in gaining malignancy. METHODS: The expression of the different components of signalling events were analyzed by QPCR, Western blot, Immunofluorescence microscopy and flow cytometry. beta-catenin promoter activity was checked by luciferase assay. RESULTS: We observed reduced EMT in ovarian cancer cells upon co-activation with TGFbeta1 and LiCl as shown by the expressions of epithelial/mesenchymal markers and the EMT promoting factor, Snail1, accompanied by decrease in the invasion and migration of the cells compared to individual pathway activation. A detailed study of the mechanism suggested reduction in the beta-catenin and p-GSK3b (Ser 9) levels to be the driving cause of this phenomenon, which was reversed upon co-activation with higher concentrations of LiCl. CONCLUSIONS: Therefore, tumourigenesis might be affected by the concentration of ligand/ growth factors for the respective signalling pathways activated in the tumour microenvironment and interaction between them might alter tumourigenesis.CI - (c) 2017 The Author(s)Published by S. Karger AG, Basel.

TMPRSS2:ERG (T/E) gene fusions are present in approximately 50% of all prostate cancer (PCa) cases. The expression of fusion mRNAs from distinct T/E variants is associated with clinicopathological parameters, while the underlying molecular processes remain unclear. We characterized the molecular mechanisms and functional implications caused by doxycycline (Dox)-inducible overexpression of the frequent T/E III and VI fusion variants in LNCaP cells. Induction of T/E expression resulted in increased cellular migratory and invasive potential, and reduced proliferation and accumulation in G1 phase. T/E overexpressing cells showed epithelial-to-mesenchymal transition (EMT), as demonstrated by upregulation of TGF-beta and WNT pathway genes, mesenchymal markers, and increased phosphorylation of the p38 MAPK. Augmented secretion of TGF-beta1 and -beta2, and T/E-mediated regulation of ALK1, a member of the TGF-beta receptor family, was detected. ALK1 inhibition in T/E overexpressing cells blocked p38 phosphorylation and reduced the expression of the TGF-beta target genes VIM, MMP1, CDH2, and SNAI2. We found a T/E variant VI-specific induction of miR-503 associated with reduced expression of SMAD7 and CDH1. Overexpression of miR-503 led to increased levels of VIM and MMP1. Our findings indicate that TGF-beta signaling is a major determinant of EMT in T/E overexpressing LNCaP cells. We provide evidence that T/E VI-specific transcriptional modulation by miR-503 accounts for differences in the activation of EMT pathway genes, promoting the aggressive phenotype of tumors expressing T/E variant VI. We suggest that ALK1-mediated TGF-beta signaling is a novel oncogenic mechanism in T/E positive PCa.

Lung cancer is one of the most lethal cancers due to its highly metastatic spreading. The motility of lung cancer cells is regulated by paracrine factors, such as TGF-beta, in the tumor microenvironment through the induction of epithelial-to-mesenchymal transition (EMT). The stability of microtubules is reported to be associated with the EMT process and the migration of cancer cells. Here, we observed that RCC1 domain-containing protein 1 (RCCD1) is highly expressed in non-small cell lung cancer (NSCLC) patients with poor prognosis, and RCCD1 is much higher expressed in tumor tissues compared with adjacent normal tissues. Depletion of RCCD1 using siRNAs significantly inhibits the migration of lung cancer cells. Subsequent studies reveal that the loss of RCCD1 results in upregulation of acetylated alpha-tubulin levels and stabilizes cytoskeletal microtubules. Mechanistically, we observed that RCCD1 modulates the stability of microtubules through interacting with JMJD5. Furthermore, RCCD1 depletion significantly attenuates the TGF-beta-induced EMT process, as assessed by altered expression of epithelial and mesenchymal markers (Occludin, Vimentin and Snail), and inhibits TGF-beta-induced cell migration. Collectively, these findings support RCCD1 as a novel regulator of TGF-beta-induced EMT in NSCLC.CI - Copyright (c) 2017 Elsevier B.V. All rights reserved.

Cathepsins B and X are lysosomal cysteine carboxypeptidases suggested as having a redundant role in cancer. They are involved in a number of processes leading to tumor progression but their role in the epithelial-mesenchymal transition (EMT) remains unknown. We have investigated the contribution of both cathepsins B and X in EMT using tumor cell lines differing in their expression of epithelial and mesenchymal markers and cell morphology. Higher levels of both cathepsins are shown to promote EMT and are associated with the mesenchymal-like cell phenotype. Moreover, simultaneous knockdown of the two peptidases triggers a reverse, mesenchymal to epithelial transition. Of the two cathepsins, cathepsin B appears to be the stronger promotor of EMT. Furthermore, we evaluated the involvement of cathepsin B and X in the transforming growth factor-beta1 (TGF-beta1) signaling pathway, one of the key signaling mechanisms triggering EMT in cancer. In MCF-7 cells the expression of cathepsin B was shown to depend on their activation with TGF-beta1 while, for cathepsin X, a TGF-beta1 independent mechanism of induction during EMT is indicated. EMT is thus shown to be another mechanism linking cathepsins B and X with tumor progression. With silencing of their expression or inhibition of enzymatic activity, the tumor cells could be reverted to less aggressive epithelial-like phenotype.CI - Copyright (c) 2017 Elsevier GmbH. All rights reserved.

