The transforming growth factor‐beta (TGF‐β) family signalling pathways play essential roles in the regulation of different cellular processes, including proliferation, differentiation, migration or cell death, which are essential for the homeostasis of tissues and organs. Because of the diverse and pleiotropic TGF‐β functions, deregulation of its pathways contributes to human disease. In the case of the liver, TGF‐β signalling participates in all stages of disease progression, from initial liver injury through inflammation and fibrosis, to cirrhosis and cancer. TGF‐β has cytostatic and apoptotic effects in hepatocytes, promoting liver differentiation during embryogenesis and physiological liver regeneration. However, high levels of TGF‐β, as a consequence of chronic liver damage, result in activation of stellate cells to myofibroblasts and massive hepatocyte cell death, which contributes to the promotion of liver fibrosis and later cirrhosis. During liver tumorigenesis, TGF‐β may behave as a suppressor factor at early stages; however, there is strong evidence that overactivation of TGF‐β signalling might contribute to later tumour progression, once cells escape from its cytostatic effects. For these reasons, targeting the TGF‐β signalling pathway is being explored to counteract liver disease progression. In this review, we aim to shed light on the state‐of‐the‐art in the signalling pathways induced by TGF‐β that are involved in different stages of liver physiology and pathology.

Different data support a role for the epidermal growth factor receptor (EGFR) pathway during liver regeneration and hepatocarcinogenesis. However, important issues, such as the precise mechanisms mediating its actions and the unique versus redundant functions, have not been fully defined. Here, we present a novel transgenic mouse model expressing a hepatocyte-specific truncated form of human EGFR, which acts as negative dominant mutant (DEGFR) and allows definition of its tyrosine kinase-dependent functions. Results indicate a critical role for EGFR catalytic activity during the early stages of liver regeneration. Thus, after two-thirds partial hepatectomy, DEGFR livers displayed lower and delayed proliferation and lower activation of proliferative signals, which correlated with overactivation of the transforming growth factor-b pathway. Altered regenerative response was associated with amplification of cytostatic effects of transforming growth factor-b through induction of cell cycle negative regulators. Interestingly, lipid synthesis was severely inhibited in DEGFR livers after partial hepatectomy, revealing a new function for EGFR kinase activity as a lipid metabolism regulator in regenerating hepatocytes. In spite of these profound alterations, DEGFR livers were able to recover liver mass by overactivating compensatory signals, such as c-Met. Our results also indicate that EGFR catalytic activity is critical in the early preneoplastic stages of the liver because DEGFR mice showed a delay in the appearance of diethyl-nitrosamine-induced tumors, which correlated with decreased proliferation and delay in the diethyl-nitrosamine-induced inflammatory process. Conclusion: These studies demonstrate that EGFR catalytic activity is critical during the initial phases of both liver regeneration and carcinogenesis and provide key mechanistic insights into how this kinase acts to regulate liver pathophysiology. (HEPATOLOGY 2016;63:604-619) See Editorial on Page 371 T he liver is a unique organ in displaying reparative response and regenerative capacity. However, prolonged liver regeneration as a consequence of Abbreviations: DEN, diethyl-nitrosamine; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; HCC, hepatocellular carcinoma; HGF, hepatocyte growth factor; mRNA, messenger RNA; NADPH, reduced nicotinamide adenine dinucleotide phosphate; PH, partial hepatectomy; qRT-PCR, quantitative reverse-transcriptase polymerase chain reaction; TGF-a/-b, transforming growth factor (a/b); TNF-a, tumor necrosis factor-a; uPA, urokinase-type plasminogen activator; WT, wild type.From the

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.

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Transforming growth factor-b (TGF-b) 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-b. We have previously shown that caveolin-1 (CAV1) is required for activation of the metalloprotease tumour necrosis factor (TNF)-a-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-b induces phosphorylation of sarcoma kinase (Src) in hepatocytes, a process that is impaired in Cav1 À/À hepatocytes, coincident with a decrease in phosphorylated Src in detergent-resistant membrane fractions. TGF-b-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-b, which was not seen in Cav1 À/À 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-b-induced cell death in Cav1 +/+ cells. In conclusion, CAV1 is required for TGF-b-mediated activation of TACE/ADAM17 through a mechanism that involves phosphorylation of Src and NOX1-mediated ROS production.Abbreviations CAV1, caveolin-1; DRM, detergent-resistant membrane; DUOX, dual oxidase; EGFR, epidermal growth factor receptor; NOX, NADPH oxidase; ROS, reactive oxygen species; Src, sarcoma kinase; TACE/ADAM17, tumour necrosis factor a-converting enzyme/a disintegrin and metalloproteinase 17; TGF-b, transforming growth factor b; TNF-a, tumour necrosis factor a. 1300

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.

Hepatocellular carcinoma (HCC) is a major health problem. In human hepatocarcinogenesis, the balance between cell death and proliferation is deregulated, tipping the scales for a situation where antiapoptotic signals are overpowering the death-triggering stimuli. HCC cells harbor a wide variety of mutations that alter the regulation of apoptosis and hence the response to chemotherapeutical drugs, making them resistant to the proapoptotic signals. Considering all these modifications found in HCC cells, therapeutic approaches need to be carefully studied in order to specifically target the antiapoptotic signals. This review deals with the recent relevant contributions reporting molecular alterations for HCC that lead to a deregulation of apoptosis, as well as the challenge of death-inducing chemotherapeutics in current HCC treatment.

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