Epithelial-mesenchymal Transition In Response Research Articles

Klotho mitigates cyclosporine A (CsA)-induced epithelial-mesenchymal transition (EMT) and renal fibrosis in rats.

Klotho deficiency is implicated in various kidney diseases, including renal fibrosis. The aim of this study was to investigate the effect of Klotho administration on epithelial-mesenchymal transition (EMT) and renal fibrosis induced by cyclosporine A (CsA) in rats. CsA-induced renal fibrosis was established by oral administration of CsA (30mg/kg) to rats on a low-salt diet for 28days. Klotho was administered to rats by intraperitoneal injection. Renal pathological changes were evaluated by hematoxylin and eosin and Masson's trichrome staining. The EMT response was assessed by measuring the level of TGF-β1, E-cadherin and α-SMA by immunohistochemistry and Western blot. Administration of CsA for 28days induced renal damage, decreased Klotho expression and activated the EMT response (demonstrated as increased TGF-β1 and α-SMA expression accompanied by decreased in E-cadherin expression). Treatment with Klotho significantly ameliorated pathological lesions of the kidney by modulating the expression of EMT-associated proteins in the kidney. Klotho inhibits CsA-induced EMT and renal fibrosis in rats. Klotho may serve as a therapeutic agent to minimize CsA-induced renal fibrosis.

Transforming growth factor-β1 stimulates hedgehog signaling to promote epithelial-mesenchymal transition after kidney injury.

The epithelial-mesenchymal transition (EMT) of tubular epithelial cells (TECs) is crucial for the induction and progression of kidney fibrosis. However, the underlying molecular mechanisms that trigger the EMT programme have not been identified. In the present study, we demonstrate that transforming growth factor (TGF)-β1 and activated hedgehog signaling mediate the EMT programme following kidney injury. Tissue samples from fibrotic kidneys show enhanced TGF-β1 levels, as well as upregulated hedgehog signaling activity, during the EMT process; these levels decrease when fibrosis is reversed. Injury promotes TGF-β1 expression and activates hedgehog signaling, thus inducing tubular EMT of TECs and extracellular matrix (ECM) accumulation in vitro. The EMT response and fibrotic appearance are also induced by enhanced TGF-β1 levels or activated hedgehog signaling. Downregulation of TGF-β1 inhibits aristolochic acid (AA)- and TGF-β1-induced EMT and ECM synthesis and correlates with decreased hedgehog signaling. Similarly, inhibiting the hedgehog pathway abolishes AA- and hedgehog-mediated EMT, resulting in reduced TGF-β1 levels. These findings highlight a key role for cross-talk between TGF-β1 and hedgehog signaling in promoting injury-induced EMT and ECM deposition in TECs.

Mechanism of suppressors of cytokine signaling 1 inhibition of epithelial-mesenchymal transition signaling through ROS regulation in colon cancer cells: suppression of Src leading to thioredoxin up-regulation.

Reactive oxygen species (ROS) participate in malignant progression of cancers including epithelial-mesenchymal transition (EMT). We have investigated the role of suppressors of cytokine signaling (SOCS)1 as an inhibitor of ROS-induced EMT using colon cancer cell lines transduced with SOCS1 and shSOCS1. Hydrogen peroxide treatment induced EMT features such as elevation of vimentin and Snail with a corresponding reduction of E-cadherin. The EMT markers are significantly decreased upon SOCS1 over-expression while increased under SOCS1 knock-down. SOCS1 inhibited ROS signaling pathways associated with EMT such as Src, Jak, and p65. Of note, strong up-regulation of Src activity in SOCS1-ablated cells was responsible for the elevated signaling leading to EMT, as shSrc or Src inhibitor abolished the shSOCS1-induced promotion of EMT response. Suppression of ROS-inducible EMT markers and invasion in SOCS1 over-expressing cells correlated with significantly low intracellular ROS levels in these cells. Analysis of antioxidant enzymes in SOCS1-transduced cells revealed a selective up-regulation of thioredoxin (Trx1), while thioredoxin ablation restored ROS levels and the associated EMT markers. As a mechanism of thioredoxin up-regulation by SOCS1, inhibition of Src activity promoting nuclear translocation of Nrf-2 is proposed. Taken together, our data strongly indicate that SOCS1 antagonizes EMT by suppressing Src activity, leading to thioredoxin expression and down-regulation of ROS levels in colon cancer cells.

