New experimental approaches to study the relevance of the TGF-β and EGFR signalling during liver tumorigenesis

Abstract

Hepatocellular Carcinoma (HCC) is one of the most common cancers. During hepatocarcinogenesis (a multistep process) there is a disruption of the balance between cell death and survival signalling pathways, where pro-apoptotic molecules are down-regulated/inactivated, and pro-survival pathways are over-activated. Some of these de-regulated signals are the Transforming Growth Factor beta (TGF-β) and the Epidermal Growth Factor Receptor (EGFR) pathway. The EGFR pathway is involved in the development and progression of hepatocarcinogenesis. EGF expression in the liver increases during cirrhosis and plays a role in both cirrhosis and HCC. In HCC cells, TGF-β can up-regulate the expression of different EGFR ligands and activate Src, Akt, and ERKs signalling pathways. The pro-tumorigenic cytokine TGF-β, is not the only pro-inflammatory molecule which EGFR interacts with. HCC is a clear example of inflammatory-related cancer and during the initial stages, exist chronic tissue damage and inflammation in the liver that result in overactivation of EGFR pathway. In addition, in HCC cells when EGFR pathway is down-regulated (by different mechanisms), the suppressor effects of TGF-β are coincident with increased up-regulation of NOX4 (NADPH-oxidases family member). In hepatocytes, NOX4 is required for the mitochondrial-mediated apoptosis induced by TGF-β and it acts as a negative regulator of cell growth in hepatocytes and liver tumour cells. Interestingly, EGF inhibits NOX4 expression acting at transcriptional level on NOX4 promoter. We have previously reported that EGFR pathway impairs the suppressor arm of TGF-β pathway in hepatocytes and liver tumour cells in vitro. Whether this effect may be relevant in vivo has not been explored yet. Furthermore, recent results have emphasized the essential role of intracellular trafficking proteins –caveolin-1 and clathrin– in the regulation of TGF-β and EGF receptors. However, less is known about how these proteins affects TGF-β and EGFR signalling pathways. Recently we reported that caveolin-1 is necessary for TGF-β induction of anti-apoptotic signalling in hepatocytes in a Src/NOX1-dependent manner. Furthermore, caveolin-1 has an essential role in switching the response to TGF-β from cytostatic to tumourigenic in liver tumour cells. Nevertheless, the role of clathrin in the crosstalk between these two signalling pathways and its relevance was barely known.

To clear up the role of the EGFR in the process of hepatocarcinogenesis our group generated a novel transgenic mouse model expressing a hepatocyte-specific truncated form of the human EGFR, which acts as negative dominant mutant (∆EGFR) and allows defining its tyrosine kinase-dependent functions. These mice were treated with carbon tetrachloride (CCl4) to induce liver fibrosis or diethylnitrosamine (DEN) to induce liver tumorigenesis. Results presented here propose that EGFR is crucial in the first stages of liver fibrosis and hepatocarcinogenesis. Interestingly, the most relevant role of EGFR during both processes seems the regulation of the inflammatory liver environment. Hepatocytes and liver tumour cells have the ability to express pro- and/or anti-inflammatory signals whose balance would determinate the liver disease progression. In this process, the crosstalk with non-parenchymal cells, such as Kupffer cells or Hepatic Stellate cells, plays an essential role. Moreover, we show that trafficking proteins could have a significant role during the tumorigenic crosstalk between EGFR and TGF-β. In this context, clathrin may favour a switch of TGF-β to its anti-apoptotic role through EGFR, promoting survival and pro-tumorigenic advantages to liver cancer cells. Indeed, HCC patients present elevated levels of clathrin in tumoral tissue, highlighting its relevance for HCC progression. Chronic liver diseases are the consequence of deregulation in multifactor and multipath networks which articulate the molecular mechanisms that underlie liver tumorigenesis. This thesis proposes new insights about the role of inflammatory process and trafficking receptor proteins in the crosstalk between EGFR and TGF-β during liver chronic diseases.