OBJECTIVE: To investigate the role of TGF-beta1 on epithelial mesenchymal transition (EMT) and invasion in oral squamous cell carcinoma cell line Tca8113. MATERIALS AND METHODS: Cultured Tca8113 cells were treated with different concentrations of TGF-beta1 for 24 h. The morphological changes were observed under phase-contrast microscopy. The mRNA and protein expression levels of EMT relative marker E-cadherin and Vimentin were detected by RT-PCR and Western blot. The effect of TGF-beta1 on migration and invasion ability of Tca8113 cells were detected using transwell method. RESULTS: The results demonstrated that TGF-beta1 could induce morphological changes in Tca8113 cells from epithelial to mesenchymal. The mRNA and protein level of epithelial marker E-cadherin was downregulated following treatment with TGF-beta1, whereas the mRNA and protein expression level of mesenchymal marker protein Vimentin was upregulated. Furthermore, TGF-beta1 significantly enhances the migration and invasiveness of Tca8113 cells, which were effectively reversed by TGF-beta1 inhibitor, LY2109761 CONCLUSIONS: TGF-beta1 enhances Tca8113 cells migration and invasion by inducing epithelial mesenchymal transition.

Epidemiological studies have shown obesity to be linked with poorer outcomes in breast cancer patients. The molecular mechanisms responsible for the increased risk of invasive/metastatic disease with obesity are complex, but may include elevated levels of adipokines such as leptin. Using physiological levels of leptin found in obesity in a novel chronic in vitro treatment model (expression changes representing epithelial-to-mesenchymal transition (EMT) and a cancer stem cell (CSC) like phenotype in breast epithelial and cancer cell lines (MCF10A, MCF10AT1, MCF7 and MDA-MB-231). Further, we have discovered that these effects were accompanied by increased expression of TGFB1, and could be significantly reduced by co-treatment with neutralizing antibody against TGFB1, indicating that the induction of these characteristics was mediated via TGFB1. Occurring in both MCF7 and MCF10AT1 cells, it suggests these actions of leptin to be independent of estrogen receptor status. By linking leptin signalling to the established TGFB1 pathway of metastasis / EMT, this study gives a direct mechanism by which leptin can contribute to the poorer outcomes of obese cancer patients. Inhibitors of TGFB1 are in currently in phase III clinical trials in other malignancies, thus identifying the connection between leptin and TGFB1 will open new therapeutic opportunities for improving outcomes for obese breast cancer patients.

BACKGROUND: Accumulating evidence indicates that N-cadherin is a cell adhesion molecule that has critical roles in tumour progression. However, the role of N-cadherin in hepatocellular carcinoma (HCC) remains controversial. METHODS: This study aims to investigate the expression status of N-cadherin and its molecular mechanisms in HCC. RESULTS: The expression of N-cadherin was markedly overexpressed in HCC tissues and cell lines. We identified that miR-199b-5p binds to the 3'-UTR of N-cadherin mRNA, thus decreasing N-cadherin expression in HCC cells. We also found the downregulation of miR-199b-5p in HCC specimens, which was inversely correlated with N-cadherin upregulation, predicted poor clinical outcomes in HCC patients. Next, we determined that miR-199b-5p overexpression promoted cell aggregation, suppressed cell migration and invasion in HCC cells, and inhibited xenografts tumour metastasis in nude mice. Moreover, we demonstrated that miR-199b-5p attenuated TGF-beta1 induced epithelial-mesenchymal transition (EMT) -associated traits, while its effects could be partially reversed by N-cadherin restoration. Finally, we examined that N-cadherin downregulation or miR-199b-5p overexpression suppressed TGF-beta1-induced Akt phosphorylation, and inhibition of PI3K/Akt pathway blocked TGF-beta1-induced N-cadherin overexpression in HCC cells. CONCLUSIONS: Our data demonstrate that N-Cadherin was markedly overexpressed and miR-199b-5p was significantly downregulated in HCC. MiR-199b-5p exerts inhibitory effects on EMT, and directly targets N-cadherin in HCC, supporting the potential utility of miR-199b-5p as a promising strategy to treat HCC. Also, a positive regulatory loop exists between N-cadherin and Akt signalling represents a novel mechanism of TGF-beta1-mediated EMT in HCC cells.