Abstract IA24: Forcing through tumor metastasis: Regulation of epithelial-mesenchymal plasticity by matrix stiffness

Abstract In cancer, the presence of dense clusters of collagen fibrils indicates increased matrix stiffness and correlates with poor survival. Previous studies have identified a functional role for matrix stiffness in driving tumor progression, but it is unclear how mechanical inputs are transduced into transcriptional outputs to drive tumor metastasis. To investigate mechanisms of tumor cell mechanotransduction, we utilized a 3D Matrigel culture of murine (Eph4Ras) or human (MCF10A, MCF10DCIS, Bt-549) mammary epithelial cells coupled with a polyacrylamide hydrogel base with calibrated elastic moduli ranging between ~150Pa of normal mammary gland and ~5700Pa of breast tumor tissues. This approach allows for the interrogation of the effects of changes in matrix stiffness independent of other biochemical variables in extracellular matrix (ECM). Using this system, we show that increasing matrix stiffness induced morphological changes resembling the Epithelial-Mesenchymal Transition (EMT) program. Therefore, we hypothesized that increasing matrix stiffness could directly signal through EMT-inducing transcription factors to induce EMT and promote invasion. Using a combination of bioengineering, cell biollogy, proteomic, imaging, and murine tumor modeling approaches, we found that the EMT-inducing transcription factor TWIST1 is an essential mechanomediator that promotes EMT in response to increasing matrix stiffness. Loss of Twist1 prevents matrix stiffness-induced EMT and tumor cell invasion. TGF-beta signaling, a major EMT-inducing biochemical signal, acts synergistically with increasing matrix stiffness, the mechanical cure to induce a complete EMT program in a Twist1-dependent manner. At the compliant matrix stiffness, TWIST1 is sequestered in the cytoplasm by its cytoplasmic binding partner G3BP2, thus preventing TWIST1 from accessing DNA to drive EMT. High matrix stiffness promotes nuclear translocation of TWIST1 by releasing TWIST1 from G3BP2. Loss of G3BP2 leads to constitutive TWIST1 nuclear localization and synergizes with increasing matrix stiffness to induce EMT and invasion in a Twist1-dependent manner. In MCF10DCIS breast tumor xenografts, loss of G3BP2 promotes local invasion and distant metastasis in the lung. In human stage-3 breast tumors, collagen fiber alignment, a marker of increasing matrix stiffness, and reduced expression of G3BP2 together predict poor survival. Together, our findings reveal a TWIST1-G3BP2 mechanotransduction pathway that responds to biomechanical signals from the tumor microenvironment to drive EMT, invasion and metastasis. Citation Format: Jing Yang. Forcing through tumor metastasis: Regulation of epithelial-mesenchymal plasticity by matrix stiffness. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr IA24.

DAB2IP regulates EMT and metastasis of prostate cancer through targeting PROX1 transcription and destabilizing HIF1α protein

Prospero-related homeobox 1 (PROX1) is an essential regulator in lymphangiogenesis and has been implicated in both oncogenic and tumor-suppressive functions in many types of human cancers. However, the role of PROX1 in prostate cancer (PCa) remains poorly understood. In this study, based on different PCa cell lines and knockout mice, we showed that PROX1 could be suppressed by DAB2IP, a novel member of the Ras GTPase-activating protein family and a critical player in control of epithelial-mesenchymal transition (EMT) and PCa metastasis. Mechanistically, PROX1 overexpression in DAB2IP-deficient PCa cells could enhance the accumulation of HIF1α protein by inhibiting ubiquitin pathway and then consequently induce an EMT response, which is characterized by repression of E-cadherin, up-regulation of vimentin and matrix metallopeptidases (MMPs) and enhancement of cell migration. Together, our data provides a new insight into mechanism that DAB2IP regulates EMT and PCa metastasis, especially points out the potential roles of its downstream PROX1/HIF1α signaling in a unique non-skeletal metastasis of PCa.

