Epigenetic Regulation of NRF2/KEAP1 by Phytochemicals

Epigenetics has provided a new dimension to our understanding of nuclear factor erythroid 2–related factor 2/Kelch-like ECH-associated protein 1 (human NRF2/KEAP1 and murine Nrf2/Keap1) signaling. Unlike the genetic changes affecting DNA sequence, the reversible nature of epigenetic alterations provides an attractive avenue for cancer interception. Thus, targeting epigenetic mechanisms in the corresponding signaling networks represents an enticing strategy for therapeutic intervention with dietary phytochemicals acting at transcriptional, post-transcriptional, and post-translational levels. This regulation involves the interplay of histone modifications and DNA methylation states in the human NFE2L2/KEAP1 and murine Nfe2l2/Keap1 genes, acetylation of lysine residues in NRF2 and Nrf2, interaction with bromodomain and extraterminal domain (BET) acetyl “reader” proteins, and non-coding RNAs such as microRNA (miRNA) and long non-coding RNA (lncRNA). Phytochemicals documented to modulate NRF2 signaling act by reversing hypermethylated states in the CpG islands of NFE2L2 or Nfe2l2, via the inhibition of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), through the induction of ten-eleven translocation (TET) enzymes, or by inducing miRNA to target the 3′-UTR of the corresponding mRNA transcripts. To date, fewer than twenty phytochemicals have been reported as NRF2 epigenetic modifiers, including curcumin, sulforaphane, resveratrol, reserpine, and ursolic acid. This opens avenues for exploring additional dietary phytochemicals that regulate the human epigenome, and the potential for novel strategies to target NRF2 signaling with a view to beneficial interception of cancer and other chronic diseases.


Introduction
The NRF2 signaling axis has received widespread attention from the research community due to its critical role in responding to xenobiotic and electrophilic stress [1]. Binding of NRF2 to antioxidant response element (ARE) sequences in gene promoters activates antioxidant and xenobiotic detoxifying enzymes [2]. Activation of NRF2 by various dietary phytochemicals provides a promising strategy to prevent cancer, and the protective role of Nrf2/NRF2 activators has been verified in preclinical models and in human clinical trials [3,4].
However, inactivating mutations in KEAP1 that lead to constitutive NRF2 activation [5,6] can provide a growth advantage in some cancer cells. The yin/yang aspect of NRF2 signaling was first elaborated by Wang et al., reporting that knockdown of NRF2 using siRNA, or stable overexpression of KEAP1, sensitized human cancer cells to chemotherapy [7]. There is growing evidence linking elevated NRF2 expression with chemoresistance and poor prognosis in various cancer types [8,9]. the Neh6 domain involving glycogen synthase kinase-3β (GSK-3β), which is recognized by β-TrCP ubiquitin ligase [22].

Epigenetic Mechanisms Regulating NRF2 Signaling
Mechanisms of NRF2 regulation discussed above can be influenced both by genetic and epigenetic alterations. Biallelic inactivation of KEAP1, resulting in NRF2 hyperactivation, was identified as a relatively common event in lung cancer [25]. Nuclear accumulation of NRF2 and low expression of KEAP1 correlated with tumor aggressiveness, although the expected phenotypic outcomes were not necessarily consistent in cases of somatic mutation in KEAP1 vs. NFE2L2 [26].
The discovery of KEAP1 promoter hypermethylation, leading to gene silencing in lung cancer [27], prompted widespread research on epigenetic regulation of the NRF2 signaling pathway. This topic has been covered in detail in several excellent review articles [28][29][30]. Prior to discussion of the various phytochemicals reported to act via epigenetic mechanisms, a brief description of the epigenetic regulation of NRF2 signaling is presented first.

DNA Methylation
Several CpG islands were identified in the promoters of NFE2L2 and Nfe2l2 [31]. Hypermethylation of these CpG sites markedly lowered NRF2 expression in prostate tumorigenesis [32]. As discussed above, KEAP1 promoter methylation was first reported in lung cancer, but similar observations have been linked to poor prognosis in glioma, breast cancer, non-small cell lung carcinoma, colorectal cancer, clear renal cell carcinoma, and pancreatic cancer [33][34][35][36][37][38].

