Potent Inducer Of Epithelial-mesenchymal Transition Research Articles

Epithelial-mesenchymal transition expression profiles as a prognostic factor for disease-free survival in hepatocellular carcinoma: Clinical significance of transforming growth factor-β signaling

Epithelial-mesenchymal transition (EMT) plays a pivotal role in cancer invasion and metastasis, and transforming growth factor (TGF)-β signaling is a potent inducer of EMT. However, the clinical significance of the correlation between EMT marker expression and TGF-β signaling in hepatocellular carcinoma (HCC) patients remains unknown. In this study, immunohistochemistry was used to analyze the expression of EMT markers and phospho-Smad2 nuclear positivity, and their association with clinicopathological features in 150 HCC patients. E-cadherin(high)/vimentin(low) and E-cadherin(low)/vimentin(high) expression profiles were determined in 55 (36.7%) and 21 (14.0%) patients, respectively. The E-cadherin(low)/vimentin(high) expression profile was significantly correlated with poor tumor differentiation (P<0.001), vascular invasion (P=0.007) and extrahepatic recurrence following curative surgery (P=0.026). Furthermore, the E-cadherin(low)/vimentin(high) expression profile was significantly correlated with shorter disease-free survival compared to E-cadherin(high)/vimentin(low) (P=0.002). Forty-one patients (27.3%) were demonstrated to have high phospho-Smad2 nuclear positivity, which was significantly correlated with the E-cadherin(low)/vimentin(high) expression profile (P<0.001). In conclusion, this study suggests that EMT expression profiles are useful prognostic markers for disease-free survival in HCC patients, and that the E-cadherin(low)/vimentin(high) expression profile is closely associated with high-grade malignant behavior such as tumoral vascular invasion and metastasis in HCC. Additionally, TGF-β-mediated EMT may play an important role in the aggressiveness of HCC.

TGF-β-induced activation of mTOR complex 2 drives epithelial–mesenchymal transition and cell invasion

In cancer progression, carcinoma cells gain invasive behavior through a loss of epithelial characteristics and acquisition of mesenchymal properties, a process that can lead to epithelial–mesenchymal transition (EMT). TGF-β is a potent inducer of EMT, and increased TGF-β signaling in cancer cells is thought to drive cancer-associated EMT. Here, we examine the physiological requirement for mTOR complex 2 (mTORC2) in cells undergoing EMT. TGF-β rapidly induces mTORC2 kinase activity in cells undergoing EMT, and controls epithelial cell progression through EMT. By regulating EMT-associated cytoskeletal changes and gene expression, mTORC2 is required for cell migration and invasion. Furthermore, inactivation of mTORC2 prevents cancer cell dissemination in vivo. Our results suggest that the mTORC2 pathway is an essential downstream branch of TGF-β signaling, and represents a responsive target to inhibit EMT and prevent cancer cell invasion and metastasis.

TGF-β-induced activation of mTOR complex 2 drives epithelial–mesenchymal transition and cell invasion

In cancer progression, carcinoma cells gain invasive behavior through a loss of epithelial characteristics and acquisition of mesenchymal properties, a process that can lead to epithelial-mesenchymal transition (EMT). TGF-β is a potent inducer of EMT, and increased TGF-β signaling in cancer cells is thought to drive cancer-associated EMT. Here, we examine the physiological requirement for mTOR complex 2 (mTORC2) in cells undergoing EMT. TGF-β rapidly induces mTORC2 kinase activity in cells undergoing EMT, and controls epithelial cell progression through EMT. By regulating EMT-associated cytoskeletal changes and gene expression, mTORC2 is required for cell migration and invasion. Furthermore, inactivation of mTORC2 prevents cancer cell dissemination in vivo. Our results suggest that the mTORC2 pathway is an essential downstream branch of TGF-β signaling, and represents a responsive target to inhibit EMT and prevent cancer cell invasion and metastasis.

