Oxidative stress in obesity-associated hepatocellular carcinoma: sources, signaling and therapeutic challenges

Abstract

Obesity affects more than 650 million individuals worldwide and is a well-established risk factor for the development of hepatocellular carcinoma (HCC). Oxidative stress can be considered as a bona fide tumor promoter, contributing to the initiation and progression of liver cancer. Indeed, one of the key events involved in HCC progression is excessive levels of reactive oxygen species (ROS) resulting from the fatty acid influx and chronic inflammation. This review provides insights into the different intracellular sources of obesity-induced ROS and molecular mechanisms responsible for hepatic tumorigenesis. In addition, we highlight recent findings pointing to the role of the dysregulated activity of BCL-2 proteins and protein tyrosine phosphatases (PTPs) in the generation of hepatic oxidative stress and ROS-mediated dysfunctional signaling, respectively. Finally, we discuss the potential and challenges of novel nanotechnology strategies to prevent ROS formation in obesity-associated HCC.

Fig. 1. Progression from nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH), fibrosis, and HCC.

NAFLD is characterized by excessive lipid accumulation in lipid droplets in the cytosol of hepatocytes. Lipotoxicity causes hepatocyte death and activation and proliferation of Kupfer cells, as well as recruitment of other immune cells to the liver causing inflammation and accelerating the progression from simple steatosis to NASH. The inflammation and tissue damage lead to pathological wound healing with an accumulation of extracellular matrix proteins characterizing the fibrosis/cirrhosis, which is usually accompanied by changes in the microenvironment of the liver affecting the genetics and cellular signaling, favoring the emergence of hepatocellular carcinoma cells and development of small tumors at the early developmental stage of HCC. Without intervention, early HCC progresses to more advanced stages of HCC. About 7% of the patients with NAFLD/NASH can develop HCC without cirrhosis, but the mechanism of this progression is currently unknown.

Fig. 2. ROS production in different cell compartments and their contributions to HCC development.

Nicotinamide adenine dinucleotide phosphate oxidases (NOX) are membrane-bound enzymes that generates ^•O₂⁻ in the cytosol. Mitochondria utilize oxygen to produce ATP during oxidative phosphorylation (OXPHOS) and a percentage of oxygen is converted to ^•O₂⁻, which is typically scavenged by a family of mitochondrial superoxide dismutase (SOD) to H₂O₂. In peroxisome, flavin-containing oxidases transfer electrons from various metabolites and reduce oxygen to H₂O₂. This class of enzymes includes acyl-CoA oxidases (ACOX) and xanthine oxidoreductase (XOD). Under certain posttranslational modifications, XOD also reduces O₂ to ^•O₂⁻. The nitric oxide synthase (iNOS) is also present in peroxisomes of hepatocytes and can generate ^•O ⁻₂. Besides, the peroxisomes environment is rich in heme-containing proteins that produce ^•OH from H₂O₂ by the Fenton reaction. In the endoplasmic reticulum (ER) the oxidative protein folding process involves enzymes as protein disulfide isomerase (PDI) and ER oxidoreductin 1 (ERO1) that produces H₂O₂. NADPH oxidase 4 (NOX4) and cytochrome P450 (CYP) are also present in the ER, representing other sources of H₂O₂, which is then scavenged by peroxidases GPX7 and GPX8. Also in the ER, the accumulation of unfolded proteins leads to ER stress and stimulates ROS production. In the nucleus, NOX is also a ^•O₂⁻ source, which can be scavenged by SOD to H₂O₂. Excessive production of ROS in these different cell compartments can overwhelm the antioxidant systems, causing oxidative damage to different cellular components, affecting the cell functions, and can eventually lead to carcinogenesis.

Fig. 3. Redox-regulated transcription factors.

