EMT-related Changes Research Articles

Troglitazone Attenuates TGF-β1-Induced EMT in Alveolar Epithelial Cells via a PPARγ-Independent Mechanism

Peroxisome proliferator activated receptor γ (PPARγ) agonists are effective antifibrotic agents in a number of tissues. Effects of these agents on epithelial-mesenchymal transition (EMT) of primary alveolar epithelial cells (AEC) and potential mechanisms underlying effects on EMT have not been well delineated. We examined effects of troglitazone, a synthetic PPARγ agonist, on transforming growth factor (TGF)-β1-induced EMT in primary rat AEC and an alveolar epithelial type II (AT2) cell line (RLE-6TN). TGF-β1 (2.5 ng/mL) induced EMT in both cell types, as evidenced by acquisition of spindle-like morphology, increased expression of the mesenchymal marker α-smooth muscle actin (α-SMA) and downregulation of the tight junctional protein zonula occludens-1 (ZO-1). Concurrent treatment with troglitazone (or rosiglitazone), ameliorated effects of TGF-β1. Furthermore, following stimulation with TGF-β1 for 6 days, troglitazone reversed EMT-related morphological changes and restored both epithelial and mesenchymal markers to control levels. Treatment with GW9662 (an irreversible PPARγ antagonist), or overexpression of a PPARγ dominant negative construct, failed to inhibit these effects of troglitazone in AEC. Troglitazone not only attenuated TGF-β1-induced phosphorylation of Akt and glycogen synthase kinase (GSK)-3β, but also inhibited nuclear translocation of β-catenin, phosphorylation of Smad2 and Smad3 and upregulation of the EMT-associated transcription factor SNAI1. These results demonstrate inhibitory actions of troglitazone on TGF-β1-induced EMT in AEC via a PPARγ-independent mechanism likely through inhibition of β-catenin-dependent signaling downstream of TGF-β1, supporting a role for interactions between TGF-β and Wnt/β-catenin signaling pathways in EMT.

IL-6 Promotes Head and Neck Tumor Metastasis by Inducing Epithelial–Mesenchymal Transition via the JAK-STAT3-SNAIL Signaling Pathway

Epithelial-mesenchymal transition (EMT) is a key process in tumor metastatic cascade that is characterized by the loss of cell-cell junctions and cell polarity, resulting in the acquisition of migratory and invasive properties. However, the precise molecular events that initiate this complex EMT process in head and neck cancers are poorly understood. Increasing evidence suggests that tumor microenvironment plays an important role in promoting EMT in tumor cells. We have previously shown that head and neck tumors exhibit significantly higher Bcl-2 expression in tumor-associated endothelial cells and overexpression of Bcl-2 alone in tumor-associated endothelial cells was sufficient to enhance tumor metastasis of oral squamous cell carcinoma in a severe combined immunodeficient (SCID) mouse model. In this study, we show that endothelial cells expressing Bcl-2 (EC-Bcl-2), when cocultured with head and neck tumor cells (CAL27), significantly enhance EMT-related changes in tumor cells predominantly by the secretion of IL-6. Treatment with recombinant IL-6 or stable IL-6 overexpression in CAL27 cells or immortalized oral epithelial cells (IOE) significantly induced the expression of mesenchymal marker, vimentin, while repressing E-cadherin expression via the JAK/STAT3/Snail signaling pathway. These EMT-related changes were further associated with enhanced tumor and IOE cell scattering and motility. STAT3 knockdown significantly reversed IL-6-mediated tumor and IOE cell motility by inhibiting FAK activation. Furthermore, tumor cells overexpressing IL-6 showed marked increase in lymph node and lung metastasis in a SCID mouse xenograft model. Taken together, these results show a novel function for IL-6 in mediating EMT in head and neck tumor cells and increasing their metastatic potential.

