Role, Targets and Regulation of (de)nitrosylation in Malignancy

Abstract

Nitric oxide (NO) is a free radical that can target cellular biomolecules directly, or by means of the activity of its metabolites (RNS) generated upon reaction with transition metals (e.g., NO⁺), oxygen (e.g., N₂O₃), or superoxide (ONOO⁻). For instance, it is well-documented that NO and RNS affect DNA integrity and mitochondrial physiology, this leading to genetic mutations (1) and damage to the mitochondrial respiratory chain (2, 3), respectively. Processes ranging from apoptosis, angiogenesis, immunity, and neuronal physiology, all show seemingly contradictory behavior in response to NO. Indeed, the relevance of the steady-state NO concentrations represents a key determinant of its biological function. In support to this assumption, it has been demonstrated that cGMP-mediated processes occur at the low nM range, whereas higher NO concentrations cause protein kinase B (PKB)/Akt phosphorylation; stabilization of hypoxia inducible factor (HIF)-1α; phosphorylation of p53 and, at the μM range, they can generate detrimental conditions usually referred as to nitrosative stress (Figure 1). Likewise, in tumor biology, it is now commonly accepted that high NO concentrations mediate apoptosis and cancer growth inhibition, whereas (relatively) low concentrations usually promote tumor growth and proliferation, this supporting the nature of “doubled-edged sword” molecule for NO (4, 5). This dichotomy originates from the observations that the inducible form of NO synthase (iNOS or NOS2) was implicated in the macrophage-mediated tumor killing process (6, 7) (Figure 1). NOS2^−/− mice develop intestinal tumors (8), thereby substantiating the protective role of NOS2 within host defense mechanisms (9, 10). In accordance, a growing body of evidence pointed out that NO-releasing drugs can be toxic for cancer cells. Figure 1. Roles of NO signaling and protein denitrosylation in cancer. Nitric oxide plays different roles in cancer biology depending on its concentration. GSNOR is the main cellular denitrosylase. Counteracting the effects induced by NOS, GSNOR finely modulates protein S-nitrosylation (second panel from the top), which is establishing as the main posttranslational modification underlying NO bioactivity. A disbalance in NO signaling can promote tumor induction, survival and progression. NOS2 deficiency impairs the capability of macrophages to kill cancer cells (Top). Conversely, in conditions of normal (or induced) NOS activity, GSNOR decrease has been linked to many cancer hallmarks, such as: (i) apoptosis and anoikis resistance (due to caspases and, reasonably, FAK-1 S-nitrosylation); (ii) genomic instability (DNA repair impairment, due to AGT S-nitrosylation and degradation); (iii) cells hyperproliferation (via the NO-mediated activation of oncoproteins, such as AKT, RAS, and Src); (iv) angiogenesis (putatively regulated by HIF-1α and TRPs S-nitrosylation). Extreme nitrosative stress conditions—induced, for instance, by NOS overexpression or by the use of NO-donors—activate cell death and are implemented (or physiologically activated in macrophages) to destroy cancer cells (Bottom). NO, nitric oxide; GSNOR, S-nitrosoglutathione reductase; NOS, nitric oxide synthase; FAK1, focal adhesion kinase 1; AGT, O⁶-methylguanine-DNA methyltransferase; HIF-1α, hypoxia-inducible factor-1α; TRP, Transient receptor potential channel. On the other hand, low rate of NO production can promote tumor growth rather than killing. In line with this assumption, the overexpression of NOS isoforms has been detected in a wide range of human tumors. In particular, NOS2 has been found to be upregulated in melanoma, estrogen receptor-negative (ER⁻)-breast cancer, as well as in pancreatic, cervical liver and ovarian cancers (10). Moreover, NOS2 seems to be involved in maintaining physiologically relevant levels of NO to sustain the progression phase of carcinogenesis; mainly it is required to promote angiogenesis and to enhance the ability of cancer cells to counteract nutrient paucity in solid tumors and to metastasize (11, 10). NOS2 is also overexpressed in glioma stem cells, and its activity is required for the expression of the cell cycle inhibitor cell division autoantigen-1 (CDA1), which sustains growth and tumorigenicity (12). NOS2 has been also found to be upregulated in hepatocellular carcinoma (HCC), and is often increased in the hepatocytes of patients with chronic hepatitis and alcoholic cirrhosis, conditions that predispose to HCC (13–15). Notwithstanding all these lines of evidence, investigations on NOS2^−/− mice, in spontaneous and fibrosis-associated models of HCC, reveal little effect of NOS2-derived NO on hepatocarcinogenesis (16), meaning that other players are also involved. Redox signal underlying both pro-survival and death pathways, is a molecular information transduced by means of reactive cysteine residues that can undergo S-hydroxylation (SOH), upon reaction with ROS (i.e., H₂O₂) or S-nitrosylation (SNO), the posttranslational modification induced by NO, which is now emerging to underlie NO bioactivity (17). In the presence of a sulfhydryl group in their close proximity, both these modifications can resolve in a more stable disulfide bridge (S-thiolation, SS) (18–20). Actually, it has been recently questioned whether S-nitrosylation—given its nature of instable posttranslational modification—is able to convey the NO-mediated signal, or just acts as mere intermediate for disulfide bridge formation (21). Whatever is the end effector (if directly the SNO group or, indirectly, the SS adduct), the extent of S-nitrosylation is determined by a delicate balance between: (i)...