Reactive oxygen species (ROS) modulate the activation of several transcription factors involved with hepatocellular carcinoma development by indirectly interacting with proteins that regulate the activity of the transcription factors (NRF2, NF-kB, and HIF) or by directly reacting with the transcription factor (p53). These redox-regulated transcription factors affect diverse biological activities, including ROS and xenobiotic detoxification, inflammation, cell proliferation, apoptosis, adaptation to hypoxia, metabolism, angiogenesis, cell cycle regulation, and DNA repair. Some of these biological activities are accompanied by changes in the ROS levels, mediated by changes in the expression of different antioxidant and prooxidant enzymes and enzymes involved in the synthesis of molecules involved in the oxidative status of the cells, as GSH and NADPH. KEAP1 kelch-like ECH-associated protein 1, NRF2 nuclear factor (erythroid-derived 2)–related factor-2, NF-κB nuclear factor kappa B, IkB NF-κB inhibitor, IKK NF-κB inhibitor kinase, HIF hypoxia-inducible factor, PHD prolyl hydroxylase, p53 tumor protein p53, NQO1 NAD(P)H quinone dehydrogenase 1, HO1 heme oxygenase 1, GPX glutathione peroxidase, TXN thioredoxin, GSH reduced glutathione, NADPH nicotinamide adenine dinucleotide phosphate, SOD superoxide dismutase, CAT catalase, TRX 1/2 thioredoxin 1/2, iNOS inducible nitric oxide synthase, COX-2 cyclooxygenase 2, Cyp7b 25-hydroxycholesterol 7-alpha-hydroxylase, Cyp2E1 cytochrome P450 2E1, Cyp2c11 cytochrome P450, subfamily 2, polypeptide 11, BAX BCL-2-like protein 4, PUMA p53 upregulated modulator of apoptosis, p66^shc Src homology/collagen (Shc) adapter protein.

Fig. 4. BCL-2 family proteins are components of programmed cell death that can also regulate cellular metabolism.

Liver-specific deletion of BCL-2 interacting mediator of cell death (BIM) attenuates mitochondrial oxidative stress, hepatic steatosis, and ameliorates hepatic fatty acid metabolism in a mouse model of diet-induced obesity. Global BIM deletion increases mitochondrial respiration and lipid oxidation. Truncated phosphorylation-mediated inactivation of BH3 interacting-domain death agonist (BID) reduces carnitine palmitoyltransferase 1 (CPT-1), impairing fatty acid oxidation and promoting the accumulation of toxic lipid metabolites. A loss-of-function study showed that the proapoptotic BCL-2-associated death promoter (BAD) directly interacts with glucokinase (GK) and regulates hepatic energy metabolism by reducing fatty acid oxidation and gluconeogenesis while promoting glycolysis. Loss of hepatic BCL-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) results in increased mitochondrial mass, impaired gluconeogenesis, and reduced β-oxidation, and this phenotype is associated with elevated ROS production.

Fig. 5. Protein tyrosine phosphatases (PTPs) are important modulators of the insulin receptor (IR) and PTP oxidation plays a role in insulin resistance.

When insulin binds to the IR it leads to autophosphorylation of tyrosine residues of the IR and activates its exogenous kinase activity, which then further phosphorylates the tyrosine residues on the insulin receptor substrate (IRS) proteins. PTPs remove phosphate groups from phosphorylated tyrosine residues on proteins and inactivate the receptor kinase thereby ceasing the insulin signaling cascade. Reactive oxygen species (ROS) can oxidize and inactivate the PTPs affecting physiologically and pathophysiological insulin signaling.

Fig. 6. Obesity contributes to the development of NASH and HCC by independent mechanisms.

A Obesity is a multifactorial disease that manifests as consequential to environmental influences, mainly excessive consumption of calories (overnutrition) and a lack of physical activity (sedentarism), associated with genetic factors that predispose body fat accumulation. One of the consequences of obesity is an unbalance between the production of oxidant species and antioxidant capacity leading to oxidative stress generated by reactive oxygen species (ROS) and reactive nitrogen species (RNS). B Protein tyrosine phosphatases (PTPs) regulate tyrosine phosphorylation-dependent signal transduction through tyrosine dephosphorylation of protein substrates. The protein structure and presence of cysteine residue in the active site of PTPs with low pKa render members of this family of proteins highly susceptible to oxidation by ROS to the reversible form of sulfenic acid or irreversible forms of sulfinic and sulfonic acid accompanied by conformational changes that inhibit PTP activity and prevent substrate binding. C Obesity is frequently associated with nonalcoholic fatty liver disease (NAFLD), a condition that favors liver oxidative stress. Under this condition, STAT1 and STAT3 phosphatase protein tyrosine phosphatase non-receptor type 2 (PTPN2) is inactivated, thereby increasing STAT1 and STAT3 signaling. While heightened STAT1 signaling is responsible for the recruitment of activated cytotoxic T cells and ensuing NASH and fibrosis, this is not essential for HCC. Rather, STAT3 signaling promotes HCC without NASH and fibrosis.

Fig. 7. Schematic illustration of nanotheronostic agents targeting oxidative stress-dependent pathways in obesity-associated HCC.

Nanomedicine can be used for the delivery of therapeutic agents such as micro(mi)RNA, small interfering(si)RNA, peptides, or antibodies. The drug delivery approach needs to overcome a series of biological barriers to successfully target the molecular mechanisms of ROS production. The nanomedicine strategies should modulate BCL-2 proteins and either enhance ROS inhibitors or decrease ROS activators to prevent dysfunctional PTP activity.