Akt1 is essential for the oncogenic transformation and tumor growth in various cancers. However, the precise role of Akt1 in advanced cancers is conflicting. Using a neuroendocrine TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model, we first show that the genetic ablation or pharmacological inhibition of Akt1 in mice blunts oncogenic transformation and prostate cancer (PCa) growth. Intriguingly, triciribine (TCBN)-mediated Akt inhibition in 25-week old, tumor-bearing TRAMP mice and Akt1 gene silencing in aggressive PCa cells enhanced epithelial to mesenchymal transition (EMT) and promoted metastasis to the lungs. Mechanistically, Akt1 suppression leads to increased expression of EMT markers such as Snail1 and N-cadherin and decreased expression of epithelial marker E-cadherin in TRAMP prostate, and in PC3 and DU145 cells. Next, we identified that Akt1 knockdown in PCa cells results in increased production of TGFbeta1 and its receptor TGFbeta RII, associated with a decreased expression of beta-catenin. Furthermore, treatment of PCa cells with ICG001 that blocks nuclear translocation of beta-catenin promoted EMT and N-cadherin expression. Together, our study demonstrates a novel role of the Akt1-beta-catenin-TGFbeta1 pathway in advanced PCa.CI - Copyright (c) 2017 Elsevier B.V. All rights reserved.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is closely associated with tissue remodeling. epithelial-to-mesenchymal transition (EMT), a process of tissue remodeling, can be a therapeutic target of CRSwNP. Glucocorticoids are a type of steroid hormone that is used primarily in medical therapy for patients with CRSwNP; however, their effects on EMT in the airway epithelium remain unknown. To investigate the effects of dexamethasone and fluticasone propionate, a class of glucocorticoids, on transforming growth factor-beta1 (TGF-beta1) -induced EMT, we used A549 cells, human primary nasal epithelial cells (hPNECs) and ex vivo organ culture of the inferior turbinate. TGF-beta1 induced changes in cell morphology, suppressed the expression of E-cadherin and enhanced the expression of a-smooth muscle actin, vimentin and fibronectin in A549 cells. However, glucocorticoids inhibited EMT, migration and invasion enhancement by TGF-beta1. We found that the induction of phosphorylated ERK, p38 and the activity of Snail and Slug transcription factors by TGF-beta1 were suppressed by glucocorticoids. Glucocorticoids also had a similar effect in hPNECs and ex vivo organ cultures of the inferior turbinate. These findings suggest that glucocorticoids might be a useful therapy for preventing tissue remodeling by blocking the EMT initiated by TGF-beta1-induced MAPK and Snail/Slug signaling pathways in CRSwNP.

BACKGROUND: Proteotoxic stress and transforming growth factor (TGFbeta)- induced epithelial-mesenchymal transition (EMT) are two main contributors of intraocular fibrotic disorders, including proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). However, how these two factors communicate with each other is not well-characterized. OBJECTIVE: The aim was to investigate the regulatory role of proteotoxic stress on TGFbeta signaling in retinal pigment epithelium. METHODS: ARPE-19 cells and primary human retinal pigment epithelial (RPE) cells were treated with proteasome inhibitor MG132 and TGFbeta. Cell proliferation was analyzed by CCK-8 assay. The levels of mesenchymal markers alpha-SMA, fibronectin, and vimentin were analyzed by real-time polymerase chain reaction (PCR), western blot, and immunofluorescence. Cell migration was analyzed by scratch wound assay. The levels of p-Smad2, total Smad2, p-extracellular signal-regulated kinase 1/2 (ERK1/2), total ERK1/2, p-focal adhesion kinase (FAK), and total FAK were analyzed by western blot. The mRNA and protein levels of TGFbeta receptor-II (TGFbetaR-II) were measured by realtime PCR and western blot, respectively. RESULTS: MG132-induced proteotoxic stress resulted in reduced cell proliferation. MG132 significantly suppressed TGFbeta-induced upregulation of alpha-SMA, fibronectin, and vimentin, as well as TGFbeta-induced cell migration. The phosphorylation levels of Smad2, ERK1/2, and FAK were also suppressed by MG132. Additionally, the mRNA level and protein level of TGFbetaR-II decreased upon MG132 treatment. CONCLUSION: Proteotoxic stress suppressed TGFbeta-induced EMT through downregulation of TGFbetaR-II and subsequent blockade of Smad2, ERK1/2, and FAK activation.CI - Copyright(c) Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

epithelialmesenchymal transition (EMT) is a key mechanism underlying metastatic breast cancer. Reactive oxygen species (ROS) play an important role in EMT. Heme oxygenase1 (HMOX1) can reduce oxidative stress. However, the effect of HMOX1 on the EMT process in breast cancer cells is unknown. We treated the MCF7 breast cancer cell line with the HMOX1 inducer hemin and observed that hemin induced HMOX1 expression and inhibited migration, invasion and ROS generation in transforming growth factorbeta (TGFbeta)treated MCF7 cells using quantitative RTqPCR, western blotting, woundhealing and cell invasion assays as well as fluorescent probe DCFDA. Hemin inhibited TGFbetainduced EMT in the MCF7 cells, whereas HMOX1 siRNA attenuated the suppressive effect of hemin as determined by the expression and cellular distribution of selected EMT markers. In summary, our results revealed that hemin treatment increased HMOX1 expression and inhibited TGFbetainduced EMT in MCF7 cells.

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.

