Regulation of NF-E2-related factor 2 signaling for cancer chemoprevention: antioxidant coupled with antiinflammatory

Abstract

Cancer chemoprevention is a process of using either natural or synthetic compounds to reduce the risk of developing cancer. Observations that NF-E2-related factor 2 (Nrf2)-deficient mice lack response to some chemopreventive agents point to the important role of Nrf2 in chemoprevention. Nrf2 is a member of basic-leucine zipper transcription factor family and has been shown to regulate gene expression by binding to a response element, antioxidant responsive element. It is generally believed that activation of Nrf2 signaling is an adaptive response to the environmental and endogenous stresses. Under homeostatic conditions, Nrf2 is suppressed by association with Kelch-like ECH-associated protein 1 (Keap1), but is stimulated upon exposure to oxidative or electrophilic stress. Once activated, Nrf2 translocates into nuclei and upregulates a group of genes that act in concert to combat oxidative stress. Nrf2 is also shown to have protective function against inflammation, a pathological process that could contribute to carcinogenesis. In this review, we will discuss the current progress in the study of Nrf2 signaling, in particular, the mechanisms of Nrf2 activation by chemopreventive agents. We will also discuss some of the potential caveats of Nrf2 in cancer treatment and future opportunity and challenges on regulation of Nrf2-mediated antioxidant and antiinflammatory signaling in the context of cancer prevention.

FIG. 1.

Chemopreventive agents that block or suppress multistage carcinogenesis. Carcinogenesis is a multistep process. The initiation step is started by the transformation of the normal cell into a cancer cell (initiated cell). These cells undergo tumor promotion into preneoplastic cells, which progress to neoplastic cells. Chemopreventive agents can interfere with different steps of this process. Some agents inhibit metabolic activation of the procarcinogens to their ultimate electrophilic species, or their subsequent interaction with DNA. These agents therefore block tumor initiation (blocking agents). Alternatively, blocking agents can stimulate the detoxification of carcinogens, leading to their secretion from the body. Other agents suppress the later steps (promotion and progression) of multistage carcinogenesis (suppressing agents). Some agents can act as both blocking and suppressing agents.

FIG. 2.

Chemical structures of selected chemopreventive agents with ability to activate the NF-E2-related factor 2–antioxidant responsive element signaling pathway.

FIG. 3.

Schematic representation of the conserved regions in Nrf2 and Keap1. (A) Conserved domains are referred to as Neh domains. The Neh2 domain contains the DLG and the ETGE motifs, which are Keap1 binding motifs. The Neh4 and Neh5 domains act cooperatively to bind the coactivator CREB-binding protein, thereby activating transcription. The Neh6 domain contains two highly conserved regions and acts as a linker domain. The Neh1 domain contains the CNC-bZIP region (CNC-bZIP), which promotes dimerization partners and confers DNA-binding specificity. (B) The Keap1 protein comprises five domains: the NTR; the BTB domain (present in actin-binding proteins and mediates Keap1 homodimerization, which is required for Nrf2 retention in the cytoplasm); the IVR (an especially cysteine-rich region); the DGR (which comprises six Kelch motifs that create multiple protein contact sites). The DGR domain of Keap1 is what combines with the Neh2 region of Nrf2, and the CTR. Functionally important cysteines are illustrated. BTB, broad complex, tramtrack, and bric a brac domain; CNC, Cap ’n’ Collar; CTR, C-terminal region; DGR, double glycine/Kelch repeat region; IVR, intervening region; Keap1, Kelch-like ECH-associated protein 1; Neh, Nrf2-ECH homology; Nrf2, NF-E2-related factor 2; NTR, N-terminal region.

FIG. 4.

Hypothetic model of Nrf2-mediated redox signaling. There are two pools of Nrf2 proteins, fNrf2 and kNrf2. Under homeostatic conditions (A), kNrf2 binds to Keap1 dimer via a high affinity ETGE (hinge) motif and a low affinity DLG (latch) motif. The two-site binding exposes the Ub-acceptor site(s) in Nrf2. Ubiquitinated Nrf2 proteins are destined to proteasomal degradation. There appears to be an equilibrium between protein synthesis and degradation. As a result, there is only a small pool of fNrf2, contributing to basal activation. When stimulated by oxidative stress or upstream kinases (B), the conformation change of the Keap1 dimer, probably via intermolecular disulfide bond formation, disrupts the two-site binding. As a result, the Ub-acceptor site(s) is/are not easily accessible. The ubiquitination and the proteasomal degradation of Nrf2 are impeded and Keap1 is saturated by kNrf2. Nrf2 protein synthesis, however, is elevated. As a consequence, the pool of fNrf2 expands and it can transmit redox signals to cell nucleus via gradient nuclear translocation. fNrf2, free-floating Nrf2; Keap1, Kelch-like ECH-associated protein 1; kNrf2, Keap1-binding Nrf2; Nrf2, NF-E2-related factor 2; Ub, ubiquitination.

