Snail mediates crosstalk between TGFβ and LXRα in hepatocellular carcinoma

Understanding the complexity of changes in differentiation and cell survival in hepatocellular carcinoma (HCC) is essential for the design of new diagnostic tools and therapeutic modalities. In this context, we have analyzed the crosstalk between transforming growth factor β (TGFβ) and liver X receptor α (LXRα) pathways. TGFβ is known to promote cytostatic and pro-apoptotic responses in HCC, and to facilitate mesenchymal differentiation. We here demonstrate that stimulation of the nuclear LXRα receptor system by physiological and clinically useful agonists controls the HCC response to TGFβ. Specifically, LXRα activation antagonizes the mesenchymal, reactive oxygen species and pro-apoptotic responses to TGFβ and the mesenchymal transcription factor Snail mediates this crosstalk. In contrast, LXRα activation and TGFβ cooperate in enforcing cytostasis in HCC, which preserves their epithelial features. LXRα influences Snail expression transcriptionally, acting on the Snail promoter. These findings propose that clinically used LXR agonists may find further application to the treatment of aggressive, mesenchymal HCCs, whose progression is chronically dependent on autocrine or paracrine TGFβ

TGF-β and the Tissue Microenvironment: Relevance in Fibrosis and Cancer

Transforming growth factor-β (TGF-β) is a cytokine essential for the induction of the fibrotic response and for the activation of the cancer stroma. Strong evidence suggests that a strong cross-talk exists among TGF-β and the tissue extracellular matrix components. TGF-β is stored in the matrix as part of a large latent complex bound to the latent TGF-β binding protein (LTBP) and matrix binding of latent TGF-β complexes, which is required for an adequate TGF-β function. Once TGF-β is activated, it regulates extracellular matrix remodelling and promotes a fibroblast to myofibroblast transition, which is essential in fibrotic processes. This cytokine also acts on other cell types present in the fibrotic and tumour microenvironment, such as epithelial, endothelial cells or macrophages and it contributes to the cancer-associated fibroblast (CAF) phenotype. Furthermore, TGF-β exerts anti-tumour activity by inhibiting the host tumour immunosurveillance. Aim of this review is to update how TGF-β and the tissue microenvironment cooperate to promote the pleiotropic actions that regulate cell responses of different cell types, essential for the development of fibrosis and tumour progression. We discuss recent evidences suggesting the use of TGF-β chemical inhibitors as a new line of defence against fibrotic disorders or cancer

Sorafenib sensitizes hepatocellular carcinoma cells to physiological apoptotic stimuli

Sorafenib increases survival rate of patients with advanced hepatocellular carcinoma (HCC). The mechanism underlying this effect is not completely understood. In this work we have analyzed the effects of sorafenib on autocrine proliferation and survival of different human HCC cell lines. Our results indicate that sorafenib in vitro counteracts autocrine growth of different tumor cells (Hep3B, HepG2, PLC-PRF-5, SK-Hep1). Arrest in S/G2/M cell cycle phases were observed coincident with cyclin D1 down-regulation. However, sorafenib's main anti-tumor activity seems to occur through cell death induction which correlated with caspase activation, increase in the percentage of hypodiploid cells, activation of BAX and BAK and cytochrome c release from mitochondria to cytosol. In addition, we observed a rise in mRNA and protein levels of the pro-apoptotic BH3-domain only PUMA and BIM, as well as decreased protein levels of the anti-apoptotic MCL1 and survivin. PUMA targeting knock-down, by using specific siRNAs, inhibited sorafenib-induced apoptotic features. Moreover, we obtained evidence suggesting that sorafenib also sensitizes HCC cells to the apoptotic activity of transforming growth factor-beta (TGF-beta) through the intrinsic pathway and to tumor necrosis factor-a (TNF) through the extrinsic pathway. Interestingly, sensitization to sorafenib-induced apoptosis is characteristic of liver tumor cells, since untransformed hepatocytes did not respond to sorafenib inducing apoptosis, either alone or in combination with TGF-beta or TNF. Indeed, sorafenib effectiveness in delaying HCC late progression might be partly related to a selectively sensitization of HCC cells to apoptosis by disrupting autocrine signals that protect them from adverse conditions and pro-apoptotic physiological cytokines. J. Cell. Physiol. 227: 1319-1325, 2012. (C) 2011 Wiley Periodicals, Inc