Histone Modifications
In addition to DNA methylation, histone methylation and acetylation, among other changes, also plays a vital role in regulating gene expression. Methylation of histones occurs primarily on the basic amino acids lysine and arginine. Gene activation or repression depends on the amino acid that is methylated and the degree of methylation, i.e., monomethylation, dimethylation, or trimethylation. Enhancer of zeste homolog 2 (EZH2), which is a member of the Polycomb group of proteins that catalyze trimethylation of histone H3 lysine 27 (H3K27me3), was downregulated in lung cancer and associated with NFE2L2 gene silencing [39].
Another study showed that increased binding of transcription factor Specificity protein 1 (Sp1) on the KEAP1 promoter increased methylation of histone H3K4 by the histone methyltransferase (HMT) SET Domain Containing 7 (SETD7, Set7/9) [40]; thus, KEAP1 also is regulated by changes in histone methylation.
Histone acetyltransferases (HATs) and histone deacetylases (HDACs) also are viewed as critical epigenetic "writers" and "erasers" that catalyze, respectively, the addition and removal of acetyl groups from histone and non-histone proteins. Acetylation of NRF2 at the Neh1 DNA binding domain by the HAT p300/CREB binding protein (CBP) is required for Nrf2-dependent gene transcription [41]. Similarly, p300/CBP was implicated in the regulation of subunit p65 in NF-κB-mediated NRF2 activation, and in recruiting HDAC3 to inhibit ARE-dependent gene transcription [42]. On the other hand, HDAC2 increased the stability of NRF2 protein by deacetylating lysine residues, thereby preventing NRF2 protein degradation [43].

Epigenetic "Readers"
In addition to the "writers" and "erasers", epigenetic "reader" proteins are gaining attention in the context of Nrf2 signaling. Bromodomain and extraterminal domain (BET) proteins interact with acetylated lysine residues in histones, regulating genes such as MYC, but also with acetylated non-histone proteins such as Nrf2 to inhibit Nrf2-dependent signaling, in species as diverse as mammals and Drosophila (reviewed in [44]).
Evidence linked upregulation of KEAP1 expression with increased levels of the BET protein BRD4, and with concomitant downregulation of NRF2 in prostate cancer cell lines, where integration of RNA-seq data with chromatin immunoprecipitation (ChIP) assays correlated NRF2-dependent gene expression with KEAP1 among the top genes interacting with BRD4 [45].

Regulation by Non-Coding RNAs
Non-coding RNAs linked to NRF2 signaling include miRNAs, which contain~22 nucleotides, and lncRNAs, which are greater than 200 nucleotides in length. Other aspects of non-coding RNA biology will not be discussed here in detail, although they represent interesting avenues for future research. For example, overexpression of small nucleolar RNA ACA11 was linked to a hyper-proliferative phenotype, reactive oxygen species generation, and NRF2 nuclear import in multiple myeloma [46]. Post-transcriptional changes to RNAs, such as 6-methyladenosine (m6A) modification of NFE2L2 mRNA, also warrant further investigation [47].

Phytochemicals and the Epigenetic Regulation of NRF2 Signaling
The role of dietary phytochemicals in cancer prevention and interception is well documented, and NRF2 signaling is considered a major target for many bioactive natural compounds [55][56][57]. Such agents affect the expression and activities of downstream target genes of NRF2, and the crosstalk with other signaling pathways, not only in cancer but also in other chronic diseases. However, the impact of phytochemicals on epigenetic mechanisms regulating NRF2 is an emerging area. In this section, we provide an update on the current state of knowledge regarding epigenetic regulation of NRF2 signaling by specific phytochemicals.
In a study designed to assess epigenetic regulation of Nrf2, DIM increased Nfe2l2 mRNA in transgenic adenocarcinoma mouse prostate (TRAMP)-C1 prostate cancer cells by reversing the methylation status of the first five CpGs in the Nfe2l2 promoter. DIM inhibited the mRNA and protein expression of Dnmt1, Dnmt3a, and Dnmt3b, and well as Hdac2 and Hdac3 in vitro. In vivo, DIM reduced 5-methylcytosine immunostaining in prostate cancer tissues of the TRAMP mouse, which mirrors the pathogenesis of human prostate cancer, and DIM-supplemented diet lowered the incidence of palpable tumors and lymph node metastasis compared to controls [79] (Table 1).

