Nrf2 in TIME: The Emerging Role of Nuclear Factor Erythroid 2-Related Factor 2 in the Tumor Immune Microenvironment

Abstract

Nuclear factor erythroid 2-related factor 2 (Nrf2) mediates the cellular antioxidant response, allowing adaptation and survival under conditions of oxidative, electrophilic and inflammatory stress, and has a role in metabolism, inflammation and immunity. Activation of Nrf2 provides broad and long-lasting cytoprotection, and is often hijacked by cancer cells, allowing their survival under unfavorable conditions. Moreover, Nrf2 activation in established human tumors is associated with resistance to chemo-, radio-, and immunotherapies. In addition to cancer cells, Nrf2 activation can also occur in tumor-associated macrophages (TAMs) and facilitate an anti-inflammatory, immunosuppressive tumor immune microenvironment (TIME). Several cancer cell-derived metabolites, such as itaconate, L-kynurenine, lactic acid and hyaluronic acid, play an important role in modulating the TIME and tumor-TAMs crosstalk, and have been shown to activate Nrf2. The effects of Nrf2 in TIME are context-depended, and involve multiple mechanisms, including suppression of pro-inflammatory cytokines, increased expression of programmed cell death ligand 1 (PD-L1), macrophage colony-stimulating factor (M-CSF) and kynureninase, accelerated catabolism of cytotoxic labile heme, and facilitating the metabolic adaptation of TAMs. This understanding presents both challenges and opportunities for strategic targeting of Nrf2 in cancer.

Keywords: Keap1; Nrf2; anti-tumor immunity; immunosuppression; tumor microenvironment.

Fig. 1. Domain structure of Nrf2 and its main negative regulators.

(A) Nrf2 consists of seven Nrf2-ECH homology (Neh) domains. Neh1 is a CNC-bZIP domain that interacts with sMafs proteins and binds DNA antioxidant/electrophile response element (ARE) sequences in DNA. The amino acid motifs DLG and ETGE in Neh2 are responsible for the negative regulation of Nrf2 by Keap1. The amino acid motifs DSGIS and DSAPGS in Neh6 are responsible for the negative regulation of Nrf2 by β-TrCP; the DSGIS motif requires prior phosphorylation by GSK3. Hrd1 interacts with both Neh4 and Neh5 for Nrf2 ubiquitination. Neh3 is a transactivation domain that contains a conserved VFLVPK motif, which is necessary for binding to the chromodomain helicase DNA-binding protein 6 (CHD6). Neh4 and 5 are transactivation domains that interact with the histone-modifying enzyme CBP/p300. The retinoid X receptor α (RXRα) binds Neh7, repressing both basal and inducible expression of Nrf2-target genes. (B) Keap1 contains four domains: Broad complex, Tramtrack and Bric-à-Brac (BTB), intervening region (IVR), double glycine repeat (DGR)/Kelch, and C-terminal region (CTR). The BTB domain mediates homodimer formation and recruitment of Cullin-3. The Kelch-repeat domain and the C-terminal domain (CTR) together form a six-bladed β-propeller structure and bind to the ETGE and DLG motifs in the Neh2 domain of Nrf2, exposing lysine residues in between the motifs for ubiquitination (McMahon et al., 2006; Tong et al., 2007). The cysteine residues (Cys-) indicated in the figure function as sensors for electrophiles and/or oxidants. Keap1 has several critical cysteine residues, including Cys-151 in the BTB domain and Cys-273 and Cys-288, within the IVR domain involved in electrophilic stress sensing, essential in regulating Nrf2 stability and activity (Dayalan Naidu and Dinkova-Kostova, 2020; Dinkova-Kostova et al., 2002; Levonen et al., 2004; McMahon et al., 2010; Zhang and Hannink, 2003). (C) In β-TrCP, the D-domain is responsible for dimerization, forming homo- and heterodimers between β-TrCP1 and β-TrCP2. The F-box region is involved in the recruitment of S-Phase Kinase-Associated Protein 1 (Skp1). The WD40 repeat domain binds to DSGIS and DSAPGS motifs in Neh6 of Nrf2 (Chowdhry et al., 2013; Rada et al., 2011). Notably, the β-TrCP-mediated degradation of Nrf2 requires phosphorylation of the DSGIS motif by glycogen synthase kinase-3 (GSK3) (Chowdhry et al., 2013). GSK3 is a kinase that is constitutively active, but requires prior phosphorylation of its substrates (Robertson et al., 2018), but the identity of this priming kinase(s) that phosphorylates Nrf2 allowing subsequent recognition and phosphorylation by GSK3 is currently unknown. (D) Hrd1 is comprised of a signal peptide, six consecutive transmembrane segments (TMs), a RING-finger region, and a proline-rich cluster (Karamali et al., 2022). Hrd1 interact with the Neh4-5 domains of Nrf2 with its C-terminal domain (Wu et al., 2014).

Fig. 2. The immunosuppressive properties of tumor-associated macrophages (TAMs) in the tumor immune microenvironment (TIME).

Numerous tumor-derived factors, some of which have been shown to activate Nrf2, induce the development of the TAMs into ‘alternatively activated M2’ phenotype, exhibiting immunosuppressive and pro-invasive characteristics. These M2-like TAMs promote immune suppression in various ways, including induction of regulatory T (T_reg) cells (through secretion of IL-10 and TGF-β), inhibition of T-cell activation and proliferation, reduction of the activity of natural killer (NK) cells and CD8⁺ T cells, and promoting T-cell apoptosis. Increased levels of the MHC class I antigens HLA-G, HLA-E and prostaglandin E₂ (PGE₂) in TAMs may affect the activity of lymphokine-activated killer (LAK) cells, NK cells and cytotoxic T cells (CTLs), and inhibit the proliferation of T cells further. Increased levels of IL-6 and IL-10 and decreased levels of the MHC class II cell surface receptor HLA-DR, co-stimulatory factors, such as CD40, CD80, CD86, and pro-inflammatory cytokines, as well as diminished phagocytic activity of TAMs also contribute to T-cell inhibition. Reduced expression of CD40, CD80 and CD86 promotes T cell anergy and apoptosis. Additionally, TAMs induce differentiation of MDSCs into M2-like macrophages and increase recruitment of cancer-associated fibroblasts (CAFs). MDSC, myeloid-derived suppressor cell; IL-10, interleukin 10; TGF-β, transforming growth factor β; MHCII, MHC class II. Adapted from Wurdinger et al. (2014).