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

Abstract

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-β (TGF-β) 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-β-induced signalling in liver cells and its relevance in liver cancer. 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-β and EGFR signalling in vitro. Bioinformatic analysis was used to determine the effect of CLTC and TGFB1 expression on prognosis and overall survival in patients with hepatocellular carcinoma (HCC). Clathrin expression increased during liver tumorigenesis in humans and mice. CLTC knockdown cells responded to TGF-β 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-β-induced apoptosis and upregulation of its pro-apoptotic target NOX4. Autocrine TGF-β signalling in invasive HCC cells upregulates CLTC expression, switching its role to pro-tumorigenic. A positive correlation between TGFB1 and CLTC was found in HCC cells and patients. Patients expressing high levels of TGFB1 and CLTC had a worse prognosis and lower overall survival. This work describes a novel role for clathrin in liver tumorigenesis, favouring non-canonical pro-tumorigenic TGF-β pathways. CLTC expression in human HCC samples could help select patients that would benefit from TGF-β-targeted therapy. Clathrin heavy-chain expression increases during liver tumorigenesis in humans (CLTC) and mice (Cltc), altering the cellular response to TGF-β in favour of anti-apoptotic/pro-tumorigenic signals. A positive correlation between TGFB1 and CLTC was found in HCC cells and patients. Patients expressing high levels of TGFB1 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-β.