Celecoxib inactivates epithelial–mesenchymal transition stimulated by hypoxia and/or epidermal growth factor in colon cancer cells

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, has been reported to exert chemopreventive and antitumor effects on colon cancer, one of the most common solid epithelial malignancy worldwide. The aim of this study was to elucidate whether celecoxib may be able to affect epithelial-mesenchymal transition (EMT), a critical process involved in cancer cell invasiveness and metastasis and then proposed to be relevant for cancer progression. Human HT-29 colon cancer cells were exposed to carefully controlled hypoxic conditions and/or epidermal growth factor (EGF) and then investigated for EMT changes and signal transduction pathways involved by using morphological, molecular, and cell biology techniques. Celecoxib inhibited basal and EGF-stimulated proliferation, hypoxia-related HIF-1α recruitment/stabilization as well as hypoxia- and EGF-dependent activation of ERK and PI3K. Interestingly, celecoxib prevented EMT-related changes, as shown by modifications of β-catenin intracellular localization or vimentin and E-cadherin levels, as well as HT-29 invasiveness induced by hypoxia, EGF, or hypoxia plus EGF. Finally, experiments performed on SW-480 colon cancer cells (i.e., cells lacking COX-2) exposed to hypoxia, used here as a stimulus able to induce EMT and invasiveness, revealed that in these cells celecoxib was ineffective. Results of the present study indicate that celecoxib has the potential to negatively affect induction of EMT and increased invasiveness of colon cancer cells as elicited by different signals originating from tumor microenvironment (i.e., hypoxia and EGF). Moreover, these effects are likely be related to the pharmacological inhibitory effect exerted on COX-2 activity.

Abstract 3362: IL-6 promotes head and neck tumor metastasis by inducing epithelial-mesenchymal transition via the JAK-STAT3-SNAIL signaling pathway

Abstract Distant metastases in head and neck cancer patients almost invariably herald a poor prognosis. Five year survival rates for early stage localized head and neck cancers are over 80% but this drop to 40% where disease has spread to neck nodes, and to below 20% for patients with distant metastatic disease. Uncontrolled cell proliferation and angiogenesis are key characteristics of initiation and early growth of epithelial origin cancers. However, it is the subsequent acquisition of motility and invasiveness that leads to metastatic dissemination of these tumor cells. A key process in this metastatic cascade that converts an adherent epithelial cell into a migratory cell which can invade through the extracellular matrix is known as epithelial-mesenchymal transition (EMT). EMT plays a critical role in epithelial tumor cell metastasis and is commonly observed in tumor samples from head and neck cancer patients. However, the precise molecular events that initiate this complex process of EMT in head and neck cancers are poorly understood. There is increasing evidence that suggest an important role of tumor microenvironment in promoting EMT changes in tumor cells. We have previously shown that head and neck tumors exhibit significantly higher levels of Bcl-2 expression in tumor associated endothelial cells and overexpression of Bcl-2 alone in tumor-associated endothelial cells was sufficient to enhance tumor metastasis of oral squamous cell carcinoma in a SCID mouse model. In this study, we show that endothelial cells expressing Bcl-2 (EC-Bcl-2) when co-cultured with head and neck tumor cells significantly enhance EMT-related changes in tumor cells and this EC-Bcl-2-induced EMT in tumor cells is mediated predominantly by IL-6 secretion. Treatment with recombinant IL-6 or stable IL-6 overexpression in CAL27 cells or normal oral epithelial cells (NOE) significantly induced the expression of vimentin while repressing E-cadherin expression via the JAK/STAT3/Snail signaling pathway. This phenotype was further associated with enhanced tumor and NOE cell scattering and motility. STAT3 knock-down significantly reversed IL-6-mediated tumor and NOE cell motility by inhibiting FAK activation. Furthermore, tumor cells overexpressing IL-6 showed marked increase in lymph node and lung metastasis, in vivo. These results suggest a novel function for IL-6 in mediating head and neck tumor EMT and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3362. doi:10.1158/1538-7445.AM2011-3362

Abstract 3424: Slug-mediated epithelial mesenchymal transition in lung cancer cells

Abstract Epithelial-Mesenchymal Transition (EMT) has been classified as a unique process by which epithelial cells undergo mesenchymal phenotype leading to increased motility and invasion. The aim of this study was to elucidate the biological functions of Slug/Snail2 in lung cancer cells. We introduced Slug gene into several lung cancer cell line (A549, Ma1, and H1299) and established stable cell lines. Overexpression of Slug clearly mediated the EMT-related morphological changes including the cell scattering and the elongation of cell-shape. Real-time RT PCR and Western blot demonstrated that overexpression of Slug markedly downregulated the mRNA levels and protein levels of E-cadherin, while mesenchymal marker, N-cadherin, fibronectin 1 and vimentin, were upregulated. In addition to these changes, Slug enhanced the cellular migration activity and the cellular anchorage independent growth activity. Finally, we examined the Slug-mediated EMT on drug sensitivity to several anti-cancer dugs. Interestingly, Slug-overexpressing cells increased the drug sensitivity to only tubulin-binding agents such as vinorelbine, vincristine, and paclitaxel in vitro. Microarray analysis revealed that Slug downregulates tubulin beta 3 or 4 expression. Luciferase promoter assay showed that Slug directly down-regulates tubulin beta 4 expression at the transcription level. These results suggested that Slug-mediated sensitivity to tubulin-binding agents may be involved in expression changes of tubulin beta 3 or 4.In conclusion, we found that overexpression of Slug mediates EMT and change of drug sensitivity to tubulin-binding agents, presumably via downregulations of tubulin beta 3 and 4 in lung cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3424. doi:10.1158/1538-7445.AM2011-3424