Caveolin-1 is required for TGF-β-induced transactivation of the EGF receptor pathway in hepatocytes through the activation of the metalloprotease TACE/ADAM17

Transforming growth factor-beta (TGF-β) plays a dual role in hepatocytes, inducing both pro- and anti-apoptotic responses, whose balance decides cell fate. Survival signals are mediated by the epidermal growth factor receptor (EGFR) pathway, which is activated by TGF-β in these cells. Caveolin-1 (Cav1) is a structural protein of caveolae linked to TGF-β receptors trafficking and signaling. Previous results have indicated that in hepatocytes, Cav1 is required for TGF-β-induced anti-apoptotic signals, but the molecular mechanism is not fully understood yet. In this work, we show that immortalized Cav1−/− hepatocytes were more sensitive to the pro-apoptotic effects induced by TGF-β, showing a higher activation of caspase-3, higher decrease in cell viability and prolonged increase through time of intracellular reactive oxygen species (ROS). These results were coincident with attenuation of TGF-β-induced survival signals in Cav1−/− hepatocytes, such as AKT and ERK1/2 phosphorylation and NFκ-B activation. Transactivation of the EGFR pathway by TGF-β was impaired in Cav1−/− hepatocytes, which correlated with lack of activation of TACE/ADAM17, the metalloprotease responsible for the shedding of EGFR ligands. Reconstitution of Cav1 in Cav1−/− hepatocytes rescued wild-type phenotype features, both in terms of EGFR transactivation and TACE/ADAM17 activation. TACE/ADAM17 was localized in detergent-resistant membrane (DRM) fractions in Cav1+/+ cells, which was not the case in Cav1−/− cells. Disorganization of lipid rafts after treatment with cholesterol-binding agents caused loss of TACE/ADAM17 activation after TGF-β treatment. In conclusion, in hepatocytes, Cav1 is required for TGF-β-mediated activation of the metalloprotease TACE/ADAM17 that is responsible for shedding of EGFR ligands and activation of the EGFR pathway, which counteracts the TGF-β pro-apoptotic effects. Therefore, Cav1 contributes to the pro-tumorigenic effects of TGF-β in liver cancer cells

Downregulation of epidermal growth factor receptor in hepatocellular carcinoma facilitates transforming growth factor-β-induced epithelial to amoeboid transition

The Epidermal Growth Factor Receptor (EGFR) and the Transforming Growth Factor-beta (TGF-β) are key regulators of hepatocarcinogenesis. Targeting EGFR was proposed as a promising therapy; however, poor success was obtained in human hepatocellular carcinoma (HCC) clinical trials. Here, we describe how EGFR is frequently downregulated in HCC patients while TGF-β is upregulated. Using 2D/3D cellular models, we show that after EGFR loss, TGF-β is more efficient in its pro-migratory and invasive effects, inducing epithelial to amoeboid transition. EGFR knock-down promotes loss of cell-cell and cell-to-matrix adhesion, favouring TGF-β-induced actomyosin contractility and acquisition of an amoeboid migratory phenotype. Moreover, TGF-β upregulates RHOC and CDC42 after EGFR silencing, promoting Myosin II in amoeboid cells. Importantly, low EGFR combined with high TGFB1 or RHOC/CDC42 levels confer poor patient prognosis. In conclusion, this work reveals a new tumour suppressor function for EGFR counteracting TGF-β-mediated epithelial to amoeboid transitions in HCC, supporting a rational for targeting the TGF-β pathway in patients with low EGFR expression. Our work also highlights the relevance of epithelial to amoeboid transition in human tumours and the need to better target this process in the clinic

Mecanismos moleculares que confieren resistencia a la apoptosis por TGF-beta en células de Hepatocarcinoma Celular Humano

