Hepatocellular carcinoma (HCC) is the most common primary liver cancer and represents a major global health problem with increasing incidence and significant cancer-related morbidity and mortality. To date, only a limited number of patients is eligible for curative treatment options, and current therapies for advanced stage HCC have limited efficacy with significant side effects. Consequently, there is an urgent medical need for additional systemic therapeutic options (1).
The development and progression of HCC is a multistage process in which a chronic insult (e.g. alcohol abuse, viral hepatitis, obesity, cholestatic liver injury) induces liver injury characterized by a micro-environment abundant of various types of cellular stress, including endoplasmic reticulum stress, cellular DNA damage, necrosis of damaged hepatocytes, and oxidative stress with reactive oxygen species (ROS) production. In the liver, ROS are generated in response to a wide variety of endogenous and exogenous stimuli (2–6). ROS species play multiple biological roles and are therefore involved in a large number of physiological phenomena, such as host defense, but also posttranslational protein processing, cellular signalling, regulation of gene expression, and cellular differentiation (7–9). Their production is strongly regulated to avoid the harmful effects of a redox imbalance. In a healthy liver, antioxidant systems such as superoxide dismutase and catalase efficiently remove excess of ROS to ensure cellular homeostasis. In contrast, during chronic liver disease, increased ROS production, as well as decreased activity of the antioxidant systems, result in oxidative stress (10, 11). Hepatic carcinogenesis is believed to involve ROS-induced DNA damage and/or mitogenic signalling. Liver injury-mediated ROS production has been shown to contribute to mutagenesis and genomic instability, resulting in the induction of apoptosis and tissue damage. However, these mutations may also have the potential to activate oncogenes and/or inactivate tumor suppressors, thereby initiating oncogenesis. Thus, chronic liver disease-mediated persistent hepatocyte death establishes a microenvironment that favors survival and proliferation of hepatocytes harboring oncogenic mutations. In addition, when activated, liver resident Kupffer cells (KCs) release an ‘oxidative burst’ of superoxide and numerous other products with cytotoxic, pro-inflammatory and growth-promoting activity, and attract circulating immune cells to the liver; all contributing to the establishment of a tumor-promoting microenvironment. ROS also contribute to cancer development and progression, by acting as second messengers in disease-driven intracellular signaling pathways controlling cell proliferation, survival, motility and invasiveness, as well as by controlling the reactivity of stromal components that are fundamental for cancer development and dissemination, inflammation, tissue repair, and de novo angiogenesis (11–18).
A number of mechanisms are involved in ROS production. NADPH oxidase (NOX) proteins represent the major non-mitochondrial source of ROS. In addition, there is a growing body of evidence demonstrating that one major effect of inflammation-induced cytokine secretion is the upregulation of ROS-producing NOX isoforms. Seven homologues of the cytochrome subunit of NOX have been described (NOX1-5 and DUOX1-2). NADPH oxidases, with the exception of NOX5, are multimeric complexes, dissociated when inactive, consisting of cytosolic factors (p47phox, NOXO1, p67phox, NOXA1, p40phox, and Rac2) and a redox membrane core. They share the capacity to transport electrons from NADPH to oxygen across the plasma membrane, and to generate superoxide and other downstream ROS. Activation mechanisms, subcellular localization and tissue distribution are highly isoform-dependent (7, 8). Various isoforms, such as NOX1, NOX2, and NOX4, are distinctively expressed in specific hepatic cell types, including Kupffer cells (KCs), hepatic stellate cells (HSCs), endothelial cells, hepatocytes, and infiltrating leukocytes. Genetic and pharmacological inhibition of NOX1 has been shown to reduce inflammation and fibrosis in experimental liver disease (5, 6). The dual NOX1/4 inhibitor GKT137831 attenuated liver fibrosis, ROS production and the expression of several fibrotic, inflammatory and proliferative genes in different mouse models (19–21). In a phase I clinical trial, GKT137831 was found to be safe and well tolerated (22). Non-phagocytic NOX1 is constitutively expressed, although its genetic transcription is upregulated by hypoxia, growth factors, growth-related agonists, inflammatory mediators and pathogen-associated molecular pattern molecules (8, 23). Recent research on human HCC samples showed that high NOX1 levels are correlated with a poor prognosis (24, 25). The involvement of NOX1 in cell proliferation, tumor growth, cell motility, epithelial-mesenchymal transition (EMT) and matrix metalloproteinase-2 production has been shown in in vitro studies (13, 14). NOX1-/- knockout mice and mice with myeloid NOX1 disruption were reported to develop fewer and smaller tumors following diethylnitrosamine (DEN) injection. DEN-injected wild type (WT) mice that received the NOX-1 inhibitor ML171 also developed fewer and smaller hepatic tumor nodules, compared to their vehicle-treated counterparts (26). This antitumor effect observed in mice treated with a NOX1-specific inhibitor may open up new avenues for HCC treatment in humans.
In order to further investigate the role of NOX1 and its potential as a therapeutic target in the treatment of HCC, we studied the effect of the NOX1 inhibitor GKT771 (provided by Genkyotex) in a DEN-induced experimental HCC mouse model, with specific focus on the inflammatory tumor microenvironment (TME).