Reactive oxygen species and cancer

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Reactive oxygen species and cancer

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Hyewon Konga,b, Navdeep S. Chandela,b a

Department of Medicine, Division of Pulmonary and Critical Care, Northwestern University Feinberg School of Medicine, Chicago, IL, United States, bDepartment of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, IL, United States

Abstract Reactive oxygen species (ROS) were once considered only as a toxic by-product of aerobic metabolism. Many studies done in the past several decades, however, revealed that ROS have necessary physiological and pathological functions. In the context of cancer, there has been a persistent interest in whether ROS have a tumor-supportive or a tumor-suppressive role. Hydrogen peroxide (H2O2) conducts signaling pathways essential for the survival, proliferation, and metastasis of cancer cells. H2O2, however, can also induce the production of cytotoxic lipid ROS and trigger cancer cell death, such as ferroptosis. As a result, cancer cells increase not only the rate of H2O2 production to hyperactivate the protumorigenic signaling but also their antioxidant capacity to evade the lipid ROS-induced cell death. This unique reliance of cancer cells on both the pro- and antioxidative capacities may provide opportunities to specifically target them via ROS manipulation. In this chapter, we review the major findings that lead to the current understanding of the redox environment in cancer cells and the strategies of redox therapies against cancer. ­Keywords: Cancer, Signaling, Ferroptosis, Lipids, Mitochondria, Therapy

­Introduction High levels of reactive oxygen species (ROS) can induce oxidative damage to cellular macromolecules, such as DNA. Therefore, it was widely considered that ROS are oncogenic by promoting genomic instability (Ames, Shigenaga, & Hagen, 1993). Indeed, patients with diseases associated with increased rates of oxidative DNA damage, including cystic fibrosis, chronic hepatitis, and Fanconi’s anemia, are exposed to significantly higher risks of cancer (Brown, McBurney, Lunec, & Kelly, 1995; Hagen et  al., 1994; Takeuchi & Morimoto, 1993). The majority of ROSinduced DNA damages involve oxidative modifications of guanine (G) to 8-oxo7-hydrodeoxyguanosine (8-oxodG), which cause G to thymine (T) transversions Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00030-4 © 2020 Elsevier Inc. All rights reserved.

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(Shibutani, Takeshita, & Grollman, 1991). Such mutagenic capacity of oxidative stress can enhance the frequency of cancer-causing mutations, including activation of oncogenes or loss of tumor suppressor genes (Du, Carmichael, & Phillips, 1994; Higinbotham et al., 1992). Though ROS facilitate cancer initiation and progression in part by acting as a mutagen, studies in the past two decades highlight a role of ROS as signaling molecules that support cancer cell proliferation, survival, and metastasis. Hydrogen peroxide (H2O2) is the most stable and membrane-penetrable form of ROS and thereby has the highest potential as a secondary messenger in cellular signaling (Reczek & Chandel, 2015). Indeed, H2O2 is essential for the sustained activation of some of the most wellestablished protumorigenic signaling cascades, including phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), mitogenactivated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), and hypoxia-inducible transcription factor (HIF) (Cao et al., 2009; Chandel et al., 1998; Weinberg et al., 2010). Such tumor-supportive functions of ROS have fueled the interest in harnessing antioxidants as therapeutic or prophylactic agents against cancer. Much of the early excitement was sparked in the 1970s when Linus Pauling and Ewan Cameron reported that administration of high dose of antioxidant, vitamin C (10 g/day), in terminal cancer patients significantly prolongs their survival (Cameron & Pauling, 1976). In a chemoprevention trial carried out in Linxian county of China, where the residents have the world’s highest risk of esophageal and gastric cancer, dietary supplementation with a combination of beta-carotene, vitamin E, and selenium significantly lowered the risk of stomach cancer (Blot et al., 1993). However, the excitement about using antioxidant in cancer therapy or prevention was short-lived, as contradictory evidence has emerged: two large-scale clinical trials conducted by Mayo clinic during the 1970s and 1980s failed to demonstrate any anticancer efficacy of vitamin C (Creagan et al., 1979; Moertel et al., 1985). Other antioxidants, including vitamin A, N-acetylcysteine (NAC), beta-carotene, vitamin E, folic acid, and vitamin D, also showed no protective effect against cancer in multiple clinical trials (Fortmann et al., 2013; van Zandwijk, Dalesio, Pastorino, de Vries, & van Tinteren, 2000). Instead, beta-carotene and vitamin E significantly increase risks of lung and prostate cancer (Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group, 1994; Fortmann et al., 2013; Goodman et al., 2004; Klein et al., 2011). Since then, many studies using animal models have demonstrated that antioxidants rather promote cancer progression. NAC and vitamin E markedly accelerates ­tumor growth in mouse models of Kras- and Braf-induced lung cancer (Sayin et al., 2014). NAC also increases lymph node metastasis of endogenous melanoma in mice (Le Gal et al., 2015). Collectively, as a result, an alternative concept on the role of ROS in cancer has risen—ROS may be tumor suppressive. After several decades of investing many resources into defining whether ROS support versus suppress cancer, there is overwhelming evidence backing either argument. This controversy presents a challenge in comprehending the redox biology of

­Introduction

cancer and importantly accessing ROS in cancer therapy: Shall we attenuate it? Or foster it? In an attempt to reconcile the dispute, we review major findings advocating the tumor-supportive or tumor-suppressive functions of ROS and discuss how the two seemingly opposite observations are indeed compatible in a context of redox homeostasis in cancer cells. We propose that mitochondria and NADPH oxidases (NOXs) generate localized production of H2O2 to activate signaling pathways that promote the development and progression of cancer. However, H2O2 can potentially generate lipid hydroperoxides that induce cell death; thus, cancer cells have high levels of enzymes that specifically decrease the levels of lipid hydroperoxides. This allows cancer cells to have elevated levels of H2O2 to promote tumor growth without succumbing to cell death (Fig. 1).

FIG. 1 The state of redox homeostasis in cancer cells is shown. Common features of tumors, including activation of oncogenes, loss of tumor suppressor genes, and adaptation to tumor microenvironment, increase the rate of hydrogen peroxide (H2O2) production by mitochondria and NADPH oxidase (NOX). The H2O2 then selectively and reversibly oxidizes target enzymes, conducting redox signaling essential for the survival, proliferation, and metastasis of cancer cells. Excess H2O2, however, can undergo Fenton reaction, giving rise to hydroxyl radical (OH•). The OH• can react with unsaturated lipid (LH), initiating lipid peroxidation, which generate cytotoxic lipid hydroperoxide (LOOH). Cancer cells, therefore, must upregulate their antioxidant capacities, including glutathione (GSH) and glutathione peroxidase 4 (GPX4) that neutralize lipid hydroperoxides.

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­H2O2 promotes tumorigenesis H2O2 has been shown to transduce cellular signaling pathways by selectively and reversibly oxidizing key cysteine residues within target proteins. In one of the proposed mechanisms of the redox signaling, H2O2 oxidizes the thiol (SH) group of cysteine residues with low acid dissociation constant (pKa), thereby existing as an oxidationsusceptible form of thiolate (S−) under the physiological pH. The process can modify the S− into sulfenic acid (SOH), disulfide bond (S–S), or sulfonamide (S–N), which would result in changes of the structure and thus the enzymatic activities of the target proteins. The oxidized forms of S− can subsequently be reduced back to its original form by antioxidant enzymes, thioredoxin (TRX) and glutaredoxin (GRX) (Finkel, 2012; Reczek & Chandel, 2015). Importantly, the best-characterized targets of such redox regulation are phosphatases and kinases, which take integral parts in protumorigenic signaling pathways (Paulsen et al., 2011; Sohn & Rudolph, 2003). The earliest molecular/cellular evidence implicating H2O2 in the genesis of cancer is an observation from the early 1990s that cancer cells, compared with nontransformed cells, have elevated levels of intracellular ROS (Szatrowski & Nathan, 1991). Such prooxidative environment in cancer cells was later discovered to be essential for tumorigenesis, as H2O2 activates mitogenic signaling pathways (Bae et al., 1997; Irani et al., 1997; Sundaresan, Yu, Ferrans, Irani, & Finkel, 1995). H2O2 is necessary and sufficient for the sustained stimulations of PI3K/AKT/mTOR and MAPK/ERK signaling in cancer cells (Cao et al., 2009; Weinberg et al., 2010). A likely mechanism is the oxidative inactivation of the negative regulators of those pathways, including phosphatase and tensin homolog (PTEN), protein tyrosine phosphatase (PTP), and MAPK phosphatase (Lee et al., 2002; Salmeen et al., 2003; Seth & Rudolph, 2006). The H2O2-induced blockade of the brakes can drive the prosurvival and proliferative PI3K/AKT/mTOR and MAPK/ERK signaling into hyperactivation. The activation of AKT, interestingly, has been shown to increase intracellular H2O2 production (Behrend, Henderson, & Zwacka, 2003; Los, Maddika, Erb, & Schulze-Osthoff, 2009), suggesting an existence of a positive feedback loop to further potentiate the protumorigenic signaling. Additionally, a recent study revealed that H2O2 facilitates another cancer-causing signaling pathway via nuclear factor κ-light chain enhancer of activated B cells (NF-κB). Elevated production of mitochondrial H2O2 is necessary for activating protein kinase D-1 (PKD1) and NF-κB to amplify the proproliferative epidermal growth factor (EGF) signaling, which leads to the development of pancreatic cancer (Liou et al., 2016). The causative impact of H2O2 on the protumorigenic cellular signaling suggests that cancer cells may be selected for an increased generation of H2O2. Indeed, oncogenic transformation leads to elevated levels of intracellular H2O2. For example, expression of HrasV12 oncogene in NIH 3 T3 mouse fibroblasts increases intracellular contents of superoxide (•O2−), which is rapidly converted to H2O2 by superoxide dismutase (SOD) (Irani et  al., 1997). Similar results were found upon transforming ovarian epithelial cells and mouse hematopoietic cells by ectopic expression of HRasV12 or Bcr-Abl, respectively (Trachootham et al., 2006). Since then, several studies have honed in on the

­H2O2 promotes tumorigenesis

sources of the oncogene-driven H2O2. Expression of various oncogenes, myristoylated Akt, HRasV12, or KRasV12, in mouse embryonic fibroblasts increases the H2O2 level specifically in mitochondria, one of the major sites of intracellular H2O2 generation (Weinberg et al., 2010). Similarly, inducing KRasV12 or KRasD12 expression in mouse acinar cells increases the production of mitochondrial H2O2 (Liou et al., 2016). The HRasV12-mediated transformation was also reported to increase H2O2 production by NOX (Ogrunc et al., 2014). Notably, such oncogene-driven increases in H2O2 were detected despite an absence of elevated mitochondrial oxygen consumption (Liou et al., 2016) or an induction of cell senescence (Ogrunc et  al., 2014). Thus, the increased generation of H2O2 by mitochondria and NOX is not just a by-product of the heightened rates of metabolism and proliferation of cancer cells, but rather attributable to the neoplastic transformation or, specifically, the acquisition of oncogenes. The H2O2 production in cancer cells is also stimulated by various elements in the tumor microenvironment, such as growth factors. Addition of EGF to A431 human epidermoid carcinoma cells results in elevated concentrations of intracellular H2O2 (Bae et al., 1997). Platelet-derived growth factor (PDGF) also increases the levels of H2O2 in vascular smooth muscle cells (Sundaresan et al., 1995). Hypoxia, another critical component of tumor microenvironment, has been shown to accelerate mitochondrial H2O2 production (Chandel et al., 1998). The H2O2-rich environment in cancer cells is further potentiated by suppression of cellular antioxidant responses. Many tumor suppressors support antioxidant pathways in nontransformed cells; hence, their loss during oncogenic transformations can lead to elevation of intracellular H2O2. Breast cancer type 1 susceptibility protein (BRCA1), whose loss strongly associates with the risk of breast and ovarian cancers, was found to be essential for activating nuclear factor (erythroid-derived 2)-like 2 (NRF2), known as the master regulator of antioxidant pathways. BRCA1 interferes with the ubiquitination and degradation of NRF2, allowing for NRF2 to stabilize and translocate into the nucleus. There, NRF2 transcribes genes required for the generation and/or utilization of many cellular antioxidants, including glutathione (GSH), peroxiredoxin, thioredoxin, and nicotinamide adenine dinucleotide phosphate (NADPH). As a result, the BRCA1-mutated cancer cells exhibit defective antioxidant responses, hence an accumulation of intracellular ROS, including H2O2 (Cao et al., 2007; Gorrini et al., 2013). Tumor suppressor p53 is another potential activator of NRF2 (Chen et al., 2009; Toledano, 2009). p53 upregulates an expression of a H2O2 scavenger, glutathione peroxidase 1 (GPX1), and production of NADPH (Bensaad et al., 2006; Hussain et al., 2004). As a result, loss of p53 in cancer cells dampens their antioxidant responses and elevates intracellular H2O2 levels. Surprisingly, ablating p53 functions of cell-cycle arrest, apoptosis, and senescence, while leaving its antioxidant function intact, allows p53 to retain its tumor suppressing capacity (Li et al., 2012). Furthermore, dietary supplementation of an antioxidant, NAC, reduces the occurrence and growth of p53-deficient tumors (Sablina et al., 2005). These data suggest that the antioxidant function of certain tumor suppressors is sufficient for their ability to prevent cancer, and the loss of such tumor suppressor drives tumorigenesis mainly as a result of increasing intracellular H2O2 levels.

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Such establishment of H2O2-rich, prooxidative environment in cancer cells is critical for the genesis of cancer. Elevated intracellular H2O2 upon the loss of an antioxidant enzyme, peroxiredoxin 1 (PRDX1), significantly increases the frequency of oncogenic malignancies in mice, including a variety of lymphomas, sarcomas, and carcinomas (Neumann et al., 2003). On the other hand, decreased intracellular H2O2 due to NRF2 overexpression abrogates the in  vivo tumorigenicity of transformed human mesenchymal stem cells (Funes et al., 2014). Interestingly, targeting just the localized H2O2 pool has been shown to be sufficient in impairing tumorigenesis. Reducing the level of mitochondrial H2O2 in transformed cells with mitochondriatargeted chemical antioxidants ablates the oncogene-induced capacity of anchorage-­ independent growth (Weinberg et  al., 2010). Mitoquinone (MitoQ), one of the mitochondria-targeted antioxidants, decreases Kras-induced pancreatic tumorigenesis in  vivo (Liou et  al., 2016). Furthermore, expression of mitochondria-targeted catalase (mCatalase), which converts mitochondrial H2O2 to H2O, in a mouse model of adenomatous polyposis coli multiple intestinal neoplasia (APC(Min/+)) significantly reduces the development of spontaneous colon cancer. In contrast, when the mitochondrial H2O2 level was increased as a result of a heterozygous mutation in mitochondrial transcription factor A, the formation of spontaneous colon cancer increased (Woo et  al., 2012). Together, these data support that the increased levels of intracellular, especially the mitochondrial, H2O2 is necessary and sufficient for tumorigenesis. H2O2 promotes cancer beyond tumorigenesis by supporting metastasis. Indeed, prooxidative status of head and neck squamous cell carcinomas correlates with the presence of lymph node metastasis in patients (Dequanter, Dok, & Nuyts, 2017). Metastasis is a multistep process involving the invasion and migration of primary tumor cells into surrounding tissues, intravasation and survival of the cells in the circulatory or lymphatic systems, extravasation into distant tissues, and establishment of metastatic colonies (Lambert, Pattabiraman, & Weinberg, 2017). The early metastatic steps of migration and invasion occur through a series of cellular changes, including cytoskeletal remodeling and degradation of extracellular matrix. These events are driven by a complex network of signaling pathways in which H2O2 has been identified as an essential conductor (Tochhawng, Deng, Pervaiz, & Yap, 2013). As noted earlier, H2O2 activates MAPK/ERK pathway by oxidizing and inactivating its negative regulators, PTP and MAPK phosphatase. Downstream of the MAPK/ERK pathway, phosphorylated activator protein 1 (AP-1) facilitates the transcription of matrix metalloproteinases, which degrade the surrounding extracellular matrix to pave the way for migrating cancer cells (Ho, Wu, Chang, & Pan, 2011). Additionally, H2O2 induces PI3K signaling, likely by suppressing PTEN as previously described. The pathway leads to activation of protein tyrosine kinase Src and focal adhesion kinase (FAK) that supports the dynamic cytoskeletal remodeling during cell migration (Basuroy, Dunagan, Sheth, Seth, & Rao, 2010). Interestingly, Src and protein tyrosine kinase 2-β (Pyk2), a member of the FAK family, can be activated by mitochondrial ROS (mROS). As a result, mROS, including mitochondrial H2O2, are necessary for the migration and metastasis of multiple types of cancer cells (Porporato et al., 2014).

­Cancer cells limit damaging lipid hydroperoxide accumulation

H2O2 also promotes tumor metastasis by activating HIF. Under the state of ROS homeostasis, HIF is constantly degraded as prolyl hydroxylase enzyme (PHD) hydroxylates critical proline residues within the HIF-α subunit, allowing the VonHippel-Lindau tumor suppressor protein (VHL) to recognize and ubiquitinate HIF-α for proteosomal degradation (Epstein et al., 2001; Kaelin & Ratcliffe, 2008). Under tumor hypoxia, which increases mROS production, mitochondrial H2O2 oxidizes and inhibits the PHD, stabilizing HIF1-α to translocate into the nucleus where it engages in transcriptional activities (Bell et al., 2007; Chandel et al., 1998). Subsequently, hundreds of HIF-driven genes are expressed to confer complex cellular changes promoting tumor metastasis (Rankin & Giaccia, 2016). In human melanoma cells, for example, the mitochondrial H2O2-driven HIF-1α stabilization activates Met protooncogene that enhances variety of prometastatic phenotypes, such as spreading on extracellular matrix, motility, invasion into 3D matrices, growth of metastatic colonies, and ability to form vasculogenic mimicry (Comito et al., 2011). As a result, the H2O2-HIF pathway facilitates multiple steps of the tumor metastasis process.

­ ancer cells limit damaging lipid hydroperoxide C accumulation Cancer cells undergo a constant prooxidative pressure. During tumorigenesis, as discussed earlier, oncogenic transformations and sustained mitogenic signaling elevate the levels of intracellular H2O2 in nascent cancer cells. Subsequently, highly proliferative tumors can outgrow the rate at which the vasculature expands, rendering regions within the tumor to become hypoxic, thereby potentiating mitochondrial H2O2 generation. Tumor cells proliferating outside their matrix niches or intravasating into the circulatory system detach from extracellular matrix, which further increases the levels of intracellular ROS (Schafer et al., 2009). Moreover, the blood and viscera are highly oxidative environments, which elevate the ROS levels in circulating cancer cells (Piskounova et al., 2015). Such accumulation of intracellular H2O2, which facilitates the prosurvival, proliferation, and metastasis signaling, can paradoxically induce cancer cell death. Excess H2O2, not neutralized by oxidizing signaling or antioxidant enzymes, can undergo Fenton reaction, where H2O2 reacts with ferrous ion (Fe2+), leading to the formation of hydroxyl radical (OH•) (Winterbourn, 1995). OH• is essential for initiating lipid peroxidation process, as it abstracts hydrogen (H) from unsaturated lipid (LH), generating lipid radical (L•). The L• rapidly reacts with oxygen (O2), giving rise to lipid peroxy radical (LOO•), which can subsequently abstract H from another LH to produce lipid hydroperoxide (LOOH) and an additional L• (Gaschler & Stockwell, 2017). Such propagation of lipid radicals and hydroperoxides within cellular membranes can substantially damage the lipid bilayers by altering their physical properties, including fluidity, permeability, and thickness (Borst, Visser, Kouptsova, & Visser, 2000; Heffern et al., 2013; Wong-ekkabut et al., 2007). Lipid hydroperoxides can also degrade into malondialdehyde (MDA) or 4-hydroxynonenal (4-HNE), which

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are highly reactive molecules capable of damaging DNA and proteins (Esterbauer, Schaur, & Zollner, 1991). Furthermore, recent studies have identified lipid peroxidation as a hallmark of ferroptosis, an iron and lipid ROS-dependent mode of regulated cell death (Dixon et al., 2012; Yagoda et al., 2007; Yang et al., 2014). Consequently, to exploit high intracellular H2O2 level while evading its downstream cytotoxic effects, cancer cells must bolster their antioxidant capacities that directly or indirectly limit the accumulation of lipid ROS. NRF2, the master regulator of cellular antioxidant responses, is therefore critical for the development and progression of cancer. Expression of oncogenes, KRasD12, Braf, or c-Myc, elevates NRF2 transcription, leading to increased activation of NRF2-regulated antioxidant pathways. Genetic targeting of NRF2 significantly inhibits the KRasD12-induced pancreatic and lung tumorigenesis in  vivo (DeNicola et al., 2011). Such oncogene-induced activation of NRF2 is also necessary for the drug resistance of cancer cells. Chemical targeting of NRF2 with brusatol enhances the efficacy of a chemotherapeutic agent, cisplatin, thereby synergistically reducing the tumor burden of mice with KRasD12-induced lung tumors (Tao et al., 2014). Importantly, inhibiting NRF2 sensitizes cancer cells to ferroptosis-inducing agents, such as erastin, RSL3, and sorafenib, suggesting that NRF2 is essential to protect cancer cells from the lipid ROS-dependent cell death (Fan et al., 2017; Shin, Kim, Lee, & Roh, 2018; Sun et al., 2016). Furthermore, NRF2 supports the mRNA translation and mitogenic signaling required for the proliferation of KRasD12-driven pancreatic cancer cells (Chio et  al., 2016). Such oncogenic advantages of the NRF2 confer many types of human cancer to adopt variety of mechanisms to hyperactivate NRF2. Besides the o­ ncogene-mediated induction, these means include gain-offunction mutations of NRF2 and loss-of-function mutations of its negative regulator, kelch-like ECH-associated protein 1 (KEAP1) (Menegon, Columbano, & Giordano, 2016). Collectively, these data have spurred investigations on targeting the NRF2dependent cancers. Indeed, a recent chemical proteomics approach identified an NRF2-regulated protein, nuclear receptor subfamily 0 group B member 1 (NROB1), as a druggable target that supports nonsmall cell lung cancers (NSCLC) with aberrant NRF2 activations (Bar-Peled et al., 2017). One of the important downstream effectors of the NRF2-activated antioxidant response is glutathione (GSH). GSH is the most abundant cellular antioxidant molecule, consisting of a glycine, a cysteine, and a glutamate. De novo GSH synthesis is first catalyzed by glutamate cysteine ligase (GCL), which ligates a cysteine with a glutamate to produce γ-glutamyl cysteine. The dipeptide is subsequently combined with a glycine by GSH synthetase (GSS), giving rise to a l- γ-glutamyll-cysteinyl-glycine, or GSH. GSH, once scavenges ROS, forms oxidized glutathione or GSSG. GSSG can be reduced back to GSH by glutathione reductase (GR) and NADPH (Bansal & Simon, 2018). Many key mediators of the GSH synthesis and regeneration, including NADPH, GR, GCL, and a cysteine importer, xCT/ SLC7A11, are regulated by NRF2 (Sasaki et al., 2002; Yates et al., 2009). Cancer cells with NRF2 hyperactivation, therefore, would have elevated levels of GSH. Indeed, NADPH, SLC7A11, and GCL are highly upregulated in human tumors

­Cancer cells limit damaging lipid hydroperoxide accumulation

(Harris et al., 2015; Jiang et al., 2015), and increased GSH levels have been observed in tumor tissues from various origins, such as breast, ovarian, head and neck, and lung (Gamcsik, Kasibhatla, Teeter, & Colvin, 2012). The increased GSH content in cancer cells is necessary for the initiation and progression of cancer. Inhibiting the de novo GSH synthesis pathway by genetically and pharmacologically targeting GCL prevents spontaneous tumorigenesis in a mammary tumor (MMTV-PyMT) mouse model (Harris et al., 2015). Limiting cysteine availability by targeting SLC7A11 or administering cysteinase enzyme suppresses the growth of a variety of carcinomas in vivo (Cramer et al., 2017; Gout, Buckley, Simms, & Bruchovsky, 2001; Guo et al., 2011). Importantly, GSH is required for cancer cells to evade ferroptosis. Impairing de novo GSH synthesis by a SLC7A11 inhibitor, erastin, or a GCL inhibitor, buthionine sulfoximine (BSO), initiates ferroptosis in cancer cells (Yang et  al., 2014). Such depletion of GSH causes loss of cellular antioxidant capacities, including glutathione peroxidases (GPXs). GPXs work in concert with GSH to relay multiple redox reactions, which scavenge H2O2: cysteine or selenocysteine residues within GPXs undergo oxidation in return for reducing H2O2 to H2O. The oxidized but inactivated GPXs can subsequently be reduced back by GSH. Among the isozymes of GPX family, GPX4, intriguingly, has high preference to reduce lipid hydroperoxides (Brigelius-Flohé & Maiorino, 2013). Targeting GPX4 in cancer cells, therefore, induces elevation of intracellular lipid ROS contents and ferroptosis, which can be rescued by a lipophilic antioxidant, vitamin E (Yang et al., 2014). Collectively, the data support that GSH facilitates GPX4, which in turn limits accumulation of lipid ROS, conferring cancer cell resistance to ferroptosis. Indeed, the GSH/GPX4 pathway has been discovered to be required for protecting therapy-resistant cancer cells from ferroptotic cell death (Viswanathan et al., 2017). NADPH is a reducing equivalent essential for the antioxidant capacities of cancer cells. Oxidation of NADPH fuels reduction and regeneration of cellular antioxidants, such as GSH. Therefore, availability of this reducing equivalent can affect the intracellular levels of lipid ROS. Indeed, NADPH abundance has been proposed as a promising biomarker of ferroptosis sensitivity across a vast number of cancer cell lines (Shimada, Hayano, Pagano, & Stockwell, 2016). One of the major sources of cytosolic NADPH is oxidative pentose phosphate pathway (PPP), a metabolic pathway branching from glycolysis. Regulatory enzymes of glycolysis, pyruvate kinase isoform M2 (PKM2) and TP53-induced glycolysis regulatory phosphatase (TIGAR), can increase the flux of glucose-derived carbons into the oxidative PPP. Such NADPH-generating capacities of PKM2 and TIGAR were found to be necessary for lung and intestinal cancer cells to suppress oxidative stress, including lipid peroxidation, and establish tumors in vivo (Anastasiou et al., 2011; Cheung et al., 2013). Furthermore, direct or indirect inhibition of glucose-6-phosphate dehydrogenase (G6PD), an enzyme that catalyzes the rate-limiting step of oxidative PPP, reduces intracellular NADPH, followed by an increase in ROS, leading to defects in survival, proliferation, and invasion of multiple types of cancer cells (Du et al., 2013; Lucarelli et al., 2015; Mele et al., 2018).

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One carbon metabolism, also branching from glycolysis, is another major source of NADPH. A glycolytic intermediate 3-phosphoglycerate is first converted to serine via phosphoglycerate dehydrogenase (PHGDH). The serine subsequently donates one carbon unit to tetrahydrofolate (THF), through a reaction catalyzed by serine hydroxymethyltransferase (SHMT). The product 5,10-methyl-THF is then oxidized by methylene THF dehydrogenase (MTHFD), converting NADP+ to NADPH. Importantly, this pathway produces NADPH not only in the cytosol but also in the mitochondria (Fan et  al., 2014; Lewis et  al., 2014). A recent study revealed that metabolic activities of mitochondria, which result in generation of H2O2, contribute to the accumulation of sufficient lipid ROS to initiate ferroptosis (Gao et  al., 2019). Therefore, one‑carbon metabolism that provides a unique opportunity to support mitochondrial antioxidants may protect cancer cells from lipid peroxidation and cell death. Indeed, MYC-transformed cells subjected to hypoxia, which elevates mitochondrial H2O2 generation, significantly upregulate mitochondrial isoform of SHMT, SHMT2. Inhibition of the SHMT2 impairs cancer cell survival under hypoxia, which can be rescued by an antioxidant, NAC (Ye et al., 2014). Additionally, targeting PHGDH in breast cancer cells was shown to disturb the mitochondrial redox homeostasis, leading to reduced rate of survival under hypoxia (Samanta et al., 2016). The cytosolic NADPH generation by one‑carbon metabolism, nonetheless, is also critical for cancer cell survival. Metastasizing human melanoma cells require cytosolic folate pathway enzymes, such as MTHFD1, to survive the prooxidative environment during circulation (Piskounova et al., 2015).

­Targeting the redox biology for cancer therapy H2O2 promotes the development and progression of cancer by mediating cellular signaling pathways necessary for the survival, proliferation, and metastasis of cancer cells. However, excess H2O2 can initiate lipid peroxidation that produces lipid ROS, which are cytotoxic. Therefore, while maintaining an elevated rate of H2O2 production and the H2O2-mediated protumorigenic signaling, cancer cells bolster their antioxidant capacities to battle accumulation of lipid ROS. A recent study demonstrated that in APC-deficient intestinal cells, Wnt signaling upregulates both Ras-related C3 botulinum toxin substrate 1 (RAC1) and TIGAR. RAC1 promotes generation of H2O2 by NOX, while TIGAR supports the NADPH-mediated antioxidant defense. Simultaneous elimination of both, compared with removing RAC1 or TIGAR alone, surprisingly induces more profound proliferative defects of the APC-null intestinal crypts in  vivo (Cheung et  al., 2016). These data are consistent with the model in which both prooxidative and antioxidative capacities are indispensable for cancer. Based on this current understanding of the redox balance in cancer cells, successful redox therapy must attenuate the signaling H2O2 while fostering the toxic lipid ROS. This reveals a challenge in using dietary antioxidants against cancer, as they have limited access to the protumorigenic signaling H2O2 yet can reduce the antitumorigenic lipid ROS (Chandel & Tuveson, 2014). Therefore, recent efforts of

­Targeting the redox biology for cancer therapy

using antioxidants for cancer therapy have focused on targeted antioxidants, which can specifically remove localized pools of the signaling H2O2. Indeed, mitochondriatargeted antioxidants have been shown to be more effective in inhibiting cancer cell proliferation than the same antioxidants that reside in the cytosol (Weinberg et al., 2010). Additionally, MitoQ and MitoTempo suppress in vivo tumorigenesis and metastasis, respectively (Liou et al., 2016; Porporato et al., 2014). This therapeutic approach is made even more probable by recent developments of precision-targeted antioxidants that scavenge ROS at specific sites of mitochondrial electron transport chain without affecting bioenergetic functions of the mitochondria (Brand et  al., 2016; Orr et al., 2015). Given the reliance of cancer cells on their antioxidant capacities to evade cell death, antioxidant pathways that directly or indirectly limit lipid ROS are attractive targets for cancer therapy. Inhibiting NADPH synthesis by genetically targeting TIGAR has been reported to elevate lipid peroxidation and suppress tumorigenesis in vivo (Cheung et al., 2013). Targeting NRF2 sensitizes various types of cancer cells to chemical agents that induce ferroptosis (Fan et al., 2017; Shin et al., 2018; Sun et al., 2016). Moreover, pharmacological targeting of the GSH/GPX4 pathway induces ferroptotic cell death in cancer cells (Yang et al., 2014). Importantly, this therapeutic approach has displayed selective lethality toward cancer cells. Erastin, which inhibits SLC7A11, induces ferroptosis in transformed cells expressing HrasV12, but not in isogenic cell lines without the oncogenic Hras (Yang et al., 2014). Additionally, SLC7A11-deficient mice are healthy (Sato et al., 2005), supporting that targeting the protein in patients may have minimal to no adverse effect. Furthermore, inhibition of GPX4 has been identified to selectively induce ferroptosis in therapy-resistant cancer cells (Hangauer et al., 2017). Collectively, the data encourage development of molecules that potentiate accumulation of the cytotoxic lipid ROS in cancer patients. Interestingly, another therapeutic strategy to amplify the antitumorigenic ROS in cancer cells is to use vitamin C, due to its newly exposed role as a prooxidant. Oxidized form of vitamin C, dehydroascorbate (DHA), enters cells through a glucose transporter, GLUT1. The intracellular DHA is then reduced back to vitamin C by GSH. This process depletes the GSH pool, resulting in elevated oxidative stress and perhaps a lipid ROS accumulation. To switch on this prooxidative mode of vitamin C, it is critical to build high, millimolar concentrations of vitamin C in the blood plasma, which is achieved only by intravenous administration and not via oral administration. Additionally, the intravenous route exposes vitamin C to the oxidizing environment of circulatory system, possibly facilitating oxidation of vitamin C to its therapeutically active form of DHA. As a result, intraperitoneal injections of high dose of vitamin C in mice were found to be effective in diminishing the growth of colon cancer (Yun et al., 2015). Moreover, in a recent phase I/IIa clinical trial, intravenous administration of vitamin C, in combination with a conventional regimen of paclitaxel and carboplatin, showed a therapeutic benefit among a small group of ovarian cancer patients, without significant toxicity (Ma et al., 2014). The data provide a strong justification to conduct larger clinical trials testing efficacy of the high-dose intravenous vitamin C as a single or combinatory agent against various types of cancer.

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­Conclusion Cancer redox therapy has long been under the spotlight of oncology. Historically, the data just appeared convoluted: Sometimes antioxidants suppress cancer; sometimes, they promote it. Based on the current understanding of the redox biology of cancer cells, the paradox has most likely stemmed from the qualitative differences between the tumor-supportive and tumor-suppressive ROS, which coexist within a cancer cell. Therefore, now an intriguing task remains: how to target the tumor-supportive ROS while fostering the tumor-suppressive ROS. Answers to this question will provide significant medical advances that reduce cancer incidence and improve therapeutic outcomes.

­Acknowledgments This work was supported by National Institute of Health grants 5P01HL071643 and 5P01AG049665 to N.S.C. H.K. is supported by National Institute of Health predoctoral training grant T32CA9560.

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