Apigenin
Apigenin is a natural flavonoid derived from chamomile flowers, oranges, parsley, celery, and other natural sources, with potential antioxidant, anti-inflammatory, and anticancer properties [102]. Anticancer properties of apigenin were exhibited in many types of malignancy, and some were linked to epigenetic mechanisms [80,81,103,104]. One study showed restoration of Nrf2 expression and activity in the murine preneoplastic epidermal JB6 P+ cell line by decreasing the methylation status of the Nfe2l2 promoter, and by inhibiting the expression of Dnmts and Hdacs [105].
Contrary to this induction of Nrf2 activity, another investigation with apigenin reported increased miR-101 levels targeting the 3 UTR of NFE2L2 mRNA, with enhanced chemosensitivity of doxorubicin-resistant human hepatocellular carcinoma BEL-7402/ADM cells [106]. These findings point to possible species-specific differential methylation signatures for the corresponding target gene(s) in mouse epidermal vs. human hepatoma cells that have become drug-resistant.

Corosolic Acid
Corosolic acid is found in medical herbs, such as Lagerstroemia speciose, Eriobotrta japonica, and Tiarella polyphylla [107], and has gained attention for its beneficial effects in the prevention or treatment of metabolic disease, including diabetes. This triterpenoid also is reported to possess anticancer activity, and one study demonstrated effects on Nrf2 via epigenetic modifications. Specifically, corosolic acid induced Nfe2l2 at the transcriptional level by decreasing CpG methylation in the corresponding promoter region of TRAMP-C1 cells. Increased histone H3 lysine 27 acetylation (H3K27ac) was also observed, as well as decreased trimethylation of H3K27 (H3K27Me3). Protein levels of Dnmts (Dnmt1, Dnmt3a and Dnmt3b) and Hdacs (Hdac1, Hdac2, Hdac3, Hdac4, Hdac7 and Hdac8) were inhibited in corosolic acid-treated TRAMP-C1 cells [82]. The study suggested that upregulation of Nrf2 was responsible for the inhibitory effects of corosolic acid on anchorage-independent growth of TRAMP-C1 cells, which was abrogated following Nrf2 knockdown [82].

Curcumin
Curcumin is the principal curcuminoid found in the rhizomes of turmeric (Curcuma longa), linked to anti-inflammatory, antioxidant, antitumor, and anti-diabetic effects. Curcumin is a known inducer of NRF2 and its downstream transcriptional targets, with emerging evidence for epigenetic regulation. Using bisulfite sequencing, Khor et al. [83] demonstrated that curcumin reversed the methylation status of the first five hypermethylated CpG islands in the Nfe2l2 promoter, thereby restoring epigenetically-silenced Nrf2 in TRAMP-C1 cells. Curcumin had negligible effects on DNA methyltransferases (Dnmts) at the RNA or protein level, but rather inhibited their enzymatic activity [83]. It was concluded that the epigenetic restoration of Nrf2 activity by curcumin might play a role in the prevention of prostate cancer in TRAMP-C1 mice [83].

Delphinidin
Delphinidin is an anthocyanidin flavonoid that contributes to the intense blue coloration in many fruits and vegetables, such as blackcurrant, eggplant, black grapes, red cabbage, and blackberries, and is abundant in pomegranate fruit extract [108,109]. Delphinidin is an anthocyanidin exhibiting potent antioxidant, anti-inflammatory, and antitumor properties. A study investigated the effects of delphinidin against skin cell neoplastic transformation by modulating the Nrf2 pathway. Delphinidin inhibited the neoplastic transformation of mouse epidermal JB6 P+ cells by 12-O-tetradecanoylphorbol-13-acetate (TPA). The anthocyanidin decreased the CpG methylation ratio at the Nfe2l2 promoter resulting in upregulated mRNA and protein expression levels of Nrf2 and its target genes. The study further demonstrated downregulation of Dnmts (Dnmt1a and Dnmt3a), and class I and II Hdacs linked to reactivation of the Nrf2 pathway in JB6 P+ cells [84].