BACKGROUND: This study was aimed to investigate the epithelial differentiation of human adipose-derived mesenchymal stem cells (ADSCs) by inhibiting glycogen synthase kinase-3 (GSK3) and transforming growth factor beta (TGFbeta) signaling. METHODS AND RESULTS: STEMPRO human ADSCs at passage 2 were treated with CHIR99021 (GSK3 inhibitor), E-616452 (TGFbeta1 receptor kinase inhibitor), A-83-01 (TGFbeta type 1 receptor inhibitor), valproic acid (histone deacetylase inhibitor), tranylcypromine (monoamine oxidase inhibitor) and all-trans retinoic acid for 72 h. The mesenchymal-epithelial transition was shown by down-regulation of mesenchymal genes (Slug, Zinc Finger E-box Binding Homeobox 1 ZEB1, integrin alpha5 ITGA5 and vimentin VIM) and up-regulation of epithelial genes (E-cadherin, epithelial Cell Adhesion Molecule EpCAM, Zonula Occludens-1 ZO-1, occludin, deltaN p63 deltaNp63, Transcription Factor 4 TCF4 and Twist Family bHLH Transcription Factor TWIST), compared to untreated ADSCs. Cell morphology and stress fiber pattern were examined and the treated cells became less migratory in scratch wound closure assay. The formation of cell junction complexes was observed under transmission electron microscopy. Global gene expression using GeneChip((R)) Human Genome U133 Array (Affymetrix) showed that the treatment up-regulated 540 genes (containing genes for cell cycle, cytoskeleton reorganization, chemotaxis, epithelium development and regulation of cell migration) and down-regulated 483 genes. CONCLUSION: Human ADSCs were transited to epithelial lineage by inhibiting GSK3 and TGFbeta signaling. It can be an adult stem cell source for epithelial cell-based therapy.CI - Copyright (c) 2017 Elsevier Inc. All rights reserved.

Histone methylation is associated with various biological and pathological processes including cancer development. KDM6A is a candidate tumor suppressor gene that encodes a histone H3 lysine 27 (H3K27) demethylase. In this study, we discovered that ectopic expression of KDM6A antagonized TGF-beta-induced epithelial-mesenchymal transition (EMT) and cell migration of lung cancer cell lines through its demethylase activity. KDM6A counteracted TGF-beta-dependent changes in the expression of EMT-related genes such as CDH1/E-cadherin, FN1/Fibronectin, ZEB family and microRNA-200 family. Mechanistic investigations revealed that KDM6A inhibited the recruitment of EZH2 histone H3K27 methyltransferase and H3K27 methylation on the regulatory regions of the target genes such as CDH1 and microRNA-200 family. Knockdown of KDM6A did not proceed EMT by itself, but influenced the expression of specific target genes critical for EMT, suggesting that endogenous KDM6A was involved in EMT-inducing transcriptional program. This study demonstrated a novel regulatory role of KDM6A histone demethylase in the epigenetic control of EMT process in lung cancer cells.CI - Copyright (c) 2017 Elsevier Inc. All rights reserved.

tumor metastasis remains a major obstacle for improving overall cancer survival in cervical cancer (CC), which may be due to the existence of tumor microenvironment-related cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT). The mechanism underlying these processes needs to be further elucidated. Here, we report that TGF-beta1, one of the key microenvironmental stimuli, can enhance CSC characteristics, facilitate the EMT, and induce CK17. Silencing CK17 expression attenuated CSC-like properties without affecting the EMT markers induced by TGF-beta1, whereas forced overexpression of CK17 promoted lymphatic metastasis in vivo even without EMT inducement. Inhibitors of ERK1/2 signaling drastically decreased the induction of CK17 mediated by TGF-beta1. By combined computational and experimental approaches, we identified and validated that MZF1 was a key transcription factor binding to the promoter of CK17. Taken together, these results demonstrate that CK17 induced by the TGF-beta1-ERK1/2-MZF1 signaling pathway facilitates metastasis by promoting the acquisition of CSC properties rather than by inducing the EMT process in CC, suggesting that this CK17-related signaling pathway might be a suitable target for the development of therapy for CC metastasis.CI - (c) 2017 Federation of European Biochemical Societies.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and an irreversible lung disorder characterized by the accumulation of fibroblasts and myofibroblasts in the extracellular matrix. The transforming growth factor-beta1 (TGF-beta1)-induced epithelial-to-mesenchymal transition (EMT) is thought to be one of the possible sources for a substantial increase in the number of fibroblasts/myofibroblasts in IPF lungs. Tannic acid (TA), a natural dietary polyphenolic compound has been shown to possess diverse pharmacological effects. However, whether TA can inhibit TGF-beta1-mediated EMT in lung epithelial cells remains enigmatic. Both the human adenocarcinomic alveolar epithelial (A549) and normal bronchial epithelial (BEAS-2B) cells were treated with TGF-beta1 with or without TA. Results showed that TA addition, markedly inhibited TGF-beta1-induced EMT as assessed by reduced expression of N-cadherin, type-1-collagen, fibronectin, and vimentin. Furthermore, TA inhibited TGF-beta1-induced cell proliferation through inducing cell cycle arrest at G0/G1 phase. TGF-beta1-induced increase in the phosphorylation of Smad (Smad2 and 3), Akt as well as that of mitogen activated protein kinase (ERK1/2, JNK1/2, and p38) mediators was effectively inhibited by TA. On the other hand, TA reduced the TGF-beta1-induced increase in TGF-beta receptors expression. Using molecular docking approach, FTIR, HPLC and Western blot analyses, we further identified the direct binding of TA to TGF-beta1. Finally, we conclude that TA might directly interact with TGF-beta1, thereby repressing TGF-beta signaling and subsequent EMT process in lung epithelial cells. Further animal studies are needed to clarify its potential therapeutic benefit in pulmonary fibrosis.CI - (c) 2017 Wiley Periodicals, Inc.