Abstract 4426: The PEAK1/ZEB1/ITGA1 pathway mediates survival, stemness and TGFbeta-induced EMT in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States with a five-year survival rate of approximately 7%. Its highly invasive characteristics, lack of biomarkers for early detection, limited therapeutic targets and inherent resistance to chemotherapy are the central factors that lead to an overall poor prognosis for this malignancy. Since most cancer-related deaths are a result of metastatic tumors, we reasoned that our previously published proteomic signature of the cell pseudopodium (PD) may be a good pool from which to identify novel protein biomarkers or therapeutic targets in pancreatic cancer. We cross-referenced PD enriched proteins with Oncomine and identified 37 genes that are upregulated in pancreatic cancer. The function of integrin alpha 1 (ITGA1), a cell surface receptor for collagen and regulator of cell adhesion, has not been previously characterized in the context of PDAC. Our results indicate that ITGA1 is required for PDAC cell viability, promoting cell survival and tumor growth. We further analyzed ITGA1 cell-surface levels and discovered that ITGA1hi cells represent a subpopulation of Aldehyde Dehyrogenase high (ALDH1hi) cells, and that ITGA1 knockdown reduces the ALDH1hi cell population. One mechanism by which PDAC cells acquire metastatic potential is via epithelial-mesenchymal transition (EMT). Thus, we searched for prominent EMT genes among the top ITGA1 co-expressors using RNA-Seq data from the Cancer Bio Portal. Interestingly, ZEB1 (a transforming growth factor beta, TGFbeta, response gene and central regulator of EMT) and PEAK1 kinase (a protein we’ve shown to be required for pancreatic cancer progression and TGFbeta/ZEB1-induced EMT) co-expresses with ITGA1. We tested the function of ITGA1 on TGFbeta-induced EMT and found that ITGA1 is upregulated by TGFbeta during EMT, and required for metastasis and a complete EMT response to TGFbeta. We also demonstrate that PEAK1 is required for TGFbeta-induced ITGA1 in PDAC cells. Finally, we demonstrate that ITGA1 is responsible for collagen-induced spreading and an EMT-like shift in PDAC cell morphology. Taken together, these results point to a novel TGFbeta/PEAK1/ZEB1/ITGA1 signaling cascade that when targeted can abrogate PDAC cell survival, stemness, EMT and tumor growth/metastasis. Citation Format: Armen Gharibi, Sa La Kim, Daniel Brambilla, Yvess Adamian, Malachia Hoover, Joy Lin, Megan Agajanian, Laurelin Wolfenden, Jonathan A. Kelber. The PEAK1/ZEB1/ITGA1 pathway mediates survival, stemness and TGFbeta-induced EMT in pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4426.

Tumor suppressor bromodomain-containing protein 7 cooperates with Smads to promote transforming growth factor-β responses.

Smad proteins are central mediators in the canonical transforming growth factor-β (TGF-β) signaling pathway in mammalian cells. We report here that bromodomain-containing protein 7 (BRD7) functions as a novel transcription coactivator for Smads in TGF-β signaling. BRD7 forms a TGF-β inducible complex with Smad3/4 through its N-terminal Smad-binding domain. BRD7 simultaneously binds to acetylated histones to promote Smad-chromatin association, and associates with histone acetyltransferase p300 to enhance Smad transcriptional activity. Ectopic expression of BRD7, but not its mutants defective in Smad binding, enhances TGF-β transcriptional, tumor suppressing and epithelial-mesenchymal transition (EMT) responses. Conversely, depletion of BRD7 inhibits TGF-β responses. Thus, our study provides compelling evidence for a new function of BRD7 in fine-tuning TGF-β physiological responses.

AGE-RAGE interaction in the TGFβ2-mediated epithelial to mesenchymal transition of human lens epithelial cells.

Basement membrane (BM) proteins accumulate chemical modifications with age. One such modification is glycation, which results in the formation of advanced glycation endproducts (AGEs). In a previous study, we reported that AGEs in the human lens capsule (BM) promote the TGFβ2-mediated epithelial-to-mesenchymal transition (EMT) of lens epithelial cells, which we proposed as a mechanism for posterior capsule opacification (PCO) or secondary cataract formation. In this study, we investigated the role of a receptor for AGEs (RAGE) in the TGFβ2-mediated EMT in a human lens epithelial cell line (FHL124). RAGE was present in FHL124 cells, and its levels were unaltered in cells cultured on either native or AGE-modified BM or upon treatment with TGFβ2. RAGE overexpression significantly enhanced the TGFβ2-mediated EMT responses in cells cultured on AGE-modified BM compared with the unmodified matrix. In contrast, treatment of cells with a RAGE antibody or EN-RAGE (an endogenous ligand for RAGE) resulted in a significant reduction in the TGFβ2-mediated EMT response. This was accompanied by a reduction in TGFβ2-mediated Smad signaling and ROS generation. These results imply that the interaction of matrix AGEs with RAGE plays a role in the TGFβ2-mediated EMT of lens epithelial cells and suggest that the blockade of RAGE could be a strategy to prevent PCO and other age-associated fibrosis.