Fucoxanthin
Fucoxanthin is a xanthophyll carotenoid abundantly available in seaweeds. Its unique chemical structure provides a variety of biological activities, and it has been ascribed health benefits against chronic diseases such as cancer, obesity, and diabetes [110]. This carotenoid was found to activate Nrf2 signaling by causing demethylation of CpG islands in the Nfe2l2 promoter, resulting in inhibition of TPA-induced transformation of JB6 P+ cells. Mechanistically, fucoxanthin decreased Dnmt1 mRNA and proteins levels, but was not found to alter Hdac expression levels [85].

Luteolin
Luteolin is a natural flavonoid present in the leaves, roots, stems, and fruits of several species of plants, such as chrysanthemum flowers, onion leaves, and celery [111]. Health benefits of luteolin have been linked to interfering with "hallmark features" of carcinogenesis such as angiogenesis, cell invasion, and metastasis. Regulation of the Nrf2 signaling pathway by luteolin is well studied and is a key mechanism through which the flavonoid is thought to exert health benefits. A few recent studies have pointed towards epigenetic modifications as a key underlying mechanism of action for luteolin. One investigation showed that luteolin inhibited proliferation of human colorectal HCT116 cells by upregulating NFE2L2 mRNA and NRF2 protein expression. Bisulfite sequencing revealed a marked reduction in CpG methylation at the NFE2L2 promoter, which was associated with significant reduction in expression and activities of DNMTs (DNMT1, DNMT3a, and DNMT3b) and HDACs (HDAC1, HDAC2, HDAC3, HDAC6, and HDAC7) [86]. Another study corroborated the epigenetic regulation of NRF2 by luteolin and showed reduced DNA methylation of the NFE2L2 promoter in luteolin-treated human colon adenocarcinoma HT29 cells. In luteolin-treated cells, ChIP assays showed reduced DNMT1 binding and increased TET1 interaction at the NFE2L2 promoter [87].

Pelargonidin
Pelargonidin is a plant anthocyanindin pigment producing a characteristic red-orange color in various fruits and vegetables, such as pomegranate, red radish, and strawberry. Pelargonidin is reported to possess antioxidant and anti-inflammatory properties and shows strong cytotoxicity towards various cancer cell lines. A molecular modeling approach in silico revealed that pelargonidin might inhibit the catalytic binding sites of human DNMT1 and DNMT3a [112]. In accordance with this work, pelargonidin reduced DNA methylation of the Nfe2l2 promoter to activate Nrf2-driven gene expression in JB6 P+ cells, leading to suppression of TPA-induced neoplastic transformation. Treatment with pelargonidin decreased the expression of Dnmt1 and Dnmt3b, and Hdac1, Hdac2, Hdac3, Hdac4, and Hdac7) [88].

Polydatin
Polydatin is isolated from the Chinese herb Polygonum cuspidatum, and is a natural precursor of resveratrol, with potent anti-inflammatory properties that are beneficial against many pathologies, including atherosclerosis, neurological disorders and cancer. Polydatin was reported to induce Nrf2 by inhibiting Keap1 [113]. To elucidate the mechanisms behind the activation of Nrf2 by polydatin, an in vivo and in vitro model of non-alcoholic fatty liver disease (NAFLD) induced by high fructose was employed. Polydatin reduced fructose-induced oxidative stress and inflammation by inhibiting Keap1 and activating Nrf2. Polydatin also caused a marked increase in the levels of miR-200a, which targeted KEAP1 to activate NRF2 signaling in response to high fructose-induced oxidative stress and inflammation in BRL-3A and HepG2 cells, and in the liver of high fructose-fed rats [89]. The study demonstrated that polydatin provided protection against fructose-induced liver inflammation and lipid deposition by activating Nrf2 and reducing oxidative stress [89].

Reserpine
Reserpine is an indole alkaloid, which is the principal active component found in the plant Rauwolfia serpentina and in other species of Rauwolfia sp. Reserpine is an anti-hypertensive drug and widely used to treat neurological disorders, and also possesses anticancer activity. A study showed that reserpine induced Nrf2-driven target genes to inhibit TPA-stimulated neoplastic transformation in mouse epidermal JB6 P+ cells. The relative methylation status of the CpG island at the Nfe2l2 promoter was found to decrease with increasing concentrations of reserpine. Expression levels of Dnmt1, Dnmt3a, and Dnmt3b were decreased by reserpine treatment in JB6 P+ cells [90].