As a Ca(2+) binding protein, calreticulin (CRT) has many functions and plays an important role in a variety of tumors. The role of CRT in TGF-beta1-induced EMT is unknown. In this study, we demonstrated in vitro that TGF-beta1-induced EMT elevated the expression of CRT in A549 lung cancer cells. Subsequently, we confirmed that overexpression CRT had no capacity to induce A549 cells EMT alone, but successfully enhanced TGF-beta1-induced-EMT. Furthermore, knockdown of CRT in A549 cells significantly suppressed changes of EMT marks expression induced by TGF-beta1. On treatment with TGF-beta1, overexpression of CRT could enhance the phosphorylation of both Smad2 and Smad3. Consistently, the knockdown of CRT by siRNA-CRT could inhibit Smad signaling pathway activated by TGF-beta1. These results indicated that CRT regulates EMT induced by TGF-beta1 through Smad signaling pathway. Finally, TGF-beta1-induced-EMT enhanced store-operated Ca(2+) influx in A549 cells. CRT knockdown was able to abolish the effect of TGF-beta1 on thapsigargin (TG) -induced Ca(2+) release, but had failed to reduce store-operated Ca(2+) influx. The alteration of intracellular Ca(2+) concentration by TG or BAPTA-AM was able to regulate EMT induced by TGF-beta1 through Smad signaling pathway. Together, these data identify that CRT regulates TGF-beta1-induced-EMT through modulating Smad signaling. Furthermore, TGF-beta1-induced-EMT is highly calcium-dependent, CRT was partly involved in it.CI - Copyright (c) 2017 Elsevier Ltd. All rights reserved.

In cancer immunology, the programmed cell death 1-programmed cell death 1/ligand 1 (PD-1/PD-L1) pathway plays a major role. Anti-PD-1 and anti-PD-L1 antibodies provide reliable immunotherapy when given as treatment for various types of malignancy including lung cancer. PD-L1 expression in cancer cells has been reported to be a predictive factor for the therapeutic effects of immunotherapy. However, the mechanism of PD-L1 expression remains unclear. Another key process in cancer progression is epithelial-mesenchymal transition (EMT). In the present study, we investigated the mechanism of PD-L1 expression as well as changes in its expression during the EMT process in non-small cell lung cancer (NSCLC). In this study, A549 cells underwent EMT by treatment with TGF-beta or chemotherapeutic agents and then PD-L1 expression was evaluated. The alterations of PD-L1 expression was also examined during the reverse EMT process; mesenchymal-epithelial transition (MET). The relationship between for PD-L1 expression and EMT status in clinical specimens with NSCLC after induction chemotherapy were analyzed by immunohistochemical staining. We found that PD-L1 expression was upregulated following TGF-beta induction; in contrast, it was downregulated by TGF-beta receptor-kinase inhibitors and the MET process. Furthermore, chemo-treatment increased TGF-beta expression and enhances PD-L1 expression via autocrine TGF-beta induced EMT. Analysis of clinical samples revealed a significant relationship between PD-L1 expression and EMT status (P<0.05). In conclusion, our results suggest that PD-L1 expression is regulated by TGF-beta induced EMT and enhanced by chemo-treatment via the chemo-induced TGF-beta signaling. The anti-PD-1/PD-L1 blockade may provide more effective anticancer activities in combination with chemotherapy in NSCLC.

Idiopathic pulmonary fibrosis (IPF) is a chronic and usually progressive lung disease and the epithelial-mesenchymal transition (EMT) may play an important role in the pathogenesis of pulmonary fibrosis. IL-17 is a proinflammatory cytokine which promotes EMT profiles in lung inflammatory diseases. In this study, we investigated the effect of IL-17 on EMT in alveolar epithelial cell line A549 and the role of TGFbeta1-Smad and ERK signaling pathways in the process. Morphological observation on the cells was performed under inverted microscope. The mRNA and protein expressions of E-cad and alpha-SMA were detected by quantitative RT-PCR and western blotting. The mRNA and protein expressions of TGF-beta1 were analyzed via quantitative RT-PCR and ELISA. expressions of Smad2/3, p-Smad2/3, ERK1/2, p-ERK1/2 and p-JNK were examined by western blotting. The results indicated that IL-17 can induce A549 cells to undergo morphological changes and phenotypic markers changes, such as down-regulated E-cad expression and up-regulated alpha-SMA expression. Additionally, IL-17 enhanced TGF-beta1 expression and stimulated Smad2/3 and ERK1/2 phosphorylation in A549 cells. However, there were no significant differences in the expression of phosphorylated JNK in A549 cells with or without IL-17 treatment. SB431542 or U0126 treated cells showed inhibited morphological changes and phenotypic markers expression, such as up-regulated E-cad expression and down-regulated alpha-SMA expression. In summary, our results suggest that IL-17 can induce A549 alveolar epithelial cells to undergo EMT via the TGF-beta1 mediated Smad2/3 and ERK1/2 activation.