Effect of Cigarette Smoking on Epithelial to Mesenchymal Transition (EMT) in Lung Cancer.

Epithelial to mesenchymal transition (EMT) is a process that allows an epithelial cell to acquire a mesenchymal phenotype through multiple biochemical changes resulting in an increased migratory capacity. During cancer progression, EMT is found to be associated with an invasive or metastatic phenotype. In this review, we focus on the discussion of recent studies about the regulation of EMT by cigarette smoking. Various groups of active compounds found in cigarette smoke such as polycyclic aromatic hydrocarbons (PAH), nicotine-derived nitrosamine ketone (NNK), and reactive oxygen specicies (ROS) can induce EMT through different signaling pathways. The links between EMT and biological responses to cigarette smoke, such as hypoxia, inflammation, and oxidative damages, are also discussed. The effect of cigarette smoke on EMT is not only limited to cancer types directly related to smoking, such as lung cancer, but has also been found in other types of cancer. Altogether, this review emphasizes the importance of understanding molecular mechanisms of the induction of EMT by cigarette smoking and will help in identifying novel small molecules for targeting EMT induced by smoking.

Abstract A30: AKT activation as a mechanism of escape from TGFβ-mediated lethal EMT

Abstract Frequent inactivation of transforming growth factor β (TGFβ) pathway core components in pancreatic ductal adenocarcinoma (PDA), especially the transcription factor SMAD4, suggests that TGFβ signaling is tumor suppressive in the pancreas. Yet, 50% of PDA retains an intact core TGFβ pathway, suggesting that there are mechanisms by which these tumors escape TGFβ-mediated tumor suppression. In unpublished work, we have delineated a mechanism for TGFβ-mediated tumor suppression via a lethal epithelial-mesenchymal transition (EMT). Based on clinical and in vitro evidence that AKT activation may suppress the lethal EMT response to TGFβ, we are investigating the role of AKT signaling in directly interfering with TGFβ-mediated tumor suppression. We have taken two major approaches: (1) to test the concept that AKT inhibition may reactivate TGFβ-mediated tumor suppression, we are chemically inhibiting AKT in PDA mouse models competent and deficient in TGFβ signaling, and (2) to understand the mechanism of AKT-TGFβ crosstalk in PDA progression, we are performing RNAseq-based gene expression profiling of SMAD4-restored PDA cell lines. We have found that in vitro, AKT signaling determines whether TGFβ induces apoptosis in SMAD4-restored PDA cell lines, and by gene expression analysis, we have identified a group of genes that are differentially regulated by TGFβ in the presence and absence of AKT signaling. Furthermore, we have characterized FOXO immunohistochemical staining as a reliable marker for AKT inhibition status and developed an in vivo system in which we are testing whether AKT inhibition may be a means to reactivate TGFβ-mediated tumor suppression in PDA. Our studies indicate that AKT signaling may alter the outcome of TGFβ-mediated tumor suppression in the pancreas, and our ongoing work is aimed at dissecting the AKT-modulated transcriptional mechanisms that determine the outcome of TGFβ signaling and the consequences of this crosstalk in tumor progression. Citation Format: Yun-Han Huang, Charles David, Joan Massague. AKT activation as a mechanism of escape from TGFβ-mediated lethal EMT. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A30.

TGFβ signaling regulates epithelial-mesenchymal plasticity in ovarian cancer ascites-derived spheroids.