Role of Smad2/3 and p38 MAP kinase in TGF‐β1‐induced epithelial–mesenchymal transition of pulmonary epithelial cells

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-β1 is an inducer of EMT in pulmonary epithelial cells in vitro and in vivo, though the mechanisms are unclear. We hypothesized that TGF-β1 induced EMT through Smad-dependent and -independent processes. To test this hypothesis, we studied the roles and mechanisms of TGF-β1-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-β1 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-β1. 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-β1-induced collagen expression. Furthermore, these data demonstrate that Smads and p38 MAPK differentially regulate EMT-related changes in pulmonary epithelial cells.

Abstract 3431: Role of tumor microenvironment in promoting tumor metastasis

Abstract Metastatic spread of tumor cells to vital organs is the major cause of mortality in cancer patients. Tumor metastasis is a complex process involving multiple steps including detachment of tumor cells from the primary tumor, interaction with vascular endothelium and invasion of target organs. Although our understanding of the molecular and biological events that contribute to tumor cell progression has increased considerably over the last decade, we still know very little about the role of tumor-accessory cells in promoting tumor cell release from the primary tumor and metastasis to distal sites. It is well established that tumor angiogenesis plays a critical role in tumor progression. We have recently demonstrated that VEGF, in addition to its pro-angiogenic function, also induces the expression of Bcl-2 in the microvascular endothelial cells. Furthermore, tumor samples from head and neck cancer patients showed significantly higher Bcl-2 expression in tumor blood vessels and this enhanced Bcl-2 expression in tumor-associated endothelial cells was directly correlated with metastatic status of these patients. Upregulated Bcl-2 expression in tumor-associated endothelial cells was sufficient to enhance tumor metastasis of oral squamous cell carcinoma in a SCID mouse model. While much is known about the role of Bcl-2 in cell survival, very little is known about the role of Bcl-2 in tumor metastasis. In this study, we examined the role and mechanism(s) by which Bcl-2 expression in tumor-associated endothelial cells may promote tumor metastasis. Our results suggest that endothelial cells expressing Bcl-2 (EC-Bcl-2) promote tumor metastasis via cell-cell interaction with tumor cells as well as by secreting chemokines including IL-6 and IL-8. EC-Bcl-2 secreted IL-6 induced EMT-related changes (downregulation of E-cadherin and upregulation of vimentin proteins) in the normal oral keratinocytes as well as head and neck tumor cells. IL-8 promoted tumor metastasis by enhancing tumor cell motility and invasiveness. In addition, EC-Bcl-2 cells exhibited significantly higher adhesion molecules expression and showed enhanced tumor cell binding. Tumor cells, when cultured alone in non-adherent conditions, showed marked increase in apoptosis (anoikis). In contrast, tumor cells showed a significant decrease in anoikis when co-cultured with EC-Bcl-2 in non-adherent conditions. Similarly, tumor cells when injected along with EC-Bcl-2 showed significantly higher lung metastasis as compared to tumor cell and EC-VC or tumor cell alone group. Taken together, these results suggest that endothelial cells expressing Bcl-2 promote tumor cell metastasis by enhancing tumor cell release and chaperoning them to distal sites. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3431.