Psychological stress has recently been described as a risk factor in the development of pancreatic cancer. Here, we reported that increased neurotransmitter adrenaline was associated with the poor survival in pancreatic cancer patients. Moreover, in the cell model study, we found adrenaline promoted pancreatic cell PANC-1 migration in a dose dependent manner. Block of the beta2-adrenoreceptor with ICI118,551, significantly reduced cell migration. Further study found that adrenaline induced a cytoplasmic translocation of RNA binding protein HuR, which in turn activated TGFbeta, as shown by the SBE luciferase assay and phosphorylation of Smad2/3. Either HuR knockdown or TGFbeta inhibition reduced cell migration induced by adrenaline. Taken together, our study here revealed that adrenaline-HuR-TGFbeta regulatory axis at least partially contributes to the psychological stress induced metastasis in PANC-1 cells, shedding light on therapeutic targeting psychological stress in improving the prognosis of pancreatic cancer.CI - Copyright (c) 2017 Elsevier Inc. All rights reserved.

BACKGROUND: Core fucosylation (addition of fucose in alpha-1,6-linkage to core N-acetylglucosamine of N-glycans) catalyzed by fucosyltransferase 8 (FUT8) is critical for signaling receptors involved in many physiological and pathological processes such as cell growth, adhesion, and tumor metastasis. Transforming growth factor-beta (TGF-beta)-induced epithelial-mesenchymal transition (EMT) regulates the invasion and metastasis of breast tumors. However, whether receptor core fucosylation affects TGF-beta signaling during breast cancer progression remains largely unknown. METHOD: In this study, gene expression profiling and western blot were used to validate the EMT-associated expression of FUT8. Lentivirus-mediated gain-of-function study, short hairpin RNA (shRNA) or CRISPR/Cas9-mediated loss-of-function studies and pharmacological inhibition of FUT8 were used to elucidate the molecular function of FUT8 during TGF-beta-induced EMT in breast carcinoma cells. In addition, lectin blot, luciferase assay, and in vitro ligand binding assay were employed to demonstrate the involvement of FUT8 in the TGF-beta1 signaling pathway. The role of FUT8 in breast cancer migration, invasion, and metastasis was confirmed using an in vitro transwell assay and mammary fat pad xenograft in vivo tumor model. RESULTS: Gene expression profiling analysis revealed that FUT8 is upregulated in TGF-beta-induced EMT; the process was associated with the migratory and invasive abilities of several breast carcinoma cell lines. Gain-of-function and loss-of-function studies demonstrated that FUT8 overexpression stimulated the EMT process, whereas FUT8 knockdown suppressed the invasiveness of highly aggressive breast carcinoma cells. Furthermore, TGF-beta receptor complexes might be core fucosylated by FUT8 to facilitate TGF-beta binding and enhance downstream signaling. Importantly, FUT8 inhibition suppressed the invasive ability of highly metastatic breast cancer cells and impaired their lung metastasis. CONCLUSIONS: Our results reveal a positive feedback mechanism of FUT8-mediated receptor core fucosylation that promotes TGF-beta signaling and EMT, thus stimulating breast cancer cell invasion and metastasis.

Thymoquinone, a major ingredient of black seed oil (Nigella sativa), has been shown to exhibit anticancer capacity in various types of cancers. However, there are few studies concerning the correlation between thymoquinone and epithelial-to-mesenchymal transition (EMT) in prostate cancer. In the present study, we firstly found that thymoquinone showed antimetastatic capacity in prostate cancer DU145 and PC3 cells. Additionally, thymoquinone reversed EMT by increasing E-cadherin expression and decreasing vimentin and Slug expression in a concentration-dependent manner. Recent studies have shown that the transforming growth factor-beta (TGF-beta) signaling pathway may be associated with EMT. Intriguingly, the expression of TGF-beta, Smad2 and Smad3 at the mRNA and protein levels was notably reduced upon thymoquinone treatment in prostate cancer DU145 and PC3 cells. Subsequently, we confirmed that thymoquinone repressed metastasis and EMT of prostate cancer through downregulation of the TGF-beta/Smad2/3 signaling pathway, which may be partially reversed by TGF-beta overexpression. In summary, our findings demonstrated that thymoquinone suppressed the metastatic phenotype and reversed EMT of prostate cancer cells by negatively regulating the TGF-beta/Smad2/3 signaling pathway. These findings suggest that thymoquinone is a potential therapeutic agent against prostate cancer which functions by targeting TGF-beta.