Epithelial-mesenchymal transition (EMT) serves as a key mechanism driving tumor cell migration, invasion, and metastasis in many carcinomas. Transforming growth factor-beta (TGFβ) signaling is implicated in several steps during cancer pathogenesis and acts as a classical inducer of EMT. Since epithelial ovarian cancer (EOC) cells have the potential to switch between epithelial and mesenchymal states during metastasis, we predicted that modulation of TGFβ signaling would significantly impact EMT and the malignant potential of EOC spheroid cells. Ovarian cancer patient ascites-derived cells naturally underwent an EMT response when aggregating into spheroids, and this was reversed upon spheroid re-attachment to a substratum. CDH1/E-cadherin expression was markedly reduced in spheroids compared with adherent cells, in concert with an up-regulation of several transcriptional repressors, i.e., SNAI1/Snail, TWIST1/2, and ZEB2. Treatment of EOC spheroids with the TGFβ type I receptor inhibitor, SB-431542, potently blocked the endogenous activation of EMT in spheroids. Furthermore, treatment of spheroids with SB-431542 upon re-attachment enhanced the epithelial phenotype of dispersing cells and significantly decreased cell motility and Transwell migration. Spheroid formation was significantly compromised by exposure to SB-431542 that correlated with a reduction in cell viability particularly in combination with carboplatin treatment. Thus, our findings are the first to demonstrate that intact TGFβ signaling is required to control EMT in EOC ascites-derived cell spheroids, and it promotes the malignant characteristics of these structures. As such, we show the therapeutic potential for targeted inhibition of this pathway in ovarian cancer patients with late-stage disease.

The Effect of Bee Venom on Animal Model of Unilateral Ureteral Obstruction

Progressive renal fibrosis is the final common pathway for kidney diseases leading to chronic renal failure. Epithelial-mesenchymal transition (EMT) is known to be the critical mechanisms of the development and aggravation of chronic kidney injury. In this study, we examined the therapeutic effects of bee venom (BV) on the progression of renal fibrosis using the unilateral ureteral obstruction (UUO) model. UUO group increased the expression of fibrosis-related genes including FSP-1, PAI-1 and Vimentin, whereas these expressions were significantly decreased by BV treatment. The number of cells positive for ILK was increased in UUO mice, but decreased by BV treatment. In addition, β-catenin, and Snail1 expression was increased in UUO mice, while this effect was significantly decreased with BV treatment. The UUO operation significantly induced expression of Vimentin, and inhibited expression of E-cadherin compared to normal kidney. However, BV treatment markedly decreases Vimentin expression, and increases E-cadherin expression. In summary, these findings suggest that BV attenuate renal fibrosis and reduce EMT responses by suppression of pro-fibrotic genes, and EMT-related genes.

Targeting epithelial-mesenchymal transition.

The epithelial–mesenchymal transition (EMT) is a process in which epithelial cells dedifferentiate to become mesenchymal cells [1–3]. The process requires them to lose their cell polarity, to relinquish their cell–cell adhesion, and to gain migratory and invasive properties. The mesenchymal cells are multipotent stromal cells (mesenchymal stem cells) that can differentiate into various cell types. EMT is essential for the developing embryo to enable mesoderm and neural tube formation. EMT is also necessary for wound healing and other physiological processes. On the other hand, EMT can also be an initiator for metastasis, cancer progression, organ fibrosis, and other pathological conditions. These are disorderly processes. The induction of EMT is complex (Fig. 1) [4]. A fundamental event appears to be the loss of E-cadherin. Transcription factors that can repress E-cadherin directly or indirectly are important to this process. SNAI1/Snail 1, SNAI2/Snail 2 (also known as Slug), ZEB1, ZEB2, E47, and Kruppel-like factor-8 (KLF8) can repress E-cadherin transcription by binding to its promoter. Indirect repressors, such as Twist, Goosecoid, TCF4 (sometimes referred to as immunoglobulin transcription factor 2), homeobox protein SIX1, and FOXC2 (fork-head box protein C2) are able to repress E-cadherin indirectly [5]. Other junction proteins, such as claudins and desmosomes, are also suppressed, facilitating EMT. Various transcription factors such as grainyhead-like protein 2 homologue (GRHL2) and ETS-related transcription factors ELF3 and ELF5 are downregulated during EMT [6, 7]. The induction of EMT is dependent on numerous signaling pathways including TGF-beta, FGF, EGF, HGF, Wnt/beta-catenin, Notch, and hypoxia-induced mechanisms. Ras-MAPK signaling activates Snail and Slug during EMT induction. Slug is involved in disrupting desmosomes, inducing cell spreading, and separating the cell–cell borders [8]. However, EMT involves a second phase that cannot be triggered by Slug. This phase includes the induction of cell motility, repression of the cytokeratin expression, and activation of vimentin expression [9]. Phosphatidylinositol 3′ kinase (PI3K)/AKT-axis activation is emerging as pivotal to EMT. Similarly, hedgehog, nuclear factorkappaB (NF-κB), and activating transcription factor-2 have been implicated [10]. Of course, the EMT processes are specific, so that while Wnt signaling pathways regulate EMT in embryonic development, cardiac valve formation, and cancer, other pathways are important towound healing and fibrosis [11]. EMT is central to two recent J Mol Med contributions. Kimura et al. report on the attenuation of EMT in retinal pigment epithelial cells and subretinal fibrosis by a retinoic-acid receptor-gamma (RAR-γ) agonist [12]. The retinal pigment epithelium belongs the neural retina and is indispensable for vision. In humans, proliferation and transformation (EMT switching) of retinal pigment epithelial cells after injury causes retinal disorders and loss of vision [13]. On the other hand, EMT could develop into a therapeutic target [14]. Subretinal fibrosis contributes to the loss of vision associated with age-related macular degeneration (AMD), a devastating and common disease in older people. The authors began with retinal epithelial cells in culture. When subjected to TGF-β2, the cells expressed mesenchymal markers including α-smooth muscle actin and fibronectin. The cells also released interleukin-6. Paxillin was phosphorylated; MAPK constituents such as ERK, p38, and JNK were activated, as were Smad2 and AKT. A RAR-γ agonist, R667, attenuated the EMT responses. Furthermore, the cells treated * Friedrich C. Luft luft@charite.de