Resveratrol
Resveratrol is a widely investigated plant polyphenol due to its antioxidant, anti-inflammatory, and antimicrobial properties. Several studies have pointed towards a role as an epigenetic modifier, most notably in anti-aging research, with resveratrol classified mechanistically as a class III HDAC/ sirtuin-activating compound [114][115][116]. Other work in HepG2 cells treated with high glucose and in high-fat models of NAFLD found that the methylation status of the NFE2L2 gene was increased, while that of KEAP1 was decreased, leading to decreased NRF2 expression and activity [91]. Effects of resveratrol on the methylation status of the Nfe2l2 promoter were shown in an earlier study involving a rat model of estrogen-induced mammary cancer [92]. Singh et al., noted "inhibition of 17β-estradiol-mediated alterations in NRF2 promoter methylation and expression of NRF2 targeting miR-93 after resveratrol treatment" as evidence for "resveratrol-mediated epigenetic regulation of NRF2 during E2-induced breast carcinogenesis" [92].
In a recent study, Loc344887 was the most highly upregulated transcript in sulforaphane-treated human HCT116 colon cancer cells [94]. This non-coding RNA was identified as a novel functional pseudogene and renamed NMRAL2P, with 62% homology to the protein-coding gene NmrA-like redox sensor 1 (NMRAL1). In addition to being a direct transcriptional target of NRF2, NMRAL2P was a downstream coactivator of NRF2-dependent NQO1 expression in human colon cancer cells. It was further shown that NMRAL2P knockout HCT116 cells were less responsive to sulforaphane-induced growth inhibitory effects. This report added a further layer of epigenetic regulation to the NRF2 network [94], which also involves other non-coding RNAs altered by sulforaphane [134][135][136][137].

Tanshinone IIA
Tanshinone IIA is a lipid-soluble natural compound isolated from the medicinal herb Salvia miltiorrhiza Burge and associated with cardiovascular and cerebrovascular protective effects. The involvement of epigenetic mechanisms in the induction of Nfe2l2 by Tanshinone IIA has been demonstrated in TPA-induced neoplastic transformation of JB6 P+ cells [95] and in in vitro and in vivo models of rifampicin-induced liver injury [96]. Hypomethylation of the Nfe2l2 promoter was mechanistically linked to potent induction of Nfe2l2 mRNA and Nrf2 protein levels by Tanshinone IIA. While one study reported decreased expression of Dnmts and Hdac1, Hdac3, and Hdac8 by Tanshinone IIA in JB6 P+ cells [95], another found no significant changes in DNMTs, but elevated expression of DNA demethylases, especially ten-eleven translocation 2 (TET2), in human hepatocyte L02 and Hepa RG cells [96]. The latter investigation showed that Tanshinone IIA could prevent rifampicin-induced liver injury by inducing the expression of bile salt efflux pump (BSEP) and Na+/taurocholate cotransporter (NTCP), which were directly regulated by NRF2 [96].

Taxifolin
Taxifolin is a flavonoid found in onion, milk thistle, and in Pinaceae plants, with diverse pharmacological activities, including antioxidant, anti-inflammatory, and antimicrobial properties. Induction of Nrf2 and its downstream target genes is considered crucial for the beneficial effects of taxifolin [138]. Taxifolin was found to inhibit TPA-induced neoplastic transformation of JB6 P+ cells by epigenetically inducing Nrf2. Bisulfite sequencing showed that the flavonoid reduced the proportion of methylated CpG sites in the Nfe2l2 promoter. At the molecular level, the protein expression levels of Dnmt1, Dnmt3a Dnmt3b, Hdac1, Hdac3, and Hdac8 were significantly decreased by taxifolin [97].

Ursolic Acid
Ursolic acid is a pentacyclic triterpenoid found ubiquitously in fruits, vegetables and herbs, such as cranberry, apple peel, basil, and rosemary [139]. Two studies have investigated the epigenetic regulation of Nrf2 by ursolic acid. In one investigation, the triterpenoid activated the Nrf2 pathway by demethylating the Nfe2l2 promoter, accompanied by a reduction in the expression levels of Dnmts and Hdacs, which was linked to inhibition of TPA-induced neoplastic transformation in mouse epidermal cells [98]. In the second study, ursolic acid induced the expression of the protein methyltransferase SETD7, and knockdown of SETD7 decreased NRF2 protein and its downstream target genes in LNCaP and PC-3 cells. Also, H3K4me1 monomethylation at the NFE2L2 promoter was reduced by SETD7 knockdown. On the other hand, treatment with ursolic acid enriched H3K4me1 at the NFE2L2 promoter, leading to increased NRF2 signaling [99]. It was hypothesized that direct methylation of the NRF2 protein by SETD7 might be mechanistically relevant.