TGF-beta's multipotent cellular effects and their relations are critical for TGF-beta's pathophysiological functions. However, these effects may appear to be paradoxical in understanding TGF-beta's functions. Apoptosis and epithelial-mesenchymal transition (EMT) are two fundamental events that are deeply linked to various physiological and disease-related processes. These two major cellular fates are subtly regulated and can be potently stimulated by TGF-beta, which profoundly contribute to the biological roles of TGF-beta. Moreover, these two events are also indirectly and directly correlated with TGF-beta-mediated growth inhibition and are relevant to the current understanding of the roles of TGF-beta in tumorigenesis and cancer progression. Although TGF-beta-induced apoptosis and EMT can be singly independent cellular events, they can also be mutually exclusive but interrelated concomitant events in various cases. Thus, the modulation of apoptosis and EMT is essential for the seemingly paradoxical functions of TGF-beta. However, the concomitant effect of TGF-beta on apoptosis and EMT, the balance and regulated alterations of them are still been ignored or underestimated. This review focuses on the TGF-beta-induced concomitant apoptosis and EMT. We aim to provide an insight in understanding their significance, balance, and modulation in TGF-beta-mediated biological functions.CI - (c) The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Acute and chronic inflammatory lung diseases are often associated with epithelial cell injury/loss and fibroproliferative responses. ResolvinD1 (RvD1) is biosynthesized during the resolution phase of inflammatory response and exerts potent anti-inflammatory and promotes resolution of inflammatory lung diseases. The aim of this study was to investigate whether RvD1 exerts protective effects on alveolar epithelial cell function/differentiation and protects against fibroproliferative stimuli. Primary human alveolar type II cells were used to model the effects of RvD1 in vitro upon wound repair, proliferation, apoptosis, transdifferentiation, and epithelial-mesenchymal transition (EMT). Effects of RvD1 upon primary human lung fibroblast proliferation, collagen production, and myofibroblast differentiation were also examined. RvD1 promoted alveolar type II (ATII) cell wound repair and proliferation. RvD1 protected ATII cells against sFas-ligand/TNF-alpha-induced apoptosis and inhibition on cell proliferation and viability. RvD1 promoted ATII cells transdifferentiation. Moreover, we demonstrate that RvD1 inhibited EMT in response to TGF-beta. Furthermore RvD1 inhibited human lung fibroblast proliferation, collagen production, and myofibroblast differentiation induced by both TGF-beta and bronchoalveolar lavage fluid from acute respiratory distress syndrome (ARDS) patients. The effects of RvD1 were PI3-kinase dependent and mediated via the resolvin receptor. RvD1 seems to promote alveolar epithelial repair by stimulating ATII cells wound repair, proliferation, reducing apoptosis, and inhibiting TGF-beta-induced EMT. While RvD1 reduced fibroproliferation, collagen production, and myofibroblast differentiation. Together, these results suggest a potential new therapeutic strategy for preventing and treating chronic diseases (such as idiopathic pulmonary fibrosis) as well as the fibroproliferative phase of ARDS by targeting RvD1 actions that emphasizes natural resolution signaling pathways.

Increased metastasis and a precipitous recurrence contribute to the lethality of ovarian cancer (OC). Several molecular mechanisms including aberrant-splicing have been closely associated with the extent of cancer progression. Numerous gene transcripts are differentially spliced in cancer cells, CD44 being one of them. CD44 splice isoforms contribute to the aggressiveness and gain of stem-like properties in different cancer types, but their role in ovarian cancer remains to be elucidated. We observed augmented CD44 levels in human ovarian cancer patient samples correlated with enhanced expression of the mesenchymal spliced variant CD44s (standard) and a concurrent decrease in the epithelial variants (CD44v). Moreover, CD44s was upregulated upon TGFbeta1-induced EMT, which was mediated through the downregulation of the splicing factor, ESRP1. Furthermore, overexpression of this mesenchymal isoform in the OC cells induced EMT and invasion, followed by the gain of stem-like characteristics and chemoresistance. Since all these phenomena render lethality to this disease type, CD44s can be attributed for playing a major role in deregulated-splicing mediated ovarian cancer progression.CI - (c) 2017 Wiley Periodicals, Inc.