TP53Mutation, Epithelial-Mesenchymal Transition, and Stemlike Features in Breast Cancer Subtypes

Altered p53 protein is prevalently associated with the pathologic class of triple-negative breast cancers and loss of p53 function has recently been linked to the induction of an epithelial-mesenchymal transition (EMT) and acquisition of stemness properties. We explored the association between TP53 mutational status and expression of some genes involved in the canonical TGF-β signaling pathway (the most potent EMT inducer) and in two early EMT associated events: loss of cell polarity and acquisition of stemness-associated features. We used a publicly accessible microarray dataset consisting of 251 p53-sequenced primary breast cancers. Statistical analysis indicated that mutant p53 tumors (especially those harboring a severe mutation) were consistent with the aggressive class of triple-negative cancers and that, differently from cell cultures, surgical tumors underexpressed some TGF-β related transcription factors known as involved in EMT (ID1, ID4, SMAD3, SMAD4, SMAD5, ZEB1). These unexpected findings suggest an interesting relationship between p53 mutation, mammary cell dedifferentiation, and the concomitant acquisition of stemlike properties (as indicated by the overexpression of PROM1 and NOTCH1 genes), which improve tumor cells aggressiveness as indicated by the overexpression of genes associated with cell proliferation (CDK4, CDK6, MKI67) and migration (CXCR4, MMP1).

IL-6-induced epithelial-mesenchymal transition promotes the generation of breast cancer stem-like cells analogous to mammosphere cultures

Recently, the inflammatory cytokine IL-6 has been reported as a potent inducer of epithelial-mesenchymal transition (EMT) in breast cancer cells with an epithelial phenotype. Furthermore, EMT induces stem cell features in normal and transformed mammary cells. We explored whether IL-6-induced EMT promoted the generation of breast cancer stem-like cells (BrCSCs) in epithelial-like breast cancer cells, and whether the cytokines EGF and bFGF, analogous to IL-6, per se induced epithelial-mesenchymal transition, resulting in the enrichment of BrCSCs in mammosphere cultures. Herein, we provide evidence that IL-6 is capable of generating CD44+ cells with stem-like properties through induction of the EMT in the epithelial-like T47D breast cancer cells. We also show that mammosphere cultures of epithelial-like breast cancer cells, T47D, MCF7, ZR-75-1 and MDA-MB-453 cells, consistently generated stem-like cancer cells solely as a result of the EGF and bFGF cytokines in the mammosphere media mediating EMT. This finding demonstrated the link between the inflammatory cytokine IL-6 and BrCSCs and identified an important mechanism for the enrichment of BrCSCs in mammosphere cultures. Thus, EMT appears to be a critical mechanism for the induction of cancer cells with stem-like properties, and EMT of non-stem cancer cells could be a source of CSCs.

Simultaneous Stimulation with TGF-β1 and TNF-α Induces Epithelial Mesenchymal Transition in Bronchial Epithelial Cells

Background: Airway remodeling is an important feature of chronic airway disease, but the mechanisms involved remain unclear. Recently, epithelial mesenchymal transition (EMT) was reported to be associated with tissue fibrosis. TGF-β1, which is a potent inducer of EMT, is thought to be related to the pathogenesis of airway remodeling. We investigated whether TGF-β1 and/or TNF-α induce EMT in bronchial epithelial cells. Methods: Cultured BEAS-2B cells and primary normal human bronchial epithelial cells (NHBE) were treated with TGF-β1 and/or TNF-α. Morphological changes and the expression of EMT-related markers were evaluated by immunocytochemical staining. Expressions of EMT-related markers, extracellular matrix (ECM) components (collagen type I and versican), and TGF-β receptors I, II, and III were analyzed by quantitative RT-PCR. Migration was evaluated using the Boyden chamber technique. Results: The TGF-β1-induced EMT in BEAS-2B cells was demonstrated on the basis of morphological changes and the downregulation of E-cadherin. Costimulation with TNF-α enhanced the TGF-β1-induced morphological changes and increased vimentin expression. Treatment with TGF-β1 increased the expression of collagen type I and versican. EMT induced with TGF-β1 plus TNF-α promoted cell migration. Stimulation of NHBE with TGF-β1 led to EMT. Conclusion: TGF-β1 induced EMT in BEAS-2B cells, and costimulation with TNF-α enhanced the EMT. As a result of the EMT process, BEAS-2B cells acquired functions of mesenchymal cells. In addition, TGF-β1 treatment induced EMT in NHBE as shown by changes in EMT-related markers. Bronchial epithelial cells might contribute to airway remodeling through EMT.

