NADPH Oxidase NOX1 Controls Autocrine Growth of Liver Tumor Cells through Up-regulation of the Epidermal Growth Factor Receptor Pathway

Patricia Sancho,Isabel Fabregat

doi:10.1074/jbc.m110.114280

Abstract

FaO rat hepatoma cells proliferate in the absence of serum through a mechanism that requires activation of the epidermal growth factor receptor (EGFR) pathway. The aim of this work was to analyze the molecular mechanisms that control EGFR activation in these and other liver tumor cells. Reactive oxygen species production is observed a short time after serum withdrawal in FaO cells, coincident with up-regulation of the NADPH oxidase NOX1. NOX1-targeted knockdown, the use of antioxidants, or pharmacological inhibition of NADPH oxidase attenuates autocrine growth, coincident with lower mRNA levels of EGFR and its ligand transforming growth factor-alpha (TGF-alpha) and a decrease in phosphorylation of EGFR. EGFR-targeted knockdown induces similar effects on cell growth and downstream signals to those observed in NOX1-depleted cells. Early NOX1 activation induces both a feedback-positive loop via an Src-ERK pathway that up-regulates its own levels, and a parallel signaling pathway through p38 MAPK and AKT resulting in EGFR and TGF-alpha up-regulation. Human hepatocellular carcinoma cell lines, but not non-tumoral hepatocytes, show autocrine growth upon serum withdrawal, which is also coincident with NOX1 up-regulation that mediates EGFR and TGF-alpha expression. The use of antioxidants, or pharmacological inhibition of NADPH oxidase, effectively attenuates autocrine growth in hepatocellular carcinoma cell lines. In summary, results presented in this study indicate that NOX1 might control autocrine cell growth of liver tumor cells through regulation of the EGFR pathway.

Highlights

Been associated with the pathogenesis of this liver cancer [2], but it is generally accepted that in all cases a deregulation in the balance between proliferation and cell death is observed [3], which is mainly caused by overactivation of survival pathways, frequently related to acquisition of autocrine growth factor signaling [4]
When cells were incubated in the presence of general antioxidants, the NADPH oxidase inhibitor apocynin, or a Rac1 inhibitor, autocrine proliferation was attenuated (Fig. 1D), indicating that reactive oxygen species (ROS) produced by an NADPH oxidase system, Rac1-dependent, are required for autocrine proliferation of FaO rat hepatoma cells
To determine whether NOX1 might play a role on the autocrine growth of these cells, we decided to knock down its expression through an siRNAtargeted approach

Summary

Introduction

Been associated with the pathogenesis of this liver cancer [2], but it is generally accepted that in all cases a deregulation in the balance between proliferation and cell death is observed [3], which is mainly caused by overactivation of survival pathways, frequently related to acquisition of autocrine growth factor signaling [4]. Many liver tumor cells, such as FaO rat hepatoma cells, express high levels of EGFR ligands as compared with normal adult hepatocytes [7]. The NOX family includes seven different members as follows: NOX1–5, DUOX1, and DUOX2 [10] These enzymes are widely expressed in numerous tissues and play different roles, including cell signaling, gene expression regulation, cell death, differentiation, and growth [11]. This variety of functions implies many different pathological situations involving NADPH oxidases in different organs, including the liver, where they have been related to fibrosis, cirrhosis, and cancer [12]. Much less is known about the role of these NOX enzymes in liver cell proliferation neither in normal nor in tumor cells

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