Pulmonary fibrosis (PF) is a chronic lung disease. The transforming growth factor-beta1 (TGF-beta1)/Smad3 signaling pathway plays an important role in the pathogenesis of pulmonary fibrosis. Bone marrow-derived mesenchymal stem cells (BMSCs) have been shown to be a modulator of the molecular aspects of the fibrosis pathway. However, it is still unknown as to whether the conditioned medium from BMSCs (BMSCs-CM) inhibits the epithelial-mesenchymal transition (EMT) process. This study confirmed the hypothesis that BMSCs-CM exerts an anti-fibrotic effect on human type II alveolar epithelial cells (A549) by suppressing the phosphorylation of Smad3. We used the A549 cells in vitro to detect morphological evidence of EMT by phase-contrast microscopy. These cells were randomly divided into 4 groups as follows: the control group, the TGF-beta1 group, the SIS3 (specific inhibitor of Smad3) group and the BMSCs-CM group. The immunofluorescence method was used to determined the location of E-cadherin (E-calcium mucins; E-cad), alpha-smooth muscle actin (alpha-SMA) and p-Smad3. The expression levels of E-cad, CK8, alpha-SMA, vimentin, p-Smad3, Snail1, collagen I (COLI) and collagen III (COLIII) were detected by western blot analysis. Following exposure to TGF-beta1, the A549 cells displayed a spindle-shaped fibroblast-like morphology. In accordance with these morphological changes, the expression levels of E-cad and CK8 were downregulated, while the expression levels of alpha-SMA and vimentin were upregulated. Along with this process, the expression levels of p-Smad3, Snail1, COLI and COLIII were increased. However, the cells in the BMSCs-CM group and SIS3 group exhibited a decrease in the levels of alpha-SMA and vimentin (which had been upregulated by TGF-beta1), and an increase in the levels of E-cad and CK8 expression (which had been downregulated by TGF-beta1). On the whole, these results indicated that BMSCs-CM suppressed the EMT which might be associated with TGF-beta1/Smad3. This study provides the theoretical basis for the research of the mechanisms responsible for pulmonary disease.

Ovarian cancer is the most common and lethal type of gynecological malignancy, due to its invasiveness. The present study aimed to analyze the molecular mechanism underlying chemoresistance in ovarian carcinoma cells, which may lead to local migration toward adjacent tissues and longdistance metastasis to other organs. A total of 12 patients with ovarian fibroma were used to evaluate chemoresistance and chemosensitivity. The sensitivity and resistance of ovarian carcinoma cells was measured using apoptosis analysis, morphological observation, survival rate analysis, immunohistochemistry and immunostaining. The mechanism underlying the interaction between the epithelialmesenchymal transition (EMT) and liver kinase B1 (LKB1)saltinducible kinase 1 (SIK1) signaling pathways was additionally investigated in ovarian carcinoma. The results of the present study demonstrated that ovarian carcinoma cells isolated from patients exhibited apoptosis resistance. Inhibition of TGFbeta expression led to an inhibition of growth, migration and invasion, in addition to a promotion of apoptosis, in ovarian carcinoma cells treated with paclitaxel. Studies have indicated that the LKB1SIK1 signaling pathway may be suppressed in ovarian carcinoma cells compared with normal ovarian cells, leading to activation of the EMT signaling pathway. The results of the present study demonstrated that upregulation of LKB1 promoted SIK1 expression and markedly suppressed the growth and aggressiveness of ovarian cancer cells. Upregulation of LKB1 additionally promoted apoptosis in ovarian carcinoma cells. In addition, the results of the present study demonstrated that the knockdown of LKB1 further promoted the expression of transforming growth factorbeta and EMT, which downregulated the chemosensitivity of ovarian carcinoma cells. Additionally, overexpression of LKB1 in ovarian carcinoma cells increased chemosensitivity, resulting in a significant inhibition of migration and invasion. The present findings indicated that the enhancement of LKB1SIK1 suppressed the growth and aggressiveness of ovarian carcinoma cells isolated from clinical patients, which subsequently contributed to an inhibition of metastatic potential. In conclusion, targeting the LKB1SIK1 signaling pathway to inhibit EMT may provide potential therapeutic benefits in ovarian carcinoma.

epithelial-mesenchymal transition (EMT) is a biological event in which epithelial cells lose their polarity and cell-cell adhesions and concomitantly acquire mesenchymal traits, and is thought to play an important role in pathological processes such as wound healing and cancer progression. In this study, we evaluated transforming growth factor (TGF)-beta1-treated human keratinocyte HaCaT cells as an in vitro model of EMT. HaCaT cells were changed into an elongated fibroblast-like morphology, which is indicative of EMT in response to TGF-beta1. Phalloidin staining demonstrated the formation of actin stress fibers in TGF-beta1-treated cells. Quantitative RT-PCR analysis revealed that TGF-beta1 increased the mRNA levels of EMT transcription factors (SNAI2, TWIST1, and ZEB1) and mesenchymal markers (CDH2, VIM, and FN1), while it decreased the transcripts of epithelial phenotypic genes (CLDN1, OCLN, KRT5, KRT15, KRT13, and TGM1). Furthermore, we found that KRT13 was drastically suppressed through the reduction of RNA polymerase II occupancy of its promoter, which was accompanied by a decrease in active histone marks (H3K4me3 and H3K27ac) and an increase in a repressive mark (H3K27me3) during EMT. These findings indicate that the TGF-beta1-induced EMT program regulates a subset of epithelial and mesenchymal marker genes, and that KRT13 is transcriptionally suppressed through the modulation of the chromatin state at the KRT13 promoter in HaCaT cells.CI - Copyright (c) 2018 Elsevier Inc. All rights reserved.