With the increasing incidence of human cancer and continued difficulty in treating metastatic tumors, there is an urgent need to identify biomarkers for tumors with poor outcome and novel therapeutic targets. Many therapeutic targets have been found in recent years. One promising biomarker and therapeutic target that is valuable for human tumor is nuclear factor erythroid 2-related factor 2 (NFE2L2, Nrf2). In this chapter, we will discuss the regulatory mechanisms and conflicting roles of Nrf2 during different stages of tumor development as well as its involvement in the drug resistance and hypoxia-induced chemoresistance. We will also discuss various positive and negative modulators of Nrf2 as reference to their potential utility as study tools and leads for further clinical development.
- oxidative stress
Nrf2 is the main regulator for the expression of antioxidant enzymes and the detoxification proteins. With these abilities of Nrf2, Nrf2 activation confers cells with more anti-stress capacity, thus resulting in more malignancy and chemoresistance of tumor cells. Therefore, targeting Nrf2 in tumor may offer therapeutic benefit by undermining its advantage on the proliferation, migration, metastasis, and drug resistance of tumor cells. Collectively, Nrf2 has the potential to serve as a good biomarker and therapeutic target to overcome the poor prognosis and chemoresistance associated with tumor or tumor hypoxia [1, 2].
2. Nrf2 is the key regulator of antioxidant and detoxification abilities
Organisms survive based on the normal and steady function of cell metabolism to maintain cellular homeostasis. However, the process of metabolism produces many metabolic wastes. If these wastes are not properly removed, they become harmful. One of these metabolic wastes is the reactive oxygen species (ROS). While ROS can serve the signaling function, uncontrolled ROS may lead to cellular damage and death. Many key enzymes involved in the removal of excess ROS are tightly regulated by a transcription factor, Nrf2.
Nrf2 activation is strictly regulated by an ubiquitin-proteosome system (UPS) . Nrf2 is negatively regulated by Keap1 (Kelch-like ECH-associated protein 1), the most important molecular switch controlling the activation and inactivation of the Nrf2 pathway. Keap1 is an adaptor for Nrf2 as the Nrf2-Keap1 complex becomes a substrate for the Cul3-dependent E3 ubiquitin ligase for proteasomal degradation. Keap1 is a cysteine-rich protein, which can be modified by many oxidants and electrophiles. Upon exposure to oxidative stress, these cysteine residues may be altered by stresses to induce conformational changes that inhibit Keap1-dependent ubiquitin ligase activity to allow Nrf2 accumulation [4, 5].
Since Nrf2 is a transcription factor, the accumulated Nrf2 proteins translocate into the nucleus to dimerize with members of the small Maf family, and bind to the antioxidant response element (ARE or electrophile response element 5′-RTGABNNNGCR-3′) in the promoter regions of cell defense genes . These Nrf2-regulated proteins include Phase II detoxification enzymes and some stress response proteins as listed below:
Antioxidant proteins: proteins that control the antioxidant specializing in neutralizing the reactive species and protecting organisms from oxidative damage, such as NAD(P)H: quinone oxidoreductase 1 (NQO1), epoxide hydrolase, heme oxygenase 1 (HO-1), glutathione S-transferase (GST), and glutathione peroxidase (GPx).
Glutathione producing enzymes: proteins that regulate the synthesis and metabolism of glutathione, such as glutamate-cysteine ligase (GCL), which consists of two subunits, a light regulatory subunit; glutamate-cysteine ligase modifier subunit (GCLM), and a heavy catalytic enzyme; GCL catalytic subunit (GCLC).
Drug-metabolizing enzymes: enzymes that regulate the metabolism of drugs, including UDP-glucuronosyl-transferase 1A1 (UGT1A), carbonyl reductase 1 (CBR1), aldo-keto reductases (AKR), and cytochrome P450 (CYPs).
Xenobiotic transporters (ATP-binding cassette (ABC) transporter): proteins that belong to ATP phosphohydrolase, some of them are involved in the exclusion of drugs, xenobiotics and their metabolites , which are named multidrug resistance proteins, such as multidrug resistance protein 1 (MRP1).
3. The conflicting roles of Nrf2
Since Nrf2 is the key regulator of antioxidant capacity and detoxified proteins, the activation of Nrf2 is expected to protect cells from stresses, such as reactive oxygen species (ROS). Therefore, the Nrf2 pathway is so called oxidative stress response pathway or cellular defense pathway. Once cells or organisms are exposed to ROS induced by physical forces or chemicals, Nrf2 is activated to increase the anti-stress capacity and cope with the ROS. Nrf2 activation can stabilize the intracellular oxidant level and maintain the redox state within cells to avoid DNA damages, genomic instability, and potentially serious sabotages caused by ROS . Although DNA repair mechanism can reduce slight DNA damage, higher presence of oxidizing base lesions in DNA leads to DNA mutation, which may cause aging , cell damage, cell death, carcinogenesis, and even cancer. Therefore, various Nrf2 activators are being pursued in chemopreventive strategies  to reduce tumor development. In addition, Nrf2 activators have been used to treat various human diseases, including diabetic nephropathy [12, 13] and sickle cell disease .
On the other hand, excess ROS may lead to numerous diseases, such as inflammation, obesity, and other metabolic diseases. For example, too much oxidative stress affects the differentiation of adipocytes and impairs the normal function of white adipose tissue , leading to inflammation and adipokine secretion that affect the whole organism [16, 17]. The activation of Nrf2 defense pathway can protect organisms from many metabolic diseases.
Therefore, Nrf2 can be the double edged sword in the organism. In normal cells, Nrf2 activation keeps the redox homeostasis and prevents cancer development. However, once cancer cells have established, Nrf2 activation may drive oncogenesis and confer chemoresistance. In many cancers, constitutive Nrf2 activation is an oncogenic mutation  and a biomarker for poor prognosis [19, 20] (Figure 2). In the TCGA data, mutations in the Nrf2 pathways constitute one of the major oncogenic pathways of lung cancers [21, 22]. The angel and devil roles of Nrf2 are discussed in the following sections.
4. The good side of Nrf2
Nrf2 activation in normal cells makes cells stronger against environmental stresses and prevents carcinogenesis. Nrf2 is able to augment a wide range of cell defense processes, thereby enhancing the overall capacity of cells to detoxify potentially harmful entities. As such, the Keap1-Nrf2 pathway is generally considered as the major cellular defense pathway that offers survival advantages.
Keap1-Nrf2 is the key cellular defense mechanism to combat oxidative stress. The activated Nrf2 protect organisms from these diseases by diminishing the ROS. Nrf2 is such a natural cytoprotective response against oxidative stress-induced inflammation. Nrf2-null mice tend to spontaneously develop various inflammatory disorders, including glomerulonephritis , immune-mediated hemolytic anemia , and multiorgan autoimmune inflammation . Also, activated Nrf2 protects many body systems, including airway, liver, gastrointestinal tract and kidney, where these systems are attacked by toxic agents very often . For example, Nrf2 activation via sulforaphane (Nrf2 inducer) protects kidney from chronic renal disease  by increasing the GCLC and glutathione level. Activation of Nrf2 also alleviates the TGF-β-induced, increased α-SMA and repressed E-cadherin , which are the markers for epithelial-mesenchymal transition (EMT), through the SMUR1-SMAD7 signaling. Another Nrf2 inducer, AST120, can restore the HO-1 and NQO1 levels and decrease the production of ROS stimulated by indoxyl sulfate-induced chronic renal disease  Nrf2 not only protects the kidney but also protects the lungs. Nrf2 protects lungs from chronic pulmonary injury , fibrosis , and acute lung injury [32, 33]. Therefore, Nrf2 is named as the “multiorgan protector” .
With cytoprotective functions and the cellular defense mechanism against exogenous and endogenous insults, Nrf2 is considered as a tumor suppressor. In one hand, in vivo tumor development data using Nrf2-knockout mice has highlighted the tumor suppression ability of Nrf2. With treatment of chemical and physical stimuli, Nrf2-null mice are more prone to develop cancer . On the other hand, Nrf2 activation can remove damaged proteins, promoting the overall survival of the cell and detoxify the cellular environment to maintain the homeostasis in the organism . The abilities of Nrf2 to combat oxidative stress and inflammation, which are conducive to initiate oncogenesis, attain a result of tumor suppression .
5. The dark side of Nrf2
Gain of Nrf2 in cancer cells: the Nrf2 pathway is a powerful sensor for cellular redox state and is activated directly by oxidative stress and/or indirectly by stress response protein kinases. Although Nrf2 is beneficial to normal cells to fight against stresses, once tumor cells get the antioxidant and detoxificative abilities of Nrf2, things go in another direction. For example, the constitutive Nrf2 activation has redirected tumor metabolism to support the biosynthetic needs of tumor proliferation [37, 38]. In addition, Nrf2 makes cancer cells stronger against chemotherapy and leads cells to become more malignant. In this case, Nrf2 serves as a target for chemotherapy. Recent researches have highlighted that persistent accumulation of Nrf2 in cancer cells is harmful, since it can promote the survival and proliferation of cells that have acquired cancer-promoting mutations, and Nrf2 is also observed beneficial for tumorigenesis [11, 39–42]. Nrf2 orchestrates the expression of various genes that help cancer cells to resist chemotherapeutic treatment, including antioxidants (NQO1, NQO2, HO-1, and GCLC), antiapoptotic (Bcl-2), drug-metabolizing enzymes (G6PD, TKT, and PPARγ), and drug efflux transporters (ABCG2, MRP3, and MRP4) genes .
A. Proliferation, tumorigenesis and poor patient survival: Nrf2 contributes to the tumorigenesis, cancer proliferation in bench and poor patient survival in clinic in various tumors, including hepatocarcinoma (HCC) , breast tumor , nonsmall cell lung cancer (NSCLC) , glioma [49, 50], pancreatic adenocarcinoma , and gastric cancer . Nrf2 is also found to involve in the maintenance of quiescence, survival, and stress resistance of cancer stem cells (CSCs), thus dedicating to tumor progression and recurrence .
B. Chemotherapeutic resistance: Nrf2 exerts the detoxification and drug export through activating multidrug resistance proteins and drug transporters. This action protects cancer cells from the damage of chemotherapy, such as 5-fluorouracil (5-FU) in gastric cancer  and in gallbladder cancer , and cisplatin (CDDP) and camptothecin in pancreatic cancer .
C. Epithelial-mesenchymal transition (EMT), tumor metastasis, and malignancy: cancer cells respond to some anti-diabetic drugs, which have antioxidant properties and inhibit the Kea1-dependent obstruction of Nrf2, with Nrf2 activation and result in increased migration and metastasis, such as hypoglycemic dipeptidyl peptidase-4 inhibitors (DPP-4i), saxagliptin and sitagliptin .
D. Hypoxia-induced drug resistance: Nrf2 contributes to the chemotherapeutic drug resistance induced by hypoxia in breast cancers . Hypoxia is a natural status in the tumor center where the cancer cells outgrow the perfusion from local blood vessels for getting enough oxygen and nutrients. Hypoxia triggers the ROS unbalance and stimulates the activation of Nrf2 . Following the Nrf2 nuclear translocation and ARE binding, antioxidant enzymes are produced to maintain the stability of intracellular redox state. Blocking ROS unbalance by ROS scavenger inhibits the Nrf2 activation and the following drug resistance. Inhibition of Nrf2 or the production of related enzymes with siRNA or specific inhibitor blocks the chemoresistance under hypoxia (Figure 3).
6. Nrf2 activation in tumors
Nrf2 gets activated in malignant tumors, such as carcinomas of skin, lung, oesophagus, and larynx . Many factors control the activation of Nrf2 in tumors, some are listed below.
a. The somatic mutation of Keap1 or Nrf2: the mutations in Keap1 or Nrf2 hurt the interaction of Keap1 and Nrf2, leading to a higher free Nrf2 level and activity .
b. The decreased level of Keap1: besides somatic mutations of Keap1, epigenetic changes, such as hypermethylation on the promoter region of Keap1, decrease the expression level of Keap1 . In addition, several studies have shown that miRNAs, including mir-200A [61, 62], miR-141  and mir-28 , also regulates the expression level of Keap1 mRNA. These events lead to the decreased level of Keap1 and the nuclear accumulation of Nrf2.
c. The increased level of Nrf2: Nrf2 can be activated by increase of some oncogenes, such as KrasG12D , or disruption of tumor suppressors, such as PTEN  in tumors, that lead to better cell survival and higher drug resistance.
d. Nrf2 polymorphism: in addition to varying Nrf2 expression, Nrf2 polymorphism also affects the Nrf2 activity. The Nrf2 polymorphism contributes to poor prognosis in cancers, including cholangiocarcinoma , lung cancer , and breast cancer . It also contributed to diseases, such as increasing the risk of acute lung injury [68, 69] as well as blood pressure and cardiovascular mortality in patients with hemodialysis .
7. Regulators of Nrf2
Many molecules and chemicals are thought to regulate the Nrf2 activation; some of them are described as following (see Figure 4):
A. Negative regulators:
a. Keap1: Keap1 is a natural intracellular molecule that negatively regulates the activation of Nrf2. Keap1 binds to Nrf2 in the cytoplasm, sends Nrf2 to proteosome digestion, and keeps a low Nrf2 level in cells.
b. Ubiquitin-specific processing protease 15 (USP15): USP15 deubiquitinates Keap1, stabilizes the Keap1-Cul3-E3 ligase complex, and enhances the E3 ligase activity, which leads to the binding between Keap1 and Nrf2 and the degradation of Nrf2 .
c. Trigonelline: trigonelline is a coffee alkaloids that reduce nuclear accumulation of the Nrf2 protein, block expression of proteasomal genes (for example, s5a/psmd4 and α5/psma5), and reduce proteasome activity regulated by Nrf2 .
d. Vitamin C (ascorbic acid) [73, 74] and Vitamin E : these two vitamins are water soluble vitamins with a high capacity to capture ROS. Since Nrf2 is activated via ROS imbalance, elimination of ROS by vitamins keeps low intracellular ROS and ends in low Nrf2 activity.
e. ROS scavenger: ROS scavenger is a common name for molecules that can balance the intracellular ROS, including dithiothreitol (DTT) , N-acetylcysteine (NAC) , catalase , 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) . They are used as standards to check the antioxidant capacity of other molecules. Nrf2 activity remains low where these ROS scavengers keep the intracellular ROS level steady.
a. ROS: ROS imbalance is the key regulator of Nrf2. Excess ROS induces the activation and nuclear translocation of Nrf2 to keep intracellular ROS balance through upregulating the level and activity of antioxidant enzymes.
♦ Disruptor proteins and transcription factors
c. P21 and p62: in addition to the conformational change of Keap1 to loss the bonding affinity to Nrf2, some proteins, such as p21 and p62, can directly bond to Keap1 or Nrf2, disrupting the interaction between Nrf2 and Keap1, thus ending in the nuclear accumulation and activation of Nrf2 [84–86].
d. AhR: Aryl hydrocarbon receptor (AhR), which is a ligand-dependent transcription factor by forming a heterodimer with the aryl hydrocarbon nuclear translocator (Arnt) as a nuclear partner protein. The heterodimeric protein complex regulates expression of Nrf2, and promotes the expression of phase I enzymes, phase II enzymes, and multidrug resistance-associated proteins [87–90]. Nrf2 can also regulate the activation of AhR and subsequently modulates downstream AhR signaling cascades, including increasing the expression of xenobiotic metabolism genes and inhibit the adipogenesis in mouse embryonic fibroblasts (MEFs) .
e. Ebselen: ebselen is a glutathione peroxidase-1 mimetic and a seleno-organic antioxidant. It attenuated cisplatin-induced oxidative stress generation through Nrf2 pathway .
♦ Natural products or extracts
f. Sulforaphane (SFN): sulforaphane, which is found in cruciferous vegetables, belongs to the isothiocyanate family (such as broccoli) and is widely used as an antioxidant supplement and applied in cancer chemoprevention . SFN reacts with Keap1 and block the binding of Keap1 and Nrf2, thus activates Nrf2 and the antioxidant function .
g. Curcumin: curcumin, which is a polyphenolic natural extract of turmeric , is reported to exhibit anti-inflammation and antitumorigenic activity and chemoprevention effect [96, 97]. To activate those protective proteins, curcumin increase the antioxidant genes through regulating the binding of Nrf2 and ARE [98, 99]. Thus, curcumin becomes one of the Nrf2 activator.
h. Resveratrol: in addition to curcumin, resveratrol is another plant extract that regulates antioxidant ability. Resveratrol, the extract from grapes, berries and peanuts, exerts antioxidant, anti-inflammation, and anti-aging effects in experimental animals. Resveratrol is also reported to upregulate Nrf2 activity in cells and organisms to elevate the protection effects toward environmental stresses .
i. Coffee: coffee is one of the most widely consumed beverages in the world. Coffee is noted for its antioxidant ability which protects against chronic liver disease, diabetes, and hepatocarcinoma development with the right amount. The antioxidant ability of coffee is through the activation of Nrf2 and AhR to protect organs from oxidative stress, at least in liver and stomach .
j. Caffeic acid phenethyl ester (CAPE): CAPE, a major component extracted from the bee product propolis in honeybee hives, is known to have antimitogenic, anticarcinogenic, anti-inflammatory activities. It activates the Nrf2 pathway to inhibit oxidative stress and inflammation .
l. Flavonoid (Chrysin, Apigenin, Luteolin): these three flavonoids can reduce the ROS level through activating Nrf2 and producing the downstream phase II enzymes .
♦ Synthesized compounds
m. Oltipraz: oltipraz is an organosulfur compound belonging to the dithiolethione class. It is also a bifunctional inducer activating both phase I and phase II drug-metabolizing enzymes via the xenobiotic responsive element . It has been used as an Nrf2 activator in recent studies [107, 108].
o.Other food and clinical drugs: many other foods or clinical drugs can also affect the expression and activity of Nrf2 in recent studies.
Nrf2 is powerful in the cell defense system toward oxidative stress caused by various physiological and chemical stresses. Nrf2 activation benefits the survival of not only normal cells but also cancer cells. With the anti-oxidation and detoxification abilities of Nrf2, the proliferation, tumorigenicity, migration, and metastasis of cancer cells are higher. The detoxification and drug export mechanism also give cancer cells the ability to fight against chemotherapeutic drugs. With all the characteristics of Nrf2, it is a good marker for both poor prognosis and drug resistance in tumors, both in the regular normoxic environment or under hypoxic environment .