Negative feedback loop between p66Shc and ZEB1 regulates fibrotic EMT response in lung cancer cells.

The epithelial-to-mesenchymal transition (EMT) program is crucial for the epithelial cancer progression and fibrotic diseases. Our previous work has demonstrated that p66Shc, a focal adhesion-associated adaptor protein, is frequently downregulated in lung cancers and its depletion promotes metastasis behavior through anoikis resistance. However, mechanism underlying loss of p66Shc and EMT response is not fully understood. Here, we showed that p66Shc deficiency enhanced the expression of ZEB1, the known mesenchymal transcription factor and consequently increased Vimentin, and decreased epithelial markers of E-cadherin and β-catenin. p66Shc depletion also increased cell invasion and migration. In addition, ChIP and luciferase assays showed that these effects were directly mediated by ZEB1 repression of p66Shc promoter. Thus, our findings define a critical role of p66Shc in the suppression of fibrotic EMT response with a negative feedback loop between p66Shc and ZEB1 in lung epithelial cancer cells.

Role of TGF-β signaling in uterine carcinosarcoma.

Uterine carcinosarcomas (UCS) are rare (3-4%) but highly aggressive, accounting for a disproportionately high (16.4%) mortality among uterine malignancies. Transforming growth factor beta (TGFβ) is a multifunctional cytokine that regulates important cellular processes including epithelial-mesenchymal transition (EMT). Existence of biphasic elements and a report demonstrating amplification of TGFβ at 19q13.1 prompted us to investigate the role of TGFβ signaling in UCS.Here we demonstrated the components of TGFβ pathway are expressed and functional in UCS. TGFβ-I induced significant Smad2/3 phosphorylation, migration and EMT responses in UCS cell lines which could be attenuated by the TGFβ receptor I (TGFβR-I) or TGFβ receptor I/II (TGFβR-I/II) inhibitor developed by Eli Lilly and company. Importantly, TGFβ-I induced proliferation was c-Myc dependent, likely through activation of cell cycle. c-Myc was induced by nuclear translocation of nuclear factor of activated T cells (NFAT-1) in response to TGFβ-I. Inhibition of NFAT-1 or TGFβR-I blocked c-Myc induction, cell cycle progression and proliferation in UCS. In corroboration, mRNA levels of c-Myc were elevated in recurrent versus the non-recurrent UCS patient samples. Interestingly, in the absence of exogenous TGFβ the TGFβR-I/II inhibitor enhanced proliferation likely through non-Smad pathways. Thus, inhibition of TGFβR-I could be efficacious in treatment of UCS.

EGF-reduced Wnt5a transcription induces epithelial-mesenchymal transition via Arf6-ERK signaling in gastric cancer cells.

