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

The level of caveolin-1 expression determines response to TGF-ß as a tumor suppressor in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is a heterogeneous tumour associated with poor prognostic outcome. Caveolin-1 (CAV1), a membrane protein involved in the formation of caveolae, is frequently overexpressed in HCC. Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine having a dual role in hepatocarcinogenesis: inducer of apoptosis at early phases, but pro-tumourigenic once cells acquire mechanisms to overcome its suppressor effects. Apoptosis induced by TGF-β is mediated by upregulation of the NADPH oxidase NOX4, but counteracted by transactivation of the epidermal growth factor receptor (EGFR) pathway. Previous data suggested that CAV1 is required for the anti-apoptotic signals triggered by TGF-β in hepatocytes. Whether this mechanism is relevant in hepatocarcinogenesis has not been explored yet. Here we analysed the TGF-β response in HCC cell lines that express different levels of CAV1. Accordingly, stable CAV1 knockdown or overexpressing cell lines were generated. We demonstrate that CAV1 is protecting HCC cells from TGF-β-induced apoptosis, which attenuates its suppressive effect on clonogenic growth and increases its effects on cell migration. Downregulation of CAV1 in HLE cells promotes TGF-β-mediated induction of the pro-apoptotic BMF, which correlates with upregulation of NOX4, whereas CAV1 overexpression in Huh7 cells shows the opposite effect. CAV1 silenced HLE cells show attenuation in TGF-β-induced EGFR transactivation and activation of the PI3K/AKT pathway. On the contrary, Huh7 cells, which do not respond to TGF-β activating the EGFR pathway, acquire the capacity to do so when CAV1 is overexpressed. Analyses in samples from HCC patients revealed that tumour tissues presented higher expression levels of CAV1 compared with surrounding non-tumoural areas. Furthermore, a significant positive correlation among the expression of CAV1 and TGFB1 was observed. We conclude that CAV1 has an essential role in switching the response to TGF-β from cytostatic to tumourigenic, which could have clinical meaning in patient stratification

Clathrin switches transforming growth factor-β role to pro-tumorigenic in liver cancer

Background & Aims: Upon ligand binding, tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR), are recruited into clathrin-coated pits for internalization by endocytosis, which is relevant for signalling and/or receptor degradation. In liver cells, transforming growth factor-beta (TGF-beta) induces both pro- and anti-apoptotic signals; the latter are mediated by the EGFR pathway. Since EGFR mainly traffics via clathrin-coated vesicles, we aimed to analyse the potential role of clathrin in TGF-beta-induced signalling in liver cells and its relevance in liver cancer. Methods: Real-Time PCR and immunohistochemistry were used to analyse clathrin heavy-chain expression in human (CLTC) and mice (Cltc) liver tumours. Transient knockdown (siRNA) or overexpression of CLTC were used to analyse its role on TGF-beta and EGFR signalling in vitro. Bioinformatic analysis was used to determine the effect of CLTC and TGEB1 expression on prognosis and overall survival in patients with hepatocellular carcinoma (HCC). Results: Clathrin expression increased during liver tumorigenesis in humans and mice. CLTC knockdown cells responded to TGF-beta phosphorylating SMADs (canonical signalling) but showed impairment in the anti-apoptotic signals (EGFR transactivation). Experiments of loss or gain of function in HCC cells reveal an essential role for clathrin in inhibiting TGF-beta-induced apoptosis and upregulation of its pro-apoptotic target NOX4. Autocrine TGF-beta signalling in invasive HCC cells upregulates CLTC expression, switching its role to pro-tumorigenic. A positive correlation between TGEB1 and CLTC was found in HCC cells and patients. Patients expressing high levels of TGEB1 and CLTC had a worse prognosis and lower overall survival. Conclusions: This work describes a novel role for clathrin in liver tumorigenesis, favouring non-canonical pro-tumorigenic TGF-beta pathways. CLTC expression in human HCC samples could help select patients that would benefit from TGF-beta-targeted therapy. Lay summary: Clathrin heavy-chain expression increases during liver tumorigenesis in humans (CLTC) and mice (Mc), altering the cellular response to TGF-beta in favour of anti-apoptotic/pro-tumorigenic signals. A positive correlation between TGEB1 and CLTC was found in HCC cells and patients. Patients expressing high levels of TGEB1 and CLTC had a worse prognosis and lower overall survival. CLTC expression in HCC human samples could help select patients that would benefit from therapies targeting TGF-beta. (C) 2019 European Association for the Study of the Liver. Published by Elsevier B

The role of Caveolin-1 in the TGF-β-induced signalling in hepatocytes

[eng] The transforming growth factor-beta (TGF-β) is a cytokine involved in many physiological and pathological processes. Its dual role has been described in the liver, where it can either induce pro- or anti-apoptotic signals. The survival signals are in great extent mediated by the epidermal growth factor (EGFR). Caveolin-1 (CAV1) is a structural protein of the plasma membrane involved in the regulation of different signalling processes and receptor trafficking, as the TGF-β ones (TβRs). Traditionally, CAV1 has been associated with downregulation of the TGF-β signalling, given its presence in SMAD7 and SMURF2 positive vesicles. The great variety of functions associated with CAV1 has generated great controversy, as it acts differently depending on cell type. Regarding its role in the liver, CAV1 has been related to the SAMD-independent signals mediated by TGF-β and as an oncogene in hepatocellular carcinoma (HCC, its expression levels are increased in less differentiated tumours). Given the previously mentioned role of CAV1 in TβRs traffic and in the modulation of distinct signalling pathways, when we started this work, we hypothesized that the levels of CAV1 could be modulating the response to TGF-β in hepatocytes and in HCC cells, regulating the balance among pro- and anti-apoptotic signals induced by TGF-β in hepatocytes. Given this hypothesis, we proposed as an objective to study the role of CAV1 in the TGF-β-induced signalling in hepatocytes and in HCC cells. We decided to use an experimental cell model based on immortalized neonatal hepatocytes from CAV1-deficient mice (Cav1-/-) and we analyzed its response to TGF-β. We also used HCC cells in which CAV1 expression had been silenced or over-expressed, in regard to its basal expression levels. We observed that the lack of CAV1 both in foetal hepatocytes and in HCC cells impairs TGF-β-mediated EGFR transactivation, and in this way, cells are more sensitive to TGF-β pro-apoptotic effects. This is due to the fact that CAV1 is required for the proper activation of the metalloprotease TACE/ADAM17 by TGF-β, being responsible for the shedding of the EGFR ligands present in the plasma membrane as pro-ligands. Another important conclusion that we got was that the localization of TACE/ADAM17 in lipid raft domains is crucial for its activation, and this also requires CAV1. In HCC cells, CAV1 expression levels also determine the response to TGF-β in terms of death induction, but not in regard to cell cycle arrest. However, the final balance results in higher inhibition of the clonal growth by TGF-β when CAV1 expression is low. Another important result is that the induction of the death mediators NOX4, BMF and BIM by TGF-β is diminished when CAV1 expression levels in HCC cells are high. Finally, we have observed that CAV1 is required by the activation of Src and the NADPH oxidase-NOX1, both necessary for the activation of TACE/ADAM17 by TGF-β, being this the mechanism that explains the deficient EGFR transactivation observed both in hepatocytes and in HCC cells when CAV1 expression is low. To sum up this work, we can say that CAV1 levels condition the TGF-β-mediated anti-apoptotic response through the EGFR pathway. In HCC cells, this might be translated into a switch in the role of TGF-β: from anti- to pro-tumourigenic.[spa] El factor de crecimiento transformante-beta (TGF-β) es una citocina involucrada en distintos procesos fisiológicos y patológicos. En hígado se ha descrito su papel dual; ya que puede inducir tanto señales pro- como anti-apoptóticas. Las señales de supervivencia son mediadas principalmente por la vía del receptor del factor de crecimiento epidérmico (EGFR). Por otro lado, caveolina-1 (CAV1) es una proteína estructural de la membrana plasmática asociada a la regulación de distintos procesos como señalización celular o tráfico de receptores, como los del TGF-β (TβRs). Tradicionalmente, CAV1 ha sido asociada con la regulación a la baja de la señalización mediada por TGF-β, dada su presencia en vesículas positivas para SMAD7 y SMURF2. En relación a su papel en el hígado, CAV1 se ha relacionado con las señales SMAD-independientes mediadas por TGF-β y como un oncogén en hepatocarcinogénesis (sus niveles de expresión se incrementan en tumores menos diferenciados y con mayor capacidad metastásica). Cuando empezamos este trabajo hipotetizamos que los niveles de CAV1 podían estar modulando la respuesta a TGF-β en hepatocitos y en células de carcinoma hepatocelular (HCC), condicionando el equilibrio entre señales pro- y anti-apoptóticas inducidas por TGF-β en hepatocitos. Nos planteamos como objetivo estudiar el papel de CAV1 en la señalización inducida por TGF-β en hepatocitos y en células tumorales de hígado. Decidimos utilizar un modelo de hepatocitos procedentes de ratones deficientes en CAV1 (Cav1-/-) y analizar su respuesta al TGF-β. También usamos células de HCC en las cuales se había silenciado o sobre-expresado CAV1, en función de su expresión basal. Como resultado obtuvimos que la falta de CAV1 tanto en hepatocitos como en células tumorales de hígado impide de transactivación del EGFR por parte del TGF-β, y de esta manera las células son más sensibles a los efectos pro-apoptóticos mediados por el TGF-β. Esto es debido a que CAV1 se requiere para la correcta activación de la metaloproteasa TACE/ADAM17 por parte de TGF-β. Otra conclusión importante a la que llegamos es que la correcta distribución de TACE/ADAM17 en dominios lipídicos de membrana es importante para su activación, y que para ello requiere CAV1. En células de HCC, los niveles de CAV1 también determinan la respuesta al TGF-β en términos de inducción de muerte, pero no en relación a sus funciones citostáticas (mediando parada de ciclo celular). Otra conclusión importante a la que hemos llegado es que la inducción de los mediadores de muerte NOX4, BMF y BIM por parte de TGF-β se ve disminuida cuando los niveles de expresión de CAV1 en células tumorales de hígado son altos. Finalmente, hemos observado que CAV1 se requiere para la activación de Src y de la NADPH oxidasa NOX1, ambos necesarios para la activación de TACE/ADAM17 por parte de TGF-β, siendo este el mecanismo de la deficiente transactivación del EGFR. Como conclusión final de este trabajo, podemos decir que los niveles de CAV1 condicionan la respuesta anti-apoptótica inducida por TGF-β a través del EGFR. En células de HCC, esto se traduce en un cambio en el papel del TGF-β: de anti- a pro-tumorigénico

Caveolin-1-dependent activation of the metalloprotease TACE/ADAM17 by TGF-β in hepatocytes requires activation of Src and the NADPH oxidase NOX1

Transforming growth factor-β (TGF-β) plays a dual role in hepatocytes, inducing both pro- and anti-apoptotic responses, the balance between which decides cell fate. Survival signals are mediated by the epidermal growth factor receptor (EGFR) pathway, which is activated by TGF-β. We have previously shown that caveolin-1 (CAV1) is required for activation of the metalloprotease tumour necrosis factor (TNF)-α-converting enzyme/a disintegrin and metalloproteinase 17 (TACE/ADAM17), and hence transactivation of the EGFR pathway. The specific mechanism by which TACE/ADAM17 is activated has not yet been determined. Here we show that TGF-β induces phosphorylation of sarcoma kinase (Src) in hepatocytes, a process that is impaired in Cav1(-/-) hepatocytes, coincident with a decrease in phosphorylated Src in detergent-resistant membrane fractions. TGF-β-induced activation of TACE/ADAM17 and EGFR phosphorylation were blocked using the Src inhibitor PP2. Cav1(+/+) hepatocytes showed early production of reactive oxygen species (ROS) induced by TGF-β, which was not seen in Cav1(-/-) cells. Production of ROS was inhibited by both the NADPH oxidase 1 (NOX1) inhibitor STK301831 and NOX1 knock-down, which also impaired TACE/ADAM17 activation and thus EGFR phosphorylation. Finally, neither STK301831 nor NOX1 silencing impaired Src phosphorylation, but PP2 blocked early ROS production, showing that Src is involved in NOX1 activation. As expected, inhibition of Src or NOX1 increased TGF-β-induced cell death in Cav1(+/+) cells. In conclusion, CAV1 is required for TGF-β-mediated activation of TACE/ADAM17 through a mechanism that involves phosphorylation of Src and NOX1-mediated ROS production.This work was supported by grants from the SpanishMinistry of Economy and Competitiveness (co-fundedby FEDER funds and the European Regional Development Fund: A Way To Build Europe) (BFU2012-35538 and ISCIII-RTICC: RD12-0036-0029 to I.F.,SAF2013-43713R to P.M. -S. and BFU2012-33932 toG.E.), the Agencia de Gestio d’Ajuts Universitaris i de Recerca, Generalitat de Catalunya (2014SGR0334 toI.F.), and the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007–2013) under REA grant agreement number PITN-GA-2012-316549 (IT LIVER) toI.F. J.M. -C. was the recipient of a pre-doctoral fellowship from the Ministry of Education, Culture and Sport, Spain (AP2010-3036). The I.F. research group is supported by the European Cooperation in Science and Technology (COST Action BM1203/EUROS).Peer Reviewe

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

The level of caveolin-1 expression determines response to TGF-ß as a tumor suppressor in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is a heterogeneous tumour associated with poor prognostic outcome. Caveolin-1 (CAV1), a membrane protein involved in the formation of caveolae, is frequently overexpressed in HCC. Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine having a dual role in hepatocarcinogenesis: inducer of apoptosis at early phases, but pro-tumourigenic once cells acquire mechanisms to overcome its suppressor effects. Apoptosis induced by TGF-β is mediated by upregulation of the NADPH oxidase NOX4, but counteracted by transactivation of the epidermal growth factor receptor (EGFR) pathway. Previous data suggested that CAV1 is required for the anti-apoptotic signals triggered by TGF-β in hepatocytes. Whether this mechanism is relevant in hepatocarcinogenesis has not been explored yet. Here we analysed the TGF-β response in HCC cell lines that express different levels of CAV1. Accordingly, stable CAV1 knockdown or overexpressing cell lines were generated. We demonstrate that CAV1 is protecting HCC cells from TGF-β-induced apoptosis, which attenuates its suppressive effect on clonogenic growth and increases its effects on cell migration. Downregulation of CAV1 in HLE cells promotes TGF-β-mediated induction of the pro-apoptotic BMF, which correlates with upregulation of NOX4, whereas CAV1 overexpression in Huh7 cells shows the opposite effect. CAV1 silenced HLE cells show attenuation in TGF-β-induced EGFR transactivation and activation of the PI3K/AKT pathway. On the contrary, Huh7 cells, which do not respond to TGF-β activating the EGFR pathway, acquire the capacity to do so when CAV1 is overexpressed. Analyses in samples from HCC patients revealed that tumour tissues presented higher expression levels of CAV1 compared with surrounding non-tumoural areas. Furthermore, a significant positive correlation among the expression of CAV1 and TGFB1 was observed. We conclude that CAV1 has an essential role in switching the response to TGF-β from cytostatic to tumourigenic, which could have clinical meaning in patient stratification

The level of caveolin-1 expression determines response to TGF-ß as a tumor suppressor in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is a heterogeneous tumour associated with poor prognostic outcome. Caveolin-1 (CAV1), a membrane protein involved in the formation of caveolae, is frequently overexpressed in HCC. Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine having a dual role in hepatocarcinogenesis: inducer of apoptosis at early phases, but pro-tumourigenic once cells acquire mechanisms to overcome its suppressor effects. Apoptosis induced by TGF-β is mediated by upregulation of the NADPH oxidase NOX4, but counteracted by transactivation of the epidermal growth factor receptor (EGFR) pathway. Previous data suggested that CAV1 is required for the anti-apoptotic signals triggered by TGF-β in hepatocytes. Whether this mechanism is relevant in hepatocarcinogenesis has not been explored yet. Here we analysed the TGF-β response in HCC cell lines that express different levels of CAV1. Accordingly, stable CAV1 knockdown or overexpressing cell lines were generated. We demonstrate that CAV1 is protecting HCC cells from TGF-β-induced apoptosis, which attenuates its suppressive effect on clonogenic growth and increases its effects on cell migration. Downregulation of CAV1 in HLE cells promotes TGF-β-mediated induction of the pro-apoptotic BMF, which correlates with upregulation of NOX4, whereas CAV1 overexpression in Huh7 cells shows the opposite effect. CAV1 silenced HLE cells show attenuation in TGF-β-induced EGFR transactivation and activation of the PI3K/AKT pathway. On the contrary, Huh7 cells, which do not respond to TGF-β activating the EGFR pathway, acquire the capacity to do so when CAV1 is overexpressed. Analyses in samples from HCC patients revealed that tumour tissues presented higher expression levels of CAV1 compared with surrounding non-tumoural areas. Furthermore, a significant positive correlation among the expression of CAV1 and TGFB1 was observed. We conclude that CAV1 has an essential role in switching the response to TGF-β from cytostatic to tumourigenic, which could have clinical meaning in patient stratification

The level of caveolin-1 expression determines response to TGF-ß as a tumor suppressor in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is a heterogeneous tumour associated with poor prognostic outcome. Caveolin-1 (CAV1), a membrane protein involved in the formation of caveolae, is frequently overexpressed in HCC. Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine having a dual role in hepatocarcinogenesis: inducer of apoptosis at early phases, but pro-tumourigenic once cells acquire mechanisms to overcome its suppressor effects. Apoptosis induced by TGF-β is mediated by upregulation of the NADPH oxidase NOX4, but counteracted by transactivation of the epidermal growth factor receptor (EGFR) pathway. Previous data suggested that CAV1 is required for the anti-apoptotic signals triggered by TGF-β in hepatocytes. Whether this mechanism is relevant in hepatocarcinogenesis has not been explored yet. Here we analysed the TGF-β response in HCC cell lines that express different levels of CAV1. Accordingly, stable CAV1 knockdown or overexpressing cell lines were generated. We demonstrate that CAV1 is protecting HCC cells from TGF-β-induced apoptosis, which attenuates its suppressive effect on clonogenic growth and increases its effects on cell migration. Downregulation of CAV1 in HLE cells promotes TGF-β-mediated induction of the pro-apoptotic BMF, which correlates with upregulation of NOX4, whereas CAV1 overexpression in Huh7 cells shows the opposite effect. CAV1 silenced HLE cells show attenuation in TGF-β-induced EGFR transactivation and activation of the PI3K/AKT pathway. On the contrary, Huh7 cells, which do not respond to TGF-β activating the EGFR pathway, acquire the capacity to do so when CAV1 is overexpressed. Analyses in samples from HCC patients revealed that tumour tissues presented higher expression levels of CAV1 compared with surrounding non-tumoural areas. Furthermore, a significant positive correlation among the expression of CAV1 and TGFB1 was observed. We conclude that CAV1 has an essential role in switching the response to TGF-β from cytostatic to tumourigenic, which could have clinical meaning in patient stratification