[spa] En los últimos años nuestro grupo ha estudiado las diferentes vías de señalización inducidas por TGF-β en hepatocitos fetales de rata. A dosis bajas, el TGF-β inhibe el crecimiento, pero a concentraciones más elevadas es capaz de inducir apoptosis (Sanchez et al. 1995; Sanchez et al. 1996). El proceso apoptótico está mediado por un incremento en el contenido intracelular de ROS, dependiente de la síntesis de novo de proteínas, y que correlaciona con una caída en los niveles intracelulares de glutatión (Sanchez et al. 1997). La muerte inducida por TGF-β en estas células se correlaciona con un descenso en los niveles de Bcl-xL, la despolarización de la membrana mitocondrial, la salida de citocromo c y posterior activación de caspasas (Herrera et al. 2001a; Herrera et al. 2001b). El incremento de ROS intracelulares se produce por activación de un sistema NADPH oxidasa y por disminución en la expresión de proteínas antioxidantes (Herrera et al. 2004). Recientemente hemos descrito que la NADPH oxidasa NOX4 se induce en condiciones pro-apoptóticas (Carmona-Cuenca et al. 2006), pero otras NADPH oxidasas podrían jugar un papel diferente en la señalización inducida por TGF-β (Murillo et al., 2007). La muerte inducida por TGF-β puede ser inhibida por EGF a través de la activación de PI3K (Fabregat et al. 2000), que contrarresta la expresión de Nox4 (Carmona-Cuenca et al. 2006). Sin embargo, el 40-50% de las células sobreviven a los efectos apoptóticos del TGF-β y adquieren una morfología fibroblastoide (Sanchez et al. 1999). Esto es debido a que el TGF-β también induce señales anti-apoptóticas en los hepatocitos fetales, proceso que requiere la activación del EGFR, producida por un aumento en los niveles de expresión de sus ligandos y activación de la metaloproteasa TACE/ADAM17 que los proteoliza y activa (Valdes et al. 2004; Murillo et al. 2005; Del Castillo et al. 2006; Murillo et al. 2007). Las células que sobreviven al TGF-β responden a esta citoquina en términos de migración e invasión, disminuyendo la expresión de marcadores hepáticos (Sanchez et al. 1999), e induciendo un proceso de EMT (Valdes et al. 2002). La población mesenquimática resultante es resistente a la muerte inducida por TGF-β, ha sufrido un proceso de desdiferenciación y expresa marcadores de célula madre (Del Castillo et al. 2006; del Castillo et al. 2008). Esta población puede rediferenciarse tanto a un linaje hepatocítico como hacia células biliares cuando se mantienen con los medios de diferenciación adecuados. Por último, resultados preliminares al inicio de esta tesis doctoral proponían que la doble respuesta al TGF-β observada en hepatocitos fetales de rata en cultivo primario era exclusiva de este estadio del desarrollo hepático, ya que en hepatocitos adultos de rata el TGF-β sólo inducía apoptosis. La incapacidad del TGF-β de inducir señales de supervivencia parece deberse a la baja expresión de AKT y de TACE observada en hepatocitos adultos. Además, el TGF-β era incapaz de inducir un proceso de EMT en hepatocitos adultos de rata. A la vista de estos resultados se consideró de gran importancia analizar cuál podría ser la respuesta al TGF-β en células tumorales hepáticas. Así, nuestro principal objetivo en esta tesis ha sido analizar si las células de carcinoma hepatocelular responden a la muerte celular inducida por el TGF-β, y en el caso de que hayan adquirido resistencia, estudiar los mecanismos moleculares que la confieren. También queríamos saber si el TGF-β induce señales de supervivencia y un proceso de EMT en células tumorales hepáticas, y la relevancia de este proceso en la progresión del tumor hepático. Aunque quisimos iniciar el estudio con células de hepatoma de rata, debido a nuestra experiencia en este modelo de celular, consideramos muy importante también analizar la situación en células tumorales de hígado humano, ya que es conocido que los niveles de TGF-β son elevados en carcinoma hepatocelular (HCC) y diferentes evidencias han sugerido que la respuesta al TGF-β está alterada en células de HCC.[eng] In the last years our research has focused on analyzing the signaling pathways induced by TGF-β in liver tumor cell lines, to understand the molecular mechanisms that confer resistance to its suppressor effects. TGF-β induces apoptosis in human fetal hepatocytes and in some liver tumor cells (FaO rat hepatoma, Hep3B and PLC/PRF/5 human hepatocarcinoma cells), which requires reactive oxygen species (ROS) production and up-regulation of the NADPH oxidase NOX4. This process is coincident with an increased expression of pro-apoptotic BCL-2 family members, such as BMF or BIM. However, in these same cells, TGF-β also induces anti-apoptotic signals, mediated by the activation of the epidermal growth factor receptor (EGFR) and coincident with up-regulation of the anti-apoptotic proteins BCL-XL, MCL1 or HIAP1. Inhibition of the EGFR, either by pharmacological inhibitors or through targeting knock-down with specific siRNA, significantly enhances the apoptotic response, which indicates that the EGFR plays a relevant role in conferring resistance to TGF-β-induced cell death. However, even when the EGFR is inhibited, some hepatocellular carcinoma cells, such as HepG2 or SK-Hep1, continue showing resistance to TGF-β-induced cell death. HepG2 cells are sensitized to TGF-β-induced apoptosis through the inhibition of the MEK pathway. MEK inhibition allows TGF-β to induce its pro-apoptotic program in these cells, which is coincident with NOX4 upregulation, modulation of the expression of BCL-2 family members and caspase-3 activation. It is worthy to note that activation of survival pathways, such as EGFR or MEK/ERK, 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. Finally, our results have indicated that TGF-β is able to induce an epithelial to mesenchymal transition (EMT) process in human fetal hepatocytes, FaO rat hepatoma cells and Hep3B human hepatocarcinoma cells. TGF-β induces Snail expression, coincident with a decrease in E-cadherin mRNA and protein levels. Furthermore, cells show an increased expression of mesenchymal genes and reorganization of the actin cytoskeleton in stress fibers. Interestingly, these cells show loss of expression of specific hepatic markers and increased expression of stem cell markers. Indeed, chronic treatment with TGF-β selects a population of mesenchymal cells with a de-differentiated phenotype, reminiscent of progenitor-like cells. In summary, TGF-β induces different signals in liver tumor cells, some of them might contribute to tumor suppression (apoptosis), but others should mediate liver tumor progression and invasion