Wnt5a, a ligand for activating the non-canonical Wnt signaling pathway, is commonly associated with Epithelial-to-mesenchymal transition (EMT) in cancer cell metastasis. Here, we show that downregulation of Wnt5a mRNA and protein by EGF is necessary for EGF-induced EMT in gastric cancer SGC-7901 cells. To further explore the mechanisms, we investigated the effect of EGF signaling on Wnt5a expression. EGF increased Arf6 and ERK activity, while blockade of Arf6 activation repressed ERK activity, up-regulated Wnt5a expression and repressed EMT in response to EGF. We also demonstrate that EGF inactivated Wnt5a transcription by direct recruitment of ERK to the Wnt5a promoter. On the other hand, inhibition of ERK phosphorylation resulted in decreased movement of ERK from the cytoplasm to the nucleus, following rescued Wnt5a mRNA and protein expression and favored an epithelial phenotype of SGC-7901 cells. In addition, we notice that kinase-dead, nuclear-localised ERK has inhibitory effect on Wnt5a transcription. Analysis of gastric cancer specimens revealed an inverse correlation between P-ERK and Wnt5a protein levels and an association between Wnt5a expression and better prognosis. These findings indicate that Wnt5a is a potential suppressor of EMT and identify a novel Arf6/ERK signaling pathway for EGF-regulated Wnt5a expression at transcriptional level of gastric cancer cells.

Bisphenol A and nonylphenol have the potential to stimulate the migration of ovarian cancer cells by inducing epithelial-mesenchymal transition via an estrogen receptor dependent pathway.

Epithelial-mesenchymal transition (EMT) is an important process appearing in embryo development and tumor migration or progression, which is influenced by 17β-estradiol (E2). Bisphenol A (BPA) and nonylphenol (NP) are suspected as endocrine disrupting chemicals (EDCs) because they can exert estrogenic properties. In this study, we examined whether E2, BPA, and NP can lead to the EMT process in BG-1 ovarian cancer cells expressing estrogen receptors (ERs). To confirm the effect of E2, BPA, and NP, BG-1 cells were cultured under treatment with E2, BPA, or NP, and the alteration of EMT markers such as vimentin was examined at mRNA levels by using real-time and reverse-transcription (RT)-PCR. The expressions of snail, slug, and vimentin were enhanced by the treatment of E2, BPA, or NP compared to a control (DMSO). In protein levels, vimentin protein was increased by E2 and two EDCs, while E-cadherin was decreased. In addition, the expression of snail protein was enhanced by the treatment of E2 and the two EDCs in comparison with that of the control. Since EMT response in cancer cells can affect metastasis, we also performed a scratch assay and Western blot assay to show the migration ability caused by E2, BPA, or NP. Consequently, E2, BPA, and NP enhanced the migration capability of BG-1 cells and increased the expression of MMP-9 protein. Furthermore, to examine whether EMT and migration of BG-1 cancer cells are induced by BPA or NP via the ER dependent pathway, we cotreated the cells with ER-antagonist, ICI 182,780, in the presence of E2, BPA, or NP. As a result, the expressions of E-cadherin, vimentin, snail, and slug were reversed following treatment with an ER antagonist. Moreover, we confirmed that ICI 182,780 reduced the migration ability of BPA and NP to the control level. Taken together, these results indicate that BPA and NP, the potential EDCs, may have the ability to influence ovarian cancer metastasis via regulating EMT markers and migration in ER-expressing BG-1 ovarian cancer cells.

Mast cells induce epithelial-to-mesenchymal transition and stem cell features in human thyroid cancer cells through an IL-8-Akt-Slug pathway.

There is increasing evidence that mast cells (MCs) and their mediators are involved in the remodeling of the tumor microenvironment and promote tumor growth, angiogenesis and metastasis. We have found that an increased density of MCs in thyroid cancer (TC) correlates with enhanced invasiveness. However, the MC-derived factors responsible for this activity and the mechanisms by which they enhance TC invasiveness remain unidentified. Here, we report that MCs, when activated by TC cells, produce soluble factors that induce epithelial-to-mesenchymal transition (EMT) and stemness features of TC cells. We identified CXCL8/interleukin (IL)-8 as the main mediator contained in activated MC conditioned media (CM) capable of inducing both EMT and stemness of TC cells. Mechanistically, MC CM or exogenous IL-8 stimulated Akt phosphorylation and Slug expression in TC cells. The inhibition of the Akt pathway or depletion of the Slug transcription factor by RNA interference, reverted EMT and stemness responses. TC cells stably transfected with exogenous IL-8 underwent EMT, displayed increased stemness and enhanced tumorigenicity with respect to control cells. The analysis of TC surgical specimens by immunohistochemical analysis demonstrated a positive correlation between MC density (Tryptase(+) cells) and stemness features (OCT4 staining). Taken together, our data identify an MC-dependent IL-8-Akt-Slug pathway that sustains EMT/stemness of TC cells. The blockade of this circuit might be exploited for the therapy of advanced TC.