Abstract 5650: Silibinin inhibits epithelial to mesenchymal transition in prostate cancer cells: Role of E-cadherin and beyond

Abstract Prostate cancer (PCA) is second leading cause of cancer-related deaths in US men. High morbidity and mortality in PCA patients are due to its metastatic spread to distant organs. Epithelial to mesenchymal transition (EMT) in PCA cells is considered a pre-requisite for acquiring advanced migratory/invasive phenotype, and for subsequent metastasis. Even though multiple signaling mechanisms and cellular changes are involved in EMT, loss of E-cadherin is considered as the single most important biological event in PCA cells for acquiring an invasive mesenchymal phenotype. Accordingly, we hypothesized that promoting E-cadherin expression in PCA cells using non-toxic phytochemicals, such as silibinin, would prevent EMT and subsequent metastasis. Recently, we have established strong efficacy of silibinin against PCA cells and currently it is in phase II clinical trial in PCA patients, but its efficacy and mechanism against EMT in PCA cells are largely unknown. Results of present study showed that silibinin strongly inhibits migratory potential of quiescent PC3 and PC3MM2 PCA cells in wound healing assay (P less than 0.001); and invasive potential of PC3 (by 11-66%), PC3MM2 (by 11-61%) and C4-2B (by 32-71%) cells in transwell matrigel invasion assay. Importantly, cell viability assay confirmed that anti-invasive effect of silibinin was not due to its cytotoxicity. Immunoblot analysis showed that silibinin increases E-cadherin expression in PC3 cells in a dose and time dependent manner, which was at cellular membrane as evidenced by sub-cellular fractional and confocal analyses. Silibinin treatment also decreased the level of transcriptional repressors (Snail1, Slug and ZEB1) and increased E-cadherin mRNA expression. Silibinin treatment also decreased the level of phosphorylated Src and Hakai (an E-3 ubiquitin ligase) suggesting post-translational stabilization of E-cadherin. In other studies, silibinin decreased nuclear and cytoplasmic level of beta-catenin but not in cellular membrane. Morphological examination supported these molecular alterations as silibinin treated PC3 cells were more adherent. Similar E-cadherin increase, beta-catenin decrease and morphological changes with silibinin were also observed in PC3MM2 and C4-2B cells. Next, we generated PC3 cells with stable knock-down of E-cadherin expression (ShEC-PC3) and respective control cells (Sh-PC3). Employing these cells, we found that the anti-migratory/anti-invasive potential of silibinin is only partially dependent on E-cadherin. Since molecules, other than E-cadherin, affected by silibinin treatment (beta-catenin, Snail1, Slug and ZEB1) could also directly influence EMT-related changes, our findings suggest multiple molecular targets of silibinin in its anti-EMT and anti-migratory/anti-invasive potential. Overall, the results of present study suggest silibinin usefulness against PCA metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5650.

Abstract 3313: Snail and slug-mediated epithelial mesenchymal transition in lung cancer cells

Abstract Epithelial-Mesenchymal Transition (EMT) has been classified as a unique process by which epithelial cells undergo remarkable morphologic changes characterized by a transition from epithelial cobblestone phenotype to elongated fibroblastic phenotype (mesenchymal phenotype) leading to increased motility and invasion. Accumulating evidence indicates that EMT-inducible transcription factors such as Snail homologues (Snail1, 2, and 3) and several basic helix-loop-helix factors Twist, are poor prognostic factors and associated with chemoresistance in oncology. The aim of this study was to investigate the involvement of SNAI-mediated EMT and its malignant potential in lung cancer cells. We introduced SNAI1 or SNAI2 gene into A549 or PC-9 lung cancer cell lines and established stable cell lines A549/GFP, /SNAI1, /SNAI2, PC-9/GFP, /SNAI1 and /SNAI2. Overexpression of SNAI1 and SNAI2 clearly mediated the EMT-related morphological changes including the cell scattering and the elongation of cell-shape. Real-time RT PCR demonstrated that overexpression of SNAI1 and SNAI2 markedly down-regulated the mRNA levels of E-cadherin about 1/2 to 1/100 compared with GFP-expressing control cell lines. The mRNA upregulation of N-cadherin, fibronectin 1 and Vimentin, which changes are characteristic of EMT, were also observed. We are now investigating its oncogenic properties, gene expression changes by microarray analysis and chemoresistance using these stable SNAI1 or SNAI2 transfectants. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3313.