Four-and-a-half LIM protein 2 promotes invasive potential and epithelial-mesenchymal transition in colon cancer

Cancer invasion and metastasis may associate with the phenotype transition called epithelial-mesenchymal transition (EMT). We aim to evaluate the impact of four-and-a-half LIM protein 2 (FHL2) on EMT and invasion of colon cancer. The functional role of FHL2 in EMT was determined by overexpression or small interfering RNA-mediated depletion of FHL2. Mechanisms of FHL2 on expression or activity of E-cadherin and beta-catenin were assessed. FHL2 was highly expressed in primary and metastatic colon cancer but not in normal tissues. FHL2 was critical for cancer cell adhesion to extracellular matrix, migration and invasion. FHL2 expression was stimulated by transforming growth factor (TGF)-beta1. Moreover, FHL2 acted as a potent EMT inducer by stimulating vimentin and matrix metalloproteinase-9 expressions and causing a loss of E-cadherin, whereas those alterations of EMT markers were not affected by silencing of Smad molecules (typical TGF-beta signal mediators) in FHL2 stable transfectant cells. Therefore, FHL2 induced EMT in a TGF-beta-dependent and Smad-independent manner. FHL2 downregulated E-cadherin expression and inhibited the formation of membrane-associated E-cadherin-beta-catenin complex. FHL2 also stabilized nuclear beta-catenin, resulting in enforcement of beta-catenin transactivation activity. FHL2 is a potent EMT inducer and might be an important mediator for invasion and/or metastasis of colon cancer.

Doxorubicin in Combination with a Small TGFβ Inhibitor: A Potential Novel Therapy for Metastatic Breast Cancer in Mouse Models

BackgroundRecent studies suggested that induction of epithelial-mesenchymal transition (EMT) might confer both metastatic and self-renewal properties to breast tumor cells resulting in drug resistance and tumor recurrence. TGFβ is a potent inducer of EMT and has been shown to promote tumor progression in various breast cancer cell and animal models.Principal FindingsWe report that chemotherapeutic drug doxorubicin activates TGFβ signaling in human and murine breast cancer cells. Doxorubicin induced EMT, promoted invasion and enhanced generation of cells with stem cell phenotype in murine 4T1 breast cancer cells in vitro, which were significantly inhibited by a TGFβ type I receptor kinase inhibitor (TβRI-KI). We investigated the potential synergistic anti-tumor activity of TβR1-KI in combination with doxorubicin in animal models of metastatic breast cancer. Combination of Doxorubicin and TβRI-KI enhanced the efficacy of doxorubicin in reducing tumor growth and lung metastasis in the 4T1 orthotopic xenograft model in comparison to single treatments. Doxorubicin treatment alone enhanced metastasis to lung in the human breast cancer MDA-MB-231 orthotopic xenograft model and metastasis to bone in the 4T1 orthotopic xenograft model, which was significantly blocked when TβR1-KI was administered in combination with doxorubicin.ConclusionsThese observations suggest that the adverse activation of TGFβ pathway by chemotherapeutics in the cancer cells together with elevated TGFβ levels in tumor microenvironment may lead to EMT and generation of cancer stem cells resulting in the resistance to the chemotherapy. Our results indicate that the combination treatment of doxorubicin with a TGFβ inhibitor has the potential to reduce the dose and consequently the toxic side-effects of doxorubicin, and improve its efficacy in the inhibition of breast cancer growth and metastasis.

Abstract 2305: Identifying inhibitors of Epithelial-mesenchymal transition to prevent metastasis using global gene expression profile and a systems biology tool

Abstract Acquisition of mesenchymal phenotype by epithelial cells by a process known as “Epithelial mesenchymal transition (EMT)” is considered an initiating event in the process of tumor metastasis. EMT confers certain fundamental abilities to cancer cells that are essential for metastasis which includes the ability to invade, induce immunosuppression, resistance to anoikis and therapeutic agents. The gene expression signature of EMT was shown to correlate with poor differentiation of tumors and patient survival. In addition, EMT was implicated in conferring stem cell like properties to resulting mesenchymal cells, highlighting the relevance of this process in metastasis. Therefore, inhibition of EMT is a rational therapeutic strategy to prevent metastasis. Among the cytokines rich in tumor microenvironment Transforming growth factor (TGF)-β is a potent inducer of EMT. Utilizing the global gene expression profile following TGF-β-induced EMT in A549 lung adenocarcinoma cell line, here we demonstrate a systems biology approach for identifying potential EMT inhibitors. In this approach, a publicly available data base (www.broad.mit.edu/cmap) comprising of gene expression profiles obtained from four different cell lines in response to 164 compounds which include both FDA approved drugs and non-drug bioactive agents, was used to derive negative correlations to EMT gene expression profile using Connectivity map, a pattern matching tool. This analysis identified a list of compounds with significant negative C-scores which may serve as potential EMT inhibitors. The molecules with the highly significant negative C-scores include inhibitors of PI3K (LY294002, Wortmanin), mTOR (Tacrolimus, Sirolimus), HSP90 (Geldanamycin) and COX1/2 (Indomethacin, Aspirin), and a PPAR-γ agonist Troglitazone. With the exception of PI3K inhibitors, the other molecules are potential new EMT inhibitors. PI3K inhibitors were previously demonstrated to inhibit EMT in breast epithelial cells. So far, we validated the ability of Troglitazone, PI3K and mTOR inhibitors to block TGF-β-induced EMT in A549 cells and in all cases we demonstrated that these molecules can selectively block acquisition of mesenchymal phenotype but have no effect on the loss of epithelial phenotype during EMT. Consistently, these molecules also inhibited EMT-induced migration and invasion of A549 cells. Validations of other molecules with significant C-scores are in progress. Above data demonstrates that connectivity maps based systems approach is a viable strategy and identifies inhibitors of PI3K and mTOR and agonists of PPAR-γ as potential therapeutic agents for prevention of 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 2305.

Cadherin–catenin complex and transcription factor Snail-1 in spindle cell carcinoma of the head and neck

Spindle cell carcinoma (SpCC) is a biphasic tumor composed of squamous cell carcinoma (SCC) and a malignant spindle cell component. There is mounting evidence that SpCC is a monoclonal neoplasm originating from a stem cell giving rise to both components. We tested the hypothesis that spindle cell phenotype might be related to the cadherin-catenin complex, which forms adherens junctions between cells. We analyzed the immunohistochemical expression of E- and N-cadherin, alpha-, beta- and gamma-catenin, and Snail-1, a transcription repressor of E-cadherin, in 30 cases of SpCC, and 30 cases of SCC of the head and neck. In SpCC, cadherin and catenin expression was similar in the SCC component, whereas in the spindle cell component, loss of E-cadherin and neo-expression of N-cadherin was found in 19 cases, loss of cadherins in seven, and their co-expression in four cases. Catenin expression were altered in 18 SpCCs. Snail-1 was found in 19 SpCC cases. In SCC, E-cadherin and catenins were expressed in all cases, and N-cadherin focally in five cases. Snail-1 was observed in the stroma. To summarize, in SpCC, there is an altered expression of the cadherin-catenin complex, associated with morphological transition from epithelial to spindle cell phenotype. These features are reminiscent of epithelial-mesenchymal transition (EMT). Our study thus indicates that EMT might play an important role in the pathogenesis of SpCC. This conclusion is further supported by our finding of Snail-1 expression, a potent inducer of EMT, in more than half SpCC cases.

Cholangiocytes with Mesenchymal Features Contribute to Progressive Hepatic Fibrosis of the Polycystic Kidney Rat

The polycystic kidney (PCK) rat is an animal model of Caroli's disease with congenital hepatic fibrosis, in which the mechanism of progressive hepatic fibrosis remains unknown. This study aimed to clarify the mechanism of hepatic fibrosis of the PCK rat from the viewpoint of the contribution of pathological cholangiocytes. In liver sections of the PCK rats, intrahepatic bile ducts were constituted by two different phenotypes: bile ducts lined by cuboidal-shaped and flat-shaped cholangiocytes. The flat-shaped cholangiocytes showed reduced immunohistochemical expression of the biliary epithelial marker cytokeratin 19 and positive immunoreactivity for vimentin and fibronectin. When cultured cholangiocytes of the PCK rat were treated with transforming growth factor (TGF)-beta1, a potent inducer of epithelial-mesenchymal transition, induction of vimentin, fibronectin, and collagen expression occurred in the PCK cholangiocytes. Although the TGF-beta1 treatment reduced cytokeratin 19 expression, the epithelial cell features characterized by the expression of E-cadherin and zonula occludens-1 was maintained, and alpha-smooth muscle actin expression was not induced in the cholangiocytes. Cholangiocytes of the PCK rat may acquire mesenchymal features in response to TGF-beta1 and participate in progressive hepatic fibrosis by producing extracellular matrix molecules, which seems to be a different event from epithelial-mesenchymal transition.