γ. Tocopherol-Rich Mixture of Tocopherols (γ-TmT)
The major forms of vitamin E comprise α-, β-, γand δ-tocopherols and related tocotrienols [140][141][142]. These lipophilic compounds are abundant in vegetable oils and nuts and are widely studied as agents with an impact on human health and disease pathogenesis [143][144][145][146][147]. Numerous reviews have covered the topic of vitamin E and cancer [148][149][150][151]. Recently, γ-TmT, a commercially available by-product of vegetable oil refinery containing 57% γ-tocopherol, was linked to induction of the Nrf2 pathway via hypomethylation of the Nfe2l2 promoter in the TRAMP mouse and in TRAMP-C1 cells in vitro. This study also noted decreased protein levels of Dnmt1, Dnmt3a, and Dnmt3b in vivo compared to controls [100].

Z-Ligustilide
Z-Ligustilide is a natural benzoquinone derivative found in Chinese medicinal herbs, including Radix Angelicae Sinensis, and is reported to possess diverse pharmacological activities. Several studies [101,[152][153][154] noted upregulation of Nrf2 and its downstream antioxidant enzymes by Z-Ligustilide. Su et al. [101] demonstrated induction of Nrf2 by Radix Angelicae Sinensis and purified Z-Ligustilide. Bisulfite sequencing revealed decreased methylation at the Nfe2l2 promoter, accompanied by inhibition of Dnmts by both the plant extract and its isolated bioactive components [101].