FIG. 5.

Schematic presentation showing that Nrf2-disrupted mice (Nrf2 KO) have a higher susceptibility to carcinogenesis. The critical role of Nrf2 in protecting mice from neoplastic transformation when subject to oxidative stress and carcinogens is intact in Nrf2 WT mice having functional Nrf2-ARE signaling, by enhancing expression of detoxifying metabolizing enzymes and maintaining oxidative stress homeostasis by producing antioxidative stress enzymes. Application of chemopreventive compounds in Nrf2 WT mice can further enhance expression of phase II detoxifying and antioxidant enzymes by regulating the Nrf2-ARE signaling. ARE, antioxidant responsive element; Nrf2, NF-E2-related factor 2; Nrf2 KO, Nrf2 knockout; Nrf2 WT mice, wild-type mice with intact Nrf2 function.

FIG. 6.

A simplified illustration shows the carcinogenesis in human CRC and the role of Nrf2 in antiinflammation and oxidative stress. (A) Pathways that connect inflammation and CRC in AOM/DSS animal model, a chemical-carcinogen-induced CRC model. (B) Role of Nrf2 in protecting inflammation-associated cancer, showing a potential strategy for prevention of inflammation-associated cancers. When the Nrf2 signaling pathway is activated, activation of expression of phase II antioxidant and detoxifying enzymes such as HO-1, NQO1, UGT, and GST alike in Nrf2 WT animals can reduce the oxidative stress, and it is postulated that Nrf2 could also regulate proinflammatory genes and cytokines such as iNOS, COX-2, and iL-6 alike, and together, they protect the animal from developing inflammation-associated cancer, such as CRC (broken arrows). In contrast, Nrf2 KO animals do not possess functional Nrf2 and Nrf2 signaling pathway; after prolonged inflammatory and oxidative stress caused by AOM/DSS, they have increased incidence, multiplicity, and size of all colorectal tumors, including adenoma and adenocarcinoma as compared with WT animals (solid arrows). ACF, aberrant crypt foci; AOM, azoxymethane; CRC, colorectal cancer; DSS, dextran sulfate sodium; GST, glutathione S-transferase; HO-1, heme oxygenase-1; Nrf2, NF-E2-related factor 2; Nrf2 KO, Nrf2 knockout; Nrf2 WT mice, wild-type mice with intact Nrf2 function; NQO1, NAD(P)H:quinone oxidoreductase 1; UGT, UDP-glucuronosyltransferase.

FIG. 7.

A simplified illustration shows the pathogenesis in human PCa and the role of Nrf2 in TRAMP PCa model. (A) Normal prostatic epithelial cells may be subjected to the chronic inflammation and oxidative stress in addition to other factors such as aging and diets. For more detailed molecular pathogenesis of PCa, please refer to Nelson et al. (117). As found in CRC, inflammation is believed to be one of the key events before the formation of PCa, and proliferative inflammatory atrophy is a precursor to PIN and PCa. (B) The TRAMP mouse is an autochthonous transgenic animal model of PCa that recapitulates the whole spectrum of human prostate tumorigenesis from the earliest PIN lesions to androgen-independent disease (71). Without chemical or hormonal treatment, 100% of male TRAMP develops PCa and progress from PIN to histological cancer to carcinoma metastasis to lymph nodes, lungs, and occasionally bones sequentially over 12–28 weeks (39, 40). A schematic illustration of the putative role of inflammation and oxidative stress and their effect on Nrf2 and related phase II detoxifying and antioxidant enzymes in prostate carcinogenesis in TRAMP mice. Effective chemopreventive compounds such as COX-2 inhibitors and gamma tocopherol-enriched mixed tocopherols significantly downregulated proinflammatory genes and upregulated expression of Nrf2 and its related detoxifying and antioxidant enzymes, respectively, preventing PCa carcinogenesis in TRAMP mice, especially when they are given early before the formation of PIN. CRC, colorectal cancer; Nrf2, NF-E2-related factor 2; PCa, prostate cancer; PIN, prostatic intraepithelial neoplasia; TRAMP, transgenic adenocarcinoma mouse prostate.

FIG. 8.

A simplified scheme illustrating the interplay between Nrf2 and NF-κB leading to carcinogenesis/chemoprevention. Stimuli from ROS, RNS, and chemopreventive compounds could directly interact with members of upstream kinase signaling pathways such as PI3K, PKC, MAPK, and PERK, especially the MAPKs family, as well as interacting with Nrf2 and NF-κB pathways concurrently, the potential cross-talk between Nrf2 and Nf-κB is denoted by double-head arrows. Such multiple interactions allow chemopreventive compounds to exert their multiple beneficial cancer preventive properties. MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-kappa-B; Nrf2, NF-E2-related factor 2; PERK, PKR-like endoplasmic reticulum kinase; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; ROS, reactive oxygen species; RNS, reactive nitrogen species.