Alcohol Stimulates Activation of Snail, Epidermal Growth Factor Receptor Signaling, and Biomarkers of Epithelial–Mesenchymal Transition in Colon and Breast Cancer Cells

Alcohol consumption is associated with the risk of progressive cancers including colon and breast cancer. The mechanisms for the alcohol-induced aggressive behavior of these epithelial cancer cells have not been fully identified. Epithelial-mesenchymal transition (EMT) is a developmental program recently shown to play a role in cancer progression and metastases. We hypothesized that alcohol might promote cancer progression by inducing EMT in cancer cells and tested this hypothesis by assessing alcohol-stimulated changes in phenotypic markers of EMT as well as the EMT transcription factor Snail and its related cell signaling. Colon and breast cancer cell lines and a normal intestinal epithelial cell line were tested as well as colonic mucosal biopsy samples from alcoholic subjects. Cells were treated with alcohol and assessed for EMT-related changes using immunofluorescent microscopy, western blotting, reporter assays, RT-PCR, and knockdown of Snail with siRNA. We show alcohol upregulated the signature EMT phenotypic marker vimentin as well as matrix metalloprotease (MMP)-2, MMP-7, and MMP-9 and cell migration in colon and breast cancer cells-all characteristics of EMT. Alcohol also stimulated nuclear localization of Snail phosphorylated at Ser246, transcription from a Snail reporter plasmid, and Snail mRNA expression by RT-PCR. Snail siRNA knockdown prevented alcohol-stimulated vimentin expression. In vivo, Snail expression was significantly elevated in colonic mucosal biopsies from alcoholics. Also, we found alcohol stimulated activation of epidermal growth factor receptor (EGFR) signaling and an EGFR inhibitor blocked alcohol-induced cell migration and Snail mRNA expression. Collectively, our data support a novel mechanism for alcohol promoting cancer progression through stimulating the EMT program in cancer cells via an EGFR-Snail mediated pathway. This study reveals new pathways for alcohol-mediated promotion of cancer that could be targeted for therapy or prevention of alcohol-related cancers.

Parathyroid Hormone–Related Protein Promotes Epithelial–Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is an important process that contributes to renal fibrogenesis. TGF-beta1 and EGF stimulate EMT. Recent studies suggested that parathyroid hormone-related protein (PTHrP) promotes fibrogenesis in the damaged kidney, apparently dependent on its interaction with vascular endothelial growth factor (VEGF), but whether it also interacts with TGF-beta and EGF to modulate EMT is unknown. Here, PTHrP(1-36) increased TGF-beta1 in cultured tubuloepithelial cells and TGF-beta blockade inhibited PTHrP-induced EMT-related changes, including upregulation of alpha-smooth muscle actin and integrin-linked kinase, nuclear translocation of Snail, and downregulation of E-cadherin and zonula occludens-1. PTHrP(1-36) also induced EGF receptor (EGFR) activation; inhibition of protein kinase C and metalloproteases abrogated this activation. Inhibition of EGFR activation abolished these EMT-related changes, the activation of ERK1/2, and upregulation of TGF-beta1 and VEGF by PTHrP(1-36). Moreover, inhibition of ERK1/2 blocked EMT induced by either PTHrP(1-36), TGF-beta1, EGF, or VEGF. In vivo, obstruction of mouse kidneys led to changes consistent with EMT and upregulation of TGF-beta1 mRNA, p-EGFR protein, and PTHrP. Taken together, these data suggest that PTHrP, TGF-beta, EGF, and VEGF might cooperate through activation of ERK1/2 to induce EMT in renal tubuloepithelial cells.

Epithelium-mesenchyme transition during neural crest development.

The neural crest is the organ system whose presence defines vertebrates. The onset of migration of neural crest cells is an archetypal epithelium to mesenchyme transition (EMT), and this event identifies the cell lineage. Little is known yet of the establishment of the neural crest, although the zinc finger gene Slug seems to be involved in specifying EMT competence. The details, especially the temporal order of events in neural crest EMT, vary between different species and between different axial levels, but several important features have emerged from observations in situ and experiments in vitro and in vivo. EMT seems to be strongly associated with decrease in cell-cell adhesion, and particularly with loss of N-cadherin on the surface of neural crest cells at the time of onset of migration. The related adhesion molecule T-cadherin is also present, but correlated changes have not yet been described, while the unrelated adhesion molecule N-CAM also declines on neural crest cells, but with a time course unrelated to EMT. The extracellular matrix is also important: EMT-related changes in matrix receptor (i.e. integrin) activity are recorded in avian crest cells, while the nature of the matrix itself changes in urodele amphibians. Changes in cell shape and in cell motility also occur at the time of EMT, consistent with changes in the cytoskeleton. These concerted changes can be triggered by TGF-beta family growth factors, of which dorsalin-I appears particularly important. These may act through pathways involving controlled alterations in phosphorylation to effect the complex of responses that make up EMT. Although much remains to be understood, the spatiotemporal definability of this system makes it a very useful model for studying EMTs in general.