Discussion
In addition to genetic alterations, i.e., mutations or chromosomal rearrangements in germline and somatic cells, epigenetic mechanisms also play a crucial role in cancer development. For instance, promoter hypermethylation is associated with the silencing of many tumor suppressor genes. The preponderance of epigenetic deregulation in cancer and the reversible nature of these alterations [155,156] has focused attention on epigenetic changes as viable targets for prevention or therapeutic strategies. Epigenetic alterations represent promising targets for cancer interception across multiple stages, from early to late disease pathogenesis [156][157][158][159]. Relatively few "epigenetic" drugs have been approved as anticancer agents, including the DNMT inhibitor 5-azacytidine (Vidaza TM , Azadine TM ) and the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA, Vorinostat TM , Zolinza TM ), with recent attention also shifting to the BET inhibitor JQ1 (clinicaltrials.gov). In general, these therapeutics are not without side-effects as monotherapies, and combination approaches with standard-of-care practices provide the most viable way forward to minimize toxicity and related concerns [157].
Many of the studies reviewed here involved analyses of CpG methylation sites in the NFE2L2 promoter and inhibition of DNMTs and/or HDACs, without probing more deeply into other mechanistic aspects, such as the involvement of chromatin coactivator/corepressor complexes, long-range chromatin interactions, and non-coding RNAs. This leaves plenty of scope for future research. KEAP1-NRF2 signaling is a key molecular target of cancer preventive agents, including an array of phytochemicals (Table 1). These phytochemicals can induce NRF2 either by acting as Michael acceptors that interact with KEAP1 sensor thiols, or by activating phosphorylation cascades that stabilize NRF2 [55,160]. However, research also has demonstrated that dietary phytochemicals can regulate NRF2 signaling by diverse epigenetic mechanisms ( Figure 1). The best known NRF2 activator that has obtained clinical approval is dimethyl fumarate (Tecfidera), for the treatment of multiple sclerosis [161]. However, Tecfidera has several side-effects, including allergic reactions and gastrointestinal disturbance (www.PDR.net). There are a few related agents in clinical trials, such as Bardoxolone and SFX-01, a synthetic derivative of sulforaphane [161], which also exhibit less than desirable outcomes. Despite the promise from preclinical models and early clinical trials, safety, specificity, and potency issues must be resolved. All of these agents act by preventing the proteasomal degradation pathway. Given the multifactorial epigenetic regulation of NRF2, exploring other modulators that target NRF2 signaling at the transcription or post-transcription level warrants further attention (Figures 1 and 2). Among the phytochemicals reviewed here, most were shown to reverse NFE2L2 promoter methylation by inhibiting DNMTs and attenuating certain HDACs. It is noteworthy that inhibitors directed against DNMTs and HDACs are used for the treatment of several malignancies, and the combination has been reported to produce synergistic growth inhibitory effects in cancer cells [162]. Therefore, it would be worthwhile to compare the combination of DNMT and HDAC inhibitors vis-à-vis Nrf2 induction by dietary phytochemicals (e.g., tea polyphenols plus sulforaphane). Moreover, a recent study showed that the combined inhibition of DNMT and HDAC activity caused de novo transcription of long-terminal repeats (LTRs) of the LTR12 family, linked to LTR-derived immunogens presented on major histocompatibility complex class I molecules [163]. Thus, phytochemical-mediated inhibition of DNMT and HDAC activities might dovetail NRF2 signaling with immunoprevention and immunotherapy.
Sulforaphane is an NRF2 inducer, as well as an inhibitor of HDAC activity and protein expression. One interesting observation is that Hdac3 expression was reduced by dietary sulforaphane in 1,2-dimethylhydrazine-treated wild-type (WT) mice, but less so in Nrf2 −/+ mice, and that WT mice were more susceptible to carcinogen-induced colon tumorigenesis [164]. Thus, Nrf2 exerted an apparent oncogenic role in the gut, and Nrf2 status dictated Hdac inhibitory responses to sulforaphane and the extent of tumor growth suppression [164]. Another interesting avenue is the synergistic combination of sulforaphane with the BET inhibitor JQ1 in colon cancer models, targeting the non-histone protein Cell cycle and apoptosis regulator 2 (CCAR2) for acetylation and altered Wnt coactivator functionality [165]. As BET proteins are reported to interact with and inhibit NRF2 [44], the prospect of combined deacetylase and bromodomain inhibition affecting NRF2 regulation is worthy of further mechanistic investigation.
The phytochemicals reviewed here typically activate murine Nfe2l2 epigenetically, coincident with Nrf2 induction, except in the case of apigenin. Whereas one report noted Nfe2l2 activation via promoter hypomethylation in mouse skin epidermal cells [80], another showed miRNA-mediated inhibition of human NFE2L2 by apigenin, leading to chemosensitization of adriamycin-resistant hepatocellular carcinoma cells [106]. It would be interesting to explore the underlying circumstances for this epigenetic regulation of NRF2 by apigenin and, for example, whether DNA methylation predominates over non-coding RNA-mediated mechanisms. Activation of NRF2 is highly regulated, both temporally and spatially, and it will be important to decipher the epigenetic mechanisms in various cell types and the respective context-specific regulation of NRF2. From the current update, the majority of studies involved a single cell type and one of two preclinical models: JB6+ mouse skin epidermal cells and TRAMP prostate tumor cells. This calls for further investigation in different cancer scenarios to better characterize the epigenetic regulation of Nrf2/Keap1 by phytochemicals.
Finally, NRF2 is a complex regulator in cancer etiology because of its yin/yang roles in prevention and promotion, dictated in part by early vs. late stages of disease pathogenesis [10,11]. Epigenetic mechanisms add an additional layer of regulation, with numerous readers, writers and erasers that interact to affect histone states and chromatin access. Epigenetic aspects of NRF2 signaling by natural phytochemicals are worthy of further investigation to better understand context-dependent mechanisms that might provide new avenues for cancer prevention and interception.

Conclusions
The use of dietary components as cancer preventive agents continues to be of great interest, and experimental evidence supports the role of nutritional factors in modulating deregulated signaling pathways during cancer initiation and progression. Our understanding of the complex mechanisms of action of dietary factors is constantly evolving. One major take-home message from the accrued literature is that no agent, dietary or otherwise, is likely to act by one mechanism alone, or to affect a single molecular target without influencing other components of a signaling network. It is increasingly documented that diet or dietary components can influence gene expression through epigenetic mechanisms, but further work is needed to truly appreciate how these mechanisms can be manipulated in a beneficial manner for disease interception in the context of NRF2 signaling.