EGF augments TGFβ-induced epithelial-mesenchymal transition by promoting SHP2 binding to GAB1.

In many epithelial cells, epidermal growth factor (EGF) augments the epithelial-mesenchymal transition (EMT) that occurs when cells are treated with transforming growth factor β (TGFβ). We demonstrate that this augmentation requires activation of SH2 domain-containing phosphatase-2 (SHP2; also known as PTPN11), a proto-oncogene. In lung and pancreatic cancer cell lines, reductions in E-cadherin expression, increases in vimentin expression and increases in cell scatter rates were larger when cells were treated with TGFβ and EGF versus TGFβ or EGF alone. SHP2 knockdown promoted epithelial characteristics basally and antagonized EMT in response to TGFβ alone or in combination with EGF. Whereas EGF promoted SHP2 binding to tyrosine phosphorylated GAB1, which promotes SHP2 activity, TGFβ did not induce SHP2 association with phosphotyrosine-containing proteins. Knockdown of endogenous SHP2 and reconstitution with an SHP2 mutant with impaired phosphotyrosine binding ability eliminated the EGF-mediated EMT augmentation that was otherwise restored with wild-type SHP2 reconstitution. These results demonstrate roles for basal and ligand-induced SHP2 activity in EMT and further motivate efforts to identify specific ways to inhibit SHP2, given the role of EMT in tumor dissemination and chemoresistance.

215 SNAIL AND SLUG, MARKERS OF EPITHELIAL MESENCHYMAL TRANSITION, APPEARED TO BE ALTERED BY ALKYL-PHENOLS, BISPHENOL A AND NONYL-PHENOL, IN OVARIAN CANCER CELLS EXPRESSING ESTROGEN RECEPTORS

The ovary is the important organ to produce oocytes. Any disorder will affect embryo production. Ovarian cancer is one of gynecologic cancers in women which can affect ovarian functions. Oestradiol (E2) may be involved in ovarian cell growth and epithelial-mesenchymal transition (EMT) for diverse functions. EMT is an important process in embryo development and tumour migration or progression. Bis-phenol A (BPA) and nonyl-phenol (NP) have an estrogenic property, which can be suspected as endocrine disrupting chemicals (EDC). In this study, it has been examined whether BPA and NP can cause EMT process and migration in BG-1 ovarian cancer cells. To confirm the effect of these EDCs, BG-1 ovarian cancer cells were cultured and treated with DMSO (0.1%), E2 (10–7 M), BPA (10–6 M) and NP (10–6 M) for 0, 6, and 24 h. The mRNAs were extracted to perform reverse-transcription (RT)-PCR and the changes in the mRNA expressions were analysed by ANOVA test. Following treatments with BPA and NP, alterations of EMT markers; that is, vimentin and E-cadherin, were examined at mRNA levels by RT-PCR. The levels of vimentin were up-regulated by E2, BPA, or NP in a time-dependent manner. In addition, transcriptional factors of EMT response, i.e. snail and slug, were enhanced by these treatments more than 2 times. BG-1 cells were exposed to these EDCs for 0, 24, and 48 h. Vimentin and snail proteins were induced by E2, BPA, or NP, while the expression of E-cadherin was decreased by them. To reveal that this EMT response is affected by oestrogen receptor (ER), the cells were treated with these EDCs in the presence of an ER antagonist, ICI 182 780 (10–6 M). Treatment with ICI 182 780 reversed EDC-induced alteration of these EMT markers, E-cadherin, vimentin, and snail. Since EMT response can cause metastasis, a scratch assay was performed to show migration caused by BPA or NP. BPA or E2 enhanced migratory capability of these BG-1 cells. Taken together, these results indicate that BPA and NP, potential EDC, may have an ability to influence ovarian cancer metastasis via regulating snail and slug genes in ER-positive ovarian cancers. In a future study, their effects in inducing EMT and migration will be tested in a xenograft mouse model.This work was supported by a grant from the Next-Generation BioGreen 21 Program (no. PJ009599), Rural Development Administration, Republic of Korea.