Protein-tyrosine phosphatase 1B (PTP1B) deficiency confers resistance to transforming growth factor-β (TGF-β)-induced suppressor effects in hepatocytes

Transforming growth factor-β (TGF-β) plays a dual role in hepatocytes, mediating both tumor suppressor and promoter effects. The suppressor effects of the cytokine can be negatively regulated by activation of survival signals, mostly dependent on tyrosine kinase activity. The aim of our work was to study the role of the protein-tyrosine phosphatase 1B (PTP1B) on the cellular responses to TGF-β, using for this purpose immortalized neonatal hepatocytes isolated from both PTP1B(+/+) and PTP1B(-/-) mice. We have found that PTP1B deficiency conferred resistance to TGF-β suppressor effects, such as apoptosis and growth inhibition, correlating with lower Smad2/Smad3 activation. Both responses were recovered in the presence of the general tyrosine kinase inhibitor genistein. PTP1B(-/-) cells showed elevated NF-κB activation in response to TGF-β. Knockdown of the NF-κB p65 subunit increased cell response in terms of Smads phosphorylation and apoptosis. Interestingly, these effects were accompanied by inhibition of Smad7 up-regulation. In addition, lack of PTP1B promoted an altered NADPH oxidase (NOX) expression pattern in response to TGF-β, strongly increasing the NOX1/NOX4 ratio, which was reverted by genistein and p65 knockdown. Importantly, NOX1 knockdown inhibited nuclear translocation of p65, promoted Smad phosphorylation, and decreased Smad7 levels. In summary, our results suggest that PTP1B deficiency confers resistance to TGF-β through Smad inhibition, an effect that is mediated by NOX1-dependent NF-κB activation, which in turn, increases the level of the Smad inhibitor Smad7 and participates in a positive feedback loop on NOX1 up-regulation

Tgf-beta And The Tissue Microenvironment: Relevance In Fibrosis And Cancer

ransforming growth factor-beta (TGF-beta) is a cytokine essential for the induction of the fibrotic response and for the activation of the cancer stroma. Strong evidence suggests that a strong cross-talk exists among TGF-beta and the tissue extracellular matrix components. TGF- is stored in the matrix as part of a large latent complex bound to the latent TGF-beta binding protein (LTBP) and matrix binding of latent TGF-beta complexes, which is required for an adequate TGF-beta function. Once TGF-beta is activated, it regulates extracellular matrix remodelling and promotes a fibroblast to myofibroblast transition, which is essential in fibrotic processes. This cytokine also acts on other cell types present in the fibrotic and tumour microenvironment, such as epithelial, endothelial cells or macrophages and it contributes to the cancer-associated fibroblast (CAF) phenotype. Furthermore, TGF-beta exerts anti-tumour activity by inhibiting the host tumour immunosurveillance. Aim of this review is to update how TGF-beta and the tissue microenvironment cooperate to promote the pleiotropic actions that regulate cell responses of different cell types, essential for the development of fibrosis and tumour progression. We discuss recent evidences suggesting the use of TGF beta- chemical inhibitors as a new line of defence against fibrotic disorders or cancer

