Epithelial-to-mesenchymal transition (EMT) is fundamental to both embryogenesis and tumor metastasis. The Notch intercellular signaling pathway regulates cell fate determination throughout metazoan evolution, and overexpression of activating alleles is oncogenic in mammals. Here we demonstrate that Notch activity promotes EMT during both cardiac development and oncogenic transformation via transcriptional induction of the Snail repressor, a potent and evolutionarily conserved mediator of EMT in many tissues and tumor types. In the embryonic heart, Notch functions via lateral induction to promote a selective transforming growth factor-β (TGFβ)-mediated EMT that leads to cellularization of developing cardiac valvular primordia. Embryos that lack Notch signaling elements exhibit severely attenuated cardiac snail expression, abnormal maintenance of intercellular endocardial adhesion complexes, and abortive endocardial EMT in vivo and in vitro. Accordingly, transient ectopic expression of activated Notch1 (N1IC) in zebrafish embryos leads to hypercellular cardiac valves, whereas Notch inhibition prevents valve development. Overexpression of N1IC in immortalized endothelial cells in vitro induces EMT accompanied by oncogenic transformation, with corresponding induction of snail and repression of VE-cadherin expression. Notch is expressed in embryonic regions where EMT occurs, suggesting an intimate and fundamental role for Notch, which may be reactivated during tumor metastasis.

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.

Objective—: Marfan’s syndrome is characterized by the formation of ascending aortic aneurysms resulting from altered assembly of extracellular matrix microfibrils and chronic tissue growth factor (TGF)-β signaling. TGF-β is a potent regulator of the vascular smooth muscle cell (VSMC) phenotype. We hypothesized that as a result of the chronic TGF-β signaling, VSMC would alter their basal differentiation phenotype, which could facilitate the formation of aneurysms. This study explores whether Marfan’s syndrome entails phenotypic alterations of VSMC and possible mechanisms at the subcellular level. Approach and Results—: Immunohistochemical and Western blotting analyses of dilated aortas from Marfan patients showed overexpression of contractile protein markers (α-smooth muscle actin, smoothelin, smooth muscle protein 22 alpha, and calponin-1) and collagen I in comparison with healthy aortas. VSMC explanted from Marfan aortic aneurysms showed increased in vitro expression of these phenotypic markers and also of myocardin, a transcription factor essential for VSMC-specific differentiation. These alterations were generally reduced after pharmacological inhibition of the TGF-β pathway. Marfan VSMC in culture showed more robust actin stress fibers and enhanced RhoA-GTP levels, which was accompanied by increased focal adhesion components and higher nuclear localization of myosin-related transcription factor A. Marfan VSMC and extracellular matrix measured by atomic force microscopy were both stiffer than their respective controls. Conclusions—: In Marfan VSMC, both in tissue and in culture, there are variable TGF-β-dependent phenotypic changes affecting contractile proteins and collagen I, leading to greater cellular and extracellular matrix stiffness. Altogether, these alterations may contribute to the known aortic rigidity that precedes or accompanies Marfan’s syndrome aneurysm formation.

The multikinase inhibitor sorafenib is the only effective drug in advanced cases of hepatocellular carcinoma (HCC). However, response differs among patients and effectiveness only implies a delay. We have recently described that sorafenib sensitizes HCC cells to apoptosis. In this work, we have explored the response to this drug of six different liver tumor cell lines to define a phenotypic signature that may predict lack of response in HCC patients. Results have indicated that liver tumor cells that show a mesenchymal‐like phenotype, resistance to the suppressor effects of transforming growth factor beta (TGF‐β) and high expression of the stem cell marker CD44 were refractory to sorafenib‐induced cell death in in vitro studies, which correlated with lack of response to sorafenib in nude mice xenograft models of human HCC. In contrast, epithelial‐like cells expressing the stem‐related proteins EpCAM or CD133 were sensitive to sorafenib‐induced apoptosis both in vitro and in vivo. A cross‐talk between the TGF‐β pathway and the acquisition of a mesenchymal‐like phenotype with up‐regulation of CD44 expression was found in the HCC cell lines. Targeted CD44 knock‐down in the mesenchymal‐like cells indicated that CD44 plays an active role in protecting HCC cells from sorafenib‐induced apoptosis. However, CD44 effect requires a TGF‐β‐induced mesenchymal background, since the only overexpression of CD44 in epithelial‐like HCC cells is not sufficient to impair sorafenib‐induced cell death. In conclusion, a mesenchymal profile and expression of CD44, linked to activation of the TGF‐β pathway, may predict lack of response to sorafenib in HCC patients.

Transforming growth factor‐beta (TGF‐β) is an important regulatory suppressor factor in hepatocytes. However, liver tumor cells develop mechanisms to overcome its suppressor effects and respond to this cytokine by inducing other processes, such as the epithelial‐mesenchymal transition (EMT), which contributes to tumor progression and dissemination. Recent studies have placed chemokines and their receptors at the center not only of physiological cell migration but also of pathological processes, such as metastasis in cancer. In particular, CXCR4 and its ligand, stromal cell‐derived factor 1α (SDF‐1α) / chemokine (C‐X‐C motif) ligand 12 (CXCL12) have been revealed as regulatory molecules involved in the spreading and progression of a variety of tumors. Here we show that autocrine stimulation of TGF‐β in human liver tumor cells correlates with a mesenchymal‐like phenotype, resistance to TGF‐β‐induced suppressor effects, and high expression of CXCR4, which is required for TGF‐β‐induced cell migration. Silencing of the TGF‐β receptor1 (TGFBR1), or its specific inhibition, recovered the epithelial phenotype and attenuated CXCR4 expression, inhibiting cell migratory capacity. In an experimental mouse model of hepatocarcinogenesis (diethylnitrosamine‐induced), tumors showed increased activation of the TGF‐β pathway and enhanced CXCR4 levels. In human hepatocellular carcinoma tumors, high levels of CXCR4 always correlated with activation of the TGF‐β pathway, a less differentiated phenotype, and a cirrhotic background. CXCR4 concentrated at the tumor border and perivascular areas, suggesting its potential involvement in tumor cell dissemination. Conclusion: A crosstalk exists among the TGF‐β and CXCR4 pathways in liver tumors, reflecting a novel molecular mechanism that explains the protumorigenic effects of TGF‐β and opens new perspectives for tumor therapy. (Hepatology 2013; 58:2032–2044)

Transforming growth factor-β (TGF-β) induces apoptosis in hepatocytes, being considered a liver tumor suppressor. However, many human hepatocellular carcinoma (HCC) cells escape from its proapoptotic effects, gaining response to this cytokine in terms of malignancy. We have recently reported that the apoptosis induced by TGF-β in hepatocytes requires up-regulation of the NADPH oxidase NOX4, which mediates reactive oxygen species (ROS) production. TGF-β–induced NOX4 expression is inhibited by antiapoptotic signals, such as the phosphatydilinositol-3-phosphate kinase or the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways. The aim of the present work was to analyze whether resistance to TGF-β–induced apoptosis in HCC cells is related to the impairment of NOX4 up-regulation due to overactivation of survival signals. Results indicate that inhibition of the MAPK/ERK kinase (MEK)/ERK pathway in HepG2 cells, which are refractory to the proapoptotic effects of TGF-β, sensitizes them to cell death through a mitochondrial-dependent mechanism, coincident with increased levels of BIM and BMF, decreased levels of BCL-XL and MCL1, and BAX/BAK activation. Regulation of BMF, BCL-XL, and MCL1 occurs at the mRNA level, whereas BIM regulation occurs post-transcriptionally. ROS production and glutathione depletion are only observed in cells treated with TGF-β and PD98059, which correlates with NOX4 up-regulation. Targeting knockdown of NOX4 impairs ROS increase and all the mitochondrial-dependent apoptotic features by a mechanism that is upstream from the regulation of BIM, BMF, BCL-XL, and MCL1 levels. In conclusion, overactivation of the MEK/ERK pathway in liver tumor cells confers resistance to TGF-β–induced cell death through impairing NOX4 up-regulation, which is required for an efficient mitochondrial-dependent apoptosis. [Cancer Res 2009;69(19):7595–602]