Differential intracellular signalling induced by TGF-β in rat adult hepatocytes and hepatoma cells: Implications in liver carcinogenesis

Abstract

The transforming growth factor-beta (TGF-β) regulates hepatocyte growth, inhibiting proliferation and inducing apoptosis. Indeed, escaping from the TGF-β suppressor actions might be a prerequisite for liver tumour progression. In this work we show that TGF-β plays a dual role in regulating apoptosis in FaO rat hepatoma cells, since, in addition to its pro-apoptotic effect, TGF-β also activates survival signals, such as AKT, the epidermal growth factor receptor (EGFR) being required for its activation. TGF-β induces the expression of the EGFR ligands transforming growth factor-alpha (TGF-α) and heparin-binding EGF-like growth factor (HB-EGF) and induces intracellular re-localization of the EGFR. Cells that overcome the apoptotic effects of TGF-β undergo morphological changes reminiscent of an epithelial-mesenchymal transition (EMT) process. In contrast, TGF-β does not activate AKT in adult hepatocytes, which correlates with lack of EGFR transactivation and no response to EGFR inhibitors. Although TGF-β induces TGF-α and HB-EGF in adult hepatocytes, these cells show very low expression of TACE/ADAM 17 (TNF-α converting enzyme), which is required for EGFR ligand proteolysis and activation. Furthermore, adult hepatocytes do not undergo EMT processes in response to TGF-β, which might be due, at least in part, to the fact that F-actin re-organization induced by TGF-β in FaO cells require the EGFR pathway. Finally, results indicate that EGFR transactivation does not block TGF-β-induced cell cycle arrest in FaO cells, but must be interfering with the pro-apoptotic signalling. In conclusion, TGF-β is a suppressor factor for adult quiescent hepatocytes, but not for hepatoma cells, where it plays a dual role, both suppressing and promoting carcinogenesis.

Introduction

Apoptosis represents a physiological way to eliminate the excess of cells during both liver development and regeneration [1]. Imbalance between cell proliferation and death pathways leads to loss of tissue homeostasis and onset of various diseases. Indeed, insufficient apoptosis has been associated with development and progression of tumours of the liver and the biliary tree [1], [2], [3]. Transforming growth factor-beta (TGF-β) is an important regulatory factor in hepatocytes, inhibiting proliferation and inducing cell death [4]. Recent studies have indicated that TGF-β dysregulation is a late event in human hepatocarcinogenesis. Indeed, the escape from the antiproliferative and pro-apoptotic actions of TGF-β might be a prerequisite for tumour progression [5]. However, perturbations at receptor or Smad levels do not appear to be as frequent as they are in colon or pancreatic cancer [4]. Thus, other possibilities to disrupt TGF-β signalling might exist and they remain unexplored. Furthermore, in addition to its role in proliferation control, TGF-β is an important modulator of cell migration, immune response and angiogenesis. It is now believed that metastasis of many different types of tumour cells would require TGF-β activity and that, in the context of an advanced stage of disease, TGF-β could play a pro-oncogenic role [6], [7], [8]. In fact, TGF-β has been described as a key regulator of epithelial-mesenchymal transitions (EMT), under defined conditions [9], [10], [11]. TGF-β up-regulates Snail and down-regulates E-cadherin, induces a replacement of cytokeratin 18 by vimentin as protein of intermediate filaments and re-organizes F-actin, changes that contribute to loss of cell adhesion and increase in cell migration [9], [10], [11]. Indeed, overcoming the TGF-β suppressor effects might allow liver tumour cells to respond to this cytokine increasing migration and contributing to cell dissemination and metastasis. However, whether or not hepatoma cells are able to respond to TGF-β inducing EMT has not been explored yet.

During the last few years, data have accumulated suggesting that TGF-β actions may be modulated by other growth factors or cytokines. The epidermal growth factor (EGF) is an important survival signal for TGF-β-induced apoptosis in hepatocytes [12]. Phosphatydilinositol-3 Kinase (PI-3K) mediates the effect of EGF on TGF-β-induced death by acting upstream from the mitochondrial changes [13], [14]. Indeed, some autocrine signals, such as EGF Receptor (EGFR) ligands, might protect liver tumour cells from TGF-β-induced apoptosis. We have previously found that TGF-β induces apoptosis in foetal rat hepatocytes, through a mechanism dependent on “de novo” protein synthesis and production of reactive oxygen species (ROS) [15]. However, a subpopulation of these cells survives, concomitant with changes in morphology and phenotype, reminiscent of an EMT process [16]. Recent works have suggested that the response to TGF-β might be very complex, involving both pro-apoptotic and survival signals [17], [18]. Interestingly, TGF-β is able to transiently activate PI-3K/Akt in foetal hepatocytes by a mechanism dependent on EGF Receptor and c-SRC activities [19]. Inhibiting EGF Receptor greatly increases the apoptosis induced by TGF-β in those cells. However, whether or not this mechanism is specific for foetal, proliferative-undifferentiated cells, is an unsolved question yet. If occurring in hepatoma cells, this dual response to TGF-β might convert this cytokine in a pro-tumorigenic factor in hepatocarcinogenesis.

Considering these results together, the aim of this work has been to analyse the intracellular pathways induced by TGF-β in hepatoma cells, comparing them with those observed in adult hepatocytes, with particular interest in the transactivation of the EGFR pathway as a mechanism of apoptotis protection. Furthermore, we have analyzed whether this protection might facilitate additional responses to TGF-β, which would favour tumour progression.