Sorafenib sensitizes hepatocellular carcinoma cells to physiological apoptotic stimuli

Sorafenib increases survival rate of patients with advanced hepatocellular carcinoma (HCC). The mechanism underlying this effect is not completely understood. In this work we have analyzed the effects of sorafenib on autocrine proliferation and survival of different human HCC cell lines. Our results indicate that sorafenib in vitro counteracts autocrine growth of different tumor cells (Hep3B, HepG2, PLC-PRF-5, SK-Hep1). Arrest in S/G2/M cell cycle phases were observed coincident with cyclin D1 down-regulation. However, sorafenib's main anti-tumor activity seems to occur through cell death induction which correlated with caspase activation, increase in the percentage of hypodiploid cells, activation of BAX and BAK and cytochrome c release from mitochondria to cytosol. In addition, we observed a rise in mRNA and protein levels of the pro-apoptotic BH3-domain only PUMA and BIM, as well as decreased protein levels of the anti-apoptotic MCL1 and survivin. PUMA targeting knock-down, by using specific siRNAs, inhibited sorafenib-induced apoptotic features. Moreover, we obtained evidence suggesting that sorafenib also sensitizes HCC cells to the apoptotic activity of transforming growth factor-beta (TGF-beta) through the intrinsic pathway and to tumor necrosis factor-a (TNF) through the extrinsic pathway. Interestingly, sensitization to sorafenib-induced apoptosis is characteristic of liver tumor cells, since untransformed hepatocytes did not respond to sorafenib inducing apoptosis, either alone or in combination with TGF-beta or TNF. Indeed, sorafenib effectiveness in delaying HCC late progression might be partly related to a selectively sensitization of HCC cells to apoptosis by disrupting autocrine signals that protect them from adverse conditions and pro-apoptotic physiological cytokines. J. Cell. Physiol. 227: 1319-1325, 2012. (C) 2011 Wiley Periodicals, Inc

Sorafenib sensitizes hepatocellular carcinoma cells to physiological apoptotic stimuli

Sorafenib increases survival rate of patients with advanced hepatocellular carcinoma (HCC). The mechanism underlying this effect is not completely understood. In this work we have analyzed the effects of sorafenib on autocrine proliferation and survival of different human HCC cell lines. Our results indicate that sorafenib in vitro counteracts autocrine growth of different tumor cells (Hep3B, HepG2, PLC-PRF-5, SK-Hep1). Arrest in S/G2/M cell cycle phases were observed coincident with cyclin D1 down-regulation. However, sorafenib's main anti-tumor activity seems to occur through cell death induction which correlated with caspase activation, increase in the percentage of hypodiploid cells, activation of BAX and BAK and cytochrome c release from mitochondria to cytosol. In addition, we observed a rise in mRNA and protein levels of the pro-apoptotic BH3-domain only PUMA and BIM, as well as decreased protein levels of the anti-apoptotic MCL1 and survivin. PUMA targeting knock-down, by using specific siRNAs, inhibited sorafenib-induced apoptotic features. Moreover, we obtained evidence suggesting that sorafenib also sensitizes HCC cells to the apoptotic activity of transforming growth factor-beta (TGF-beta) through the intrinsic pathway and to tumor necrosis factor-a (TNF) through the extrinsic pathway. Interestingly, sensitization to sorafenib-induced apoptosis is characteristic of liver tumor cells, since untransformed hepatocytes did not respond to sorafenib inducing apoptosis, either alone or in combination with TGF-beta or TNF. Indeed, sorafenib effectiveness in delaying HCC late progression might be partly related to a selectively sensitization of HCC cells to apoptosis by disrupting autocrine signals that protect them from adverse conditions and pro-apoptotic physiological cytokines. J. Cell. Physiol. 227: 1319-1325, 2012. (C) 2011